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Abstract of thesis entitled
Mitochondrial transfer from induced pluripotent stem cell-derived
mesenchymal stem cells to airway epithelial and smooth muscle cells
attenuates oxidative stress-induced injury
Submitted by
LI, Xiang
for the degree of Doctor of Philosophy
jointly at the University of Hong Kong and Imperial College London
in June, 2016
Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory
disease characterized by persistent airflow limitation that is not fully reversible and is
usually caused by cigarette smoke (CS). The disease is predicted to be the fourth
leading cause of death by 2030, but none of the currently available treatments can
alleviate the progressive decline in lung function.
Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that
can be isolated from various tissues such as bone marrow (BM-MSCs). Despite
numerous reports of their efficacy in COPD-related pre-clinical models, BM-MSCs
have not demonstrated efficacy in a clinical trial of COPD, highlighting the need for
improved MSC-based therapy. The in vitro derivation of MSCs from induced
pluripotent stem cells (iPSCs) has provided a new source of MSCs. Compared to
BM-MSCs, iPSC-derived MSCs (iPSC-MSCs) are a more abundant source, have a
higher expanding capacity and are possibly not subject to the ageing-associated
dysfunction seen in BM-MSCs.
In this study I determined the effects of human iPSC-MSCs in a rat COPD model
using BM-MSCs as comparison. Rats were exposed to CS for 1 hr/day for 56 days.
iPSC-MSCs or BM-MSCs were administrated at days 29 and 43. iPSC-MSCs
demonstrated superior effects over BM-MSCs in attenuating CS-induced lung
airspace enlargement, fibrosis, inflammation and apoptosis. In a mouse model of
ozone-induced lung damage, intravenous administration of iPSC-MSCs 24 hours
before ozone exposure for 3 hours alleviated airway hyper-responsiveness,
inflammation and apoptosis in the lung.
There is increasing evidence demonstrating that mitochondrial dysfunction may
play an important role in COPD pathogenesis, indicating mitochondria as a potential
therapeutic target. Meanwhile, mitochondrial transfer from MSCs to injured airway
cells has been reported as a novel mechanism of action for MSCs.
In this study mitochondrial transfer from iPSC-MSCs to the airway epithelium of
CS-exposed rats was detected. iPSC-MSCs also transferred mitochondria to bronchial
epithelial BEAS-2B cells and primary airway smooth muscle cell (ASMCs) in vitro in
a direct co-culture system, an effect that was enhanced by CS medium (CSM). Direct
co-culture with iPSC-MSCs alleviated CSM-induced ATP deprivation in BEAS-2B
cells, as well as CSM-induced mitochondrial reactive oxygen species (ROS),
apoptosis and reduction of mitochondrial membrane potential (ΔΨm) in ASMCs.
Administration of iPSC-MSCs also prevented ozone-induced mitochondrial ROS and
ΔΨm reduction in mouse lungs.
The paracrine effects of iPSC-MSCs were also investigated. iPSC-MSC-derived
conditioned medium (iPSC-MSCs-CdM) protected BEAS2-B cells from
CSM-induced apoptosis. The effect was reduced by depleting stem cell factor (SCF)
from iPSC-MSCs-CdM. However, both iPSC-MSCs-CdM and trans-well inserts
containing iPSC-MSCs were only able to alleviate CSM-induced mitochondrial ROS,
but not ΔΨm reduction and apoptosis, in ASMCs.
I demonstrated the capacity of iPSC-MSCs to alleviate oxidative stress-induced
COPD phenotype in vivo. Mitochondrial transfer from iPSC-MSCs was able to
alleviate oxidative stress-induced mitochondrial dysfunction and apoptosis in target
cells. The full capacity of iPSC-MSCs to achieve these effects may rely on a
combination of cell-cell contact and release of paracrine factors. These findings define
iPSC-MSCs as a promising candidate for the development of MSCs-based therapy of
COPD.
Word count: 496
Mitochondrial transfer from induced pluripotent
stem cell-derived mesenchymal stem cells to airway
epithelial and smooth muscle cells attenuates
oxidative stress-induced injury
By
LI, Xiang
M.Phil. H.K., B.Sc. H.K.
A thesis submitted in fulfilment of the requirements for
the Degree of Doctor of Philosophy
jointly at The University of Hong Kong and Imperial College London
June, 2016
The copyright of this thesis rests with the author and is made available
under a Creative Commons Attribution Non-Commercial No Derivatives
licence. Researchers are free to copy, distribute or transmit the thesis on
the condition that they attribute it, that they do not use it for commercial
purposes and that they do not alter, transform or build upon it. For any
reuse or redistribution, researchers must make clear to others the licence
terms of this work.
i
Declaration
I declare that this thesis represents my own work, except where due
acknowledgement is made, and that it has not been previously included in a thesis,
dissertation or report submitted to The University of Hong Kong or Imperial College
London, or to any other institution for a degree, diploma or other qualifications.
LI, Xiang
ii
Acknowledgements
I would like to express my greatest gratitude to my primary supervisors Dr. Judith
C.W. Mak, The University of Hong Kong (HKU) and Professor Kian Fan Chung,
Imperial College London (ICL) for giving me the opportunity to do this joint Ph.D.
between the two universities, and for their guidance and support during the period of
study. I would also like to thank my co-supervisor Dr. Pankaj K Bhavsar (ICL) for his
precious supports and help especially on the progress management of the projects.
Special thanks are given to my assistant supervisor Dr. Charalambos Michaeloudes
(ICL) for his valuable suggestions on the projects and help on laboratory skills. I
would also like to thank Dr. Coen H Wiegman (ICL) for his generous help on the
animal study.
Sincere thanks are given to Professor Mary S.M. Ip from the Division of
Respiratory Medicine, Dr. Qizhou Lian and Professor Hung-fat Tse from the Division
of Cardiology, HKU for the initiation of the collaborative work on mesenchymal stem
cells and lung diseases in HKU. I would like to thank Dr. Yuelin Zhang from the
Division of Cardiology for his intense collaboration and for providing the
mesenchymal stem cells used in this study.
Grateful thanks are given to my dear labmates in HKU, Mr Dave Yeung, Dr.
Yingmin Liang, Ms Wang Yan, Ms Yuting Cui, Dr. Chunyan Zheng, Dr. Yuanyuan Li
and Dr. Sze-Kwan Lam for their friendship and support.
Finally, I would like to give my deepest thanks to my family. I thank my parents
iii
for their love and encouragement and my wife Mengke for her trust, patience and love
across the long distance. I would not have achieved this Ph.D without her support.
iv
Part of the results in this thesis has been published in the following
publications:
Journal article
1. Li X, Zhang Y, Yeung SC, Liang Y, Liang X, Ding Y, Ip MS, Tse HF, Mak JC,
Lian Q. (2014) Mitochondrial transfer of induced pluripotent stem cell-derived
mesenchymal stem cells to airway epithelial cells attenuates cigarette
smoke-induced damage. Am J Respir Cell Mol Biol. 51(3):455-65
Abstracts
1. Li X, Michaeloudes C, Zhang Y, Lian Q, Mak JCW, Bhavsar PK, Chung KF.
(2015) Oxidative Stress-Induced Mitochondria Alteration in Human Airway
Smooth Muscle Cells and Mesenchymal Stem Cells. Am J Respir Crit Care Med
191;2015:A5544 (Abstract)
2. Li X, Zhang Y, Yeung SC, Liang Y, Liang X, Ding Y, Ip MS, Tse HF, Mak JC,
Lian Q. (2014) Mitochondrial transfer of induced pluripotent stem cell-derived
mesenchymal stem cells to airway epithelial cells attenuates cigarette
smoke-induced damage. Am J Respir Crit Care Med 189;2014:A5295 (Abstract)
3. Li X, Zhang Y, Yeung SC, Lian Q, Tse HF, Ip MSM, Mak JCW. (2013) Effects of
induced pluripotent stem cell- and bone marrow-derived mesenchymal stem cells
on cigarette smoke-induced lung damage in rats. Am J Respir Crit Care Med
187;2013:A4850 (Abstract)
v
Table of Contents
Chapter 1. Introduction ............................................................................................... 1
1.1. Introduction to Chronic Obstructive Pulmonary Disease (COPD) .................................. 2
1.1.1. Definition and diagnosis of COPD .................................................................................. 2
1.1.2. Epidemiology of COPD .................................................................................................. 5
1.1.3. Risk factors of COPD ...................................................................................................... 7
1.1.4. Pathogenesis of COPD .................................................................................................... 9
1.1.4.1. Oxidative stress ..................................................................................................... 11
1.1.4.2. Chronic airway inflammation ............................................................................... 13
1.1.4.3. Protease and anti-protease imbalance ................................................................... 15
1.1.4.4. Apoptosis .............................................................................................................. 16
1.1.4.5. Ageing ................................................................................................................... 19
1.1.4.6. Bronchial epithelial cells and airway smooth muscle cells in COPD ................... 20
1.1.4.7. Mitochondria and COPD ...................................................................................... 22
1.1.5. Current therapies ........................................................................................................... 27
1.2. Introduction to Stem Cells .................................................................................................. 29
1.2.1. Definition and classification .......................................................................................... 29
1.2.2. Endogenous stem cells in lung ...................................................................................... 30
1.2.3. Mesenchymal stem cells (MSCs) .................................................................................. 32
1.2.4. Induced-pluripotent stem cells (iPSCs) ......................................................................... 33
1.2.5. iPSC-derived MSCs ...................................................................................................... 34
1.3. MSCs-based Therapy .......................................................................................................... 37
1.3.1. Efficacy of MSCs in animal models .............................................................................. 37
1.3.2. Clinical trials ................................................................................................................. 44
1.3.3. Mechanisms ................................................................................................................... 46
1.3.3.1. Engraftment and regeneration ............................................................................... 46
1.3.3.2. Immunoregulation................................................................................................. 47
1.3.3.3. Mitochondrial transfer .......................................................................................... 48
1.4. Aims of Study ....................................................................................................................... 52
Chapter 2 Material and Methods ............................................................................. 54
2.1 Materials ............................................................................................................................... 55
2.1.1 Reagents and chemicals ................................................................................................. 55
2.1.2 Experimental animals ..................................................................................................... 55
2.1.3 Human iPSC-MSCs ........................................................................................................ 56
2.1.4 Human bone marrow – derived MSCs (BM-MSCs) ...................................................... 57
2.1.5 BEAS-2B cells ............................................................................................................... 57
2.1.6 Primary human airway smooth muscle cells (ASMCs).................................................. 58
2.2 Methods ................................................................................................................................. 59
2.2.1 MSCs treatment to cigarette smoke-exposed rats .......................................................... 59
2.2.2 Histology and morphometric analysis of rat lungs ......................................................... 61
2.2.3 iPSC-MSCs treatment to ozone-exposed mice ............................................................... 62
2.2.4 Measurement of airways hyper-responsiveness (AHR) of mice .................................... 65
2.2.5 Measurement of cells and cytokines in BAL of mice ..................................................... 65
vi
2.2.6 Isolation of intact mitochondria from mouse lungs ........................................................ 66
2.2.7 Generation of cigarette smoke medium (CSM) .............................................................. 67
2.2.8 Co-culture of BEAS-2B cells with MSCs. ..................................................................... 67
2.2.9 Determination of ATP content in BEAS-2B cells following co-culture ......................... 68
2.2.10 BM-MSC and iPSC-MSC conditioned medium treatment of CSM-treated BEAS-2B
cells ......................................................................................................................................... 69
2.2.11 Immunohistochemistry ................................................................................................. 70
2.2.12 Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)
assay ........................................................................................................................................ 71
2.2.13 Total protein extraction from cell culture ..................................................................... 72
2.2.14 Western blot .................................................................................................................. 73
2.2.15 Enzyme-linked immunosorbent assay (ELISA) ........................................................... 73
2.2.16 Treatment of CSM or H2O2 to ASMCs or iPSC-MSCs ................................................ 74
2.2.17 Cell viability assay ....................................................................................................... 75
2.2.18 Measurement of mitochondrial ROS ............................................................................ 75
2.2.19 Mitochondrial membrane potential .............................................................................. 76
2.2.20 Annexin V staining ....................................................................................................... 76
2.2.21 Direct co-culture of ASMCs and iPSC-MSCs .............................................................. 77
2.2.22 Detection of mitochondrial transfer in co-culture of ASMCs and iPSC-MSCs ........... 78
2.2.23 iPSC-MSCs-conditioned medium treatment to ASMCs .............................................. 80
2.2.24 Transwell co-culture between ASMCs and iPSC-MSCs .............................................. 80
2.2.25 Statistical analysis ........................................................................................................ 80
Chapter 3. iPSC-MSCs Attenuate CS-induced Damage in Airway Epithelial Cells
through Mitochondrial Transfer and Paracrine Effect .......................................... 82
3.1. Introduction ......................................................................................................................... 83
3.2. Effects of iPSC-MSCs on CS-induced lung damage in rats. ............................................ 85
3.2.1. iPSC-MSCs attenuated CS-induced airspace enlargement, fibrosis and inflammation in
rat lung .................................................................................................................................... 85
3.2.2. iPSC-MSCs attenuated CS-induced apoptosis/proliferation imbalance in rat lung
epithelium. .............................................................................................................................. 90
3.2.3. Mitochondrial transfer from MSCs to adjacent lung cells ............................................ 91
3.3. Mitochondrial Transfer from MSCs to BEAS-2B cells in vitro ....................................... 94
3.3.1. Mitochondrial transfer from MSCs to BEAS-2B cells .................................................. 94
3.3.2. CSM promoted mitochondrial transfer from MSCs to BEAS-2B cells ........................ 96
3.3.3. Co-culture with iPSC-MSCs elevated ATP levels in BEAS-2B cells ........................... 96
3.4. iPSC-MSCs Attenuated CSM-induced Apoptosis and Proliferation Imbalance in
BEAS-2B Cells by Paracrine Effect .......................................................................................... 98
3.4.1. iPSC-MSCs-CdM attenuates CSM-induced apoptosis in BEAS-2B cells .................... 98
3.4.2. Effects of iPSC-MSCs-CdM on apoptosis and proliferation of BEAS-2B cells were
SCF-dependent ...................................................................................................................... 101
3.5. Discussion ........................................................................................................................... 105
Chapter 4. iPSC-MSCs attenuated CSM-induced Mitochondrial Dysfunction in
Human Airway Smooth Muscle Cells .................................................................... 115
4.1. Introduction ....................................................................................................................... 116
vii
4.2. Oxidative Stress-induced Mitochondrial Dysfunction in ASMCs and iPSC-MSCs .... 119
4.2.1. Oxidative stress reduced the viability of ASMCs ........................................................ 119
4.2.2. Oxidative stress induced mitochondrial ROS in ASMCs ............................................ 121
4.2.3. Oxidative stress induced reduction of ΔΨm in ASMCs .............................................. 123
4.2.4. Oxidative stress reduced cell viability of iPSC-MSCs ................................................ 125
4.2.5. Oxidative stress induced mitochondrial ROS in iPSC-MSCs ..................................... 127
4.2.6. Oxidative stress induced reduction of ΔΨm in iPSC-MSCs ....................................... 129
4.3. Direct Co-culture with iPSC-MSCs Attenuated CSM-induced Mitochondrial
Dysfunction and Apoptosis in ASMCs .................................................................................... 131
4.3.1. iPSC-MSCs prevented and reversed CSM-induced mitochondrial ROS in ASMCs .. 131
4.3.2. iPSC-MSCs prevented but did not reverse CSM-induced reduction in ΔΨm in ASMCs
.............................................................................................................................................. 134
4.3.3. iPSC-MSCs prevented CSM-induced apoptosis of ASMCs ....................................... 136
4.4. Paracrine Effects are only Partly Involved in the Protective Effects of iPSC-MSCs on
CSM-induced Mitochondrial Dysfunction and Apoptosis in ASMCs .................................. 138
4.4.1. Effects of conditioned medium from iPSC-MSCs on CSM-induced mitochondrial
dysfunction in ASMCs .......................................................................................................... 138
4.4.2. Effect of iPSC-MSCs-CdM on CSM-induce apoptosis of ASMCs ............................. 139
4.4.3. Effects of trans-well co-culture with iPSC-MSCs on CSM-induced mitochondrial
alterations in ASMCs ............................................................................................................ 142
4.4.4. Effect of trans-well co-culture with iPSC-MSCs on CSM-induced apoptosis in ASMCs
.............................................................................................................................................. 144
4.5. Mitochondrial Transfer from iPSC-MSCs to ASMCs .................................................... 145
4.5.1. Mitochondrial transfer from iPSC-MSCs to ASMCs .................................................. 145
4.5.2. CSM increased mitochondrial transfer from iPSC-MSCs to ASMCs ......................... 145
4.6. Discussion ........................................................................................................................... 149
Chapter 5. Effects of iPSC-MSCs on Ozone-induced Airway
Hyper-responsiveness, Lung Inflammation, Apoptosis and Mitochondrial
Dysfunction in Mice ................................................................................................. 157
5.1. Introduction ....................................................................................................................... 158
5.2. Results ................................................................................................................................ 159
5.2.1. iPSC-MSCs prevented ozone-induced AHR ............................................................... 159
5.2.2. Effect of iPSC-MSCs on ozone-induced infiltration of inflammatory cells in the lung
.............................................................................................................................................. 161
5.2.3. Effect of iPSC-MSCs on cytokine contents of BALF ................................................. 164
5.2.4. Effects of iPSC-MSCs on apoptosis and proliferation in ozone-exposed lung tissue . 166
5.2.5. Effect of iPSC-MSCs on ozone-induced cellular ROS and mitochondrial dysfunction in
mouse lungs .......................................................................................................................... 169
5.3. Discussion ........................................................................................................................... 172
Chapter 6. General Discussion ................................................................................ 177
6.1. General Discussion ............................................................................................................ 178
6.2. Conclusion .......................................................................................................................... 190
6.3. Future Studies .................................................................................................................... 192
References ................................................................................................................. 194
viii
List of Figures
Figure 1.1 Cause of airflow limitation in airways of patients with COPD.. ................. 3
Figure 1.2 Measurement of airflow limitation by spirometry. ...................................... 3
Figure 1.3 Prevalence of COPD (GOLD stage 2 or higher) in 2007 ............................ 7
Figure 1.4 Interplay of multiple events leading to emphysema .................................. 10
Figure 1.5 Generation of mitochondrial ROS from electron transport chain. ............ 27
Figure 1.6 Change of potency: from somatic cells to iPSC-MSCs ............................. 37
Figure 1.7 Proposed mechanism of mitochondrial transfer. ....................................... 51
Figure 2.1 Schematic diagram of BM-/iPSC-MSCs treatments in a CS-exposed rat
model ..................................................................................................................... 60
Figure 2.2 The set-up of the ventilated smoking chamber .......................................... 61
Figure 2.3 The set-up of the ventilated chamber for ozone exposure ......................... 63
Figure 2.4 Schematic diagram of iPSC-MSCs treatments in an ozone-exposed mouse
model ..................................................................................................................... 64
Figure 2.5 Protocols of direct co-culture of iPSC-MSCs and ASMCs ....................... 79
Figure 3.1 Human iPSC-MSCs and BM-MSCs alleviated CS-induced airspace
enlargement in rats ................................................................................................ 87
Figure 3.2 BM-MSCs and iPSC-MSCs attenuated CS-induced lung fibrosis in rats . 88
Figure 3.3 BM-MSCs and iPSC-MSCs ameliorated CS-induced inflammation in rat
lungs ...................................................................................................................... 89
Figure 3.4 BM-MSCs and iPSC-MSCs attenuated CS-induced increase in apoptosis
ix
and reduction of proliferation in rat lung epithelium ............................................ 92
Figure 3.5 Mitochondria transfer from MSCs to lung epithelium in rats ................... 93
Figure 3.6 Mitochondrial transfer from BM-MSCs and iPSC-MSCs to BEAS-2B
cells in co-culture .................................................................................................. 95
Figure 3.7 CSM promoted mitochondrial transfer ...................................................... 97
Figure 3.8 iPSC-MSCs restored intracellular ATP levels of BEAS-2B cells ............. 98
Figure 3.9 CdM from BM-MSCs and iPSC-MSCs ameliorated CSM-induced
apoptosis/proliferation imbalance in BEAS-2B cells. ........................................ 100
Figure 3.10 Secretion of SCF by BM-MSCs and iPSC-MSCs and expression of C-kit
by BEAS-2B cells ............................................................................................... 103
Figure 3.11 The anti-apoptotic and pro-proliferation activity of iPSC-MSCs-CdM was
partly through SCF .............................................................................................. 104
Figure 4.1 Effects of H2O2 and CSM on the viability of ASMCs ............................. 120
Figure 4.2 H2O2 and CSM induced mitochondrial ROS in ASMCs ......................... 122
Figure 4.3 H2O2 and CSM reduced ΔΨm in ASMCs ................................................ 124
Figure 4.4 Effects of H2O2 and CSM on viability of iPSC-MSCs. ........................... 126
Figure 4.5 Effects of H2O2 and CSM on mitochondrial ROS of iPSC-MSCs .......... 128
Figure 4.6 Effects of H2O2 and CSM on ΔΨm of iPSC-MSCs ................................. 130
Figure 4.7 Effects of direct co-culture with iPSC-MSCs on mitochondrial ROS in
ASMCs ................................................................................................................ 133
Figure 4.8 Effects of direct co-culture with iPSC-MSCs on ΔΨm in ASMCs.......... 135
Figure 4.9 Effects of preventive co-culture with iPSC-MSCs on apoptosis in ASMCs
x
treated with CSM ................................................................................................ 137
Figure 4.10 Effects of iPSC-MSCs-CdM on mitochondrial ROS and ΔΨm in ASMCs
............................................................................................................................. 140
Figure 4.11 Effects of iPSC-MSCs-CdM on CSM-induced apoptosis in ASMCs ... 141
Figure 4.12 Effects of trans-well co-culture with iPSC-MSCs on mitochondrial ROS
and ΔΨm in ASMCs ............................................................................................ 143
Figure 4.13 Effects of trans-well co-culture with iPSC-MSCs on apoptosis in ASMCs
............................................................................................................................. 144
Figure 4.14 Mitochondrial transfer from iPSC-MSCs to ASMCs ............................ 147
Figure 4.15 CSM enhanced mitochondrial transfer from iPSC-MSCs to ASMCs ... 148
Figure 5.1 iPSC-MSCs prevented ozone-induced AHR ........................................... 160
Figure 5.2 Effects of iPSC-MSCs on cell counting of BAL from ozone-exposed mice
............................................................................................................................. 163
Figure 5.3 Effects of iPSC-MSCs on cytokine levels in BALF after ozone exposure
............................................................................................................................. 165
Figure 5.4 Effects of iPSC-MSCs on ozone-induced apoptosis in mouse lung ........ 167
Figure 5.5 Effects of ozone and iPSC-MSCs on cellular proliferation in lung ......... 168
Figure 5.6 Effects of iPSC-MSCs on ozone-induced ROS and mitochondrial
dysfunction in lung ............................................................................................. 171
Figure 6.1 Summary of the effects of iPSC-MSCs on oxidative stress induced lung
cell damage ......................................................................................................... 191
xi
List of abbreviations
AEC1s type 1 alveolar epithelial cells
AEC2s type 2 alveolar epithelial cells
AHR airway hyper-responsiveness
ASM airway smooth muscle
ASMC airway smooth muscle cell
ATP adenosine triphosphate
BAL bronchoalveolar lavage
BALF bronchoalveolar lavage fluid
bFGF basic fibroblast growth factor
BOLD Burden of Obstructive Lung Disease
BM bone marrow
BM-MSC bone marrow-derived mesenchymal stem cell
BSA bovine serum albumin
cAMP cyclic adenosine monophosphate
CAT COPD assessment test
CC10 clara cell secretory protein 10
CCL chemokine ligand
CdM conditioned medium
COPD chronic obstructive pulmonary disease
CRP C-reactive protein
CS cigarette smoke
CSM cigarette smoke medium
DAB 3,3'-diaminobenzidine
DAMP danger-associated molecular pattern
DAPI 4,6-diamidino-2-phenylindole
DMEM Dulbecco‟s modified Eagle medium
EGF epidermal growth factor
xii
ELISA enzyme-linked immunosorbent assay
ESC embryonic stem cell
FACS fluorescence-activated cell sorting
FEV₁ forced expiratory volume in 1 s
FISH fluorescent in situ hybridization
FVC forced vital capacity
GBD Global Burden of Disease
GM-CSF granulocyte macrophage-colony stimulating factor
GOLD global initiative for chronic obstructive lung disease
GPx glutathione peroxidase
GRO growth-related oncogene
GSH glutathione
GSSG oxidized glutathione
GVHD graft-versus-host disease
HBSS Hank‟s Balanced Salt Solution
HNA human nuclear antigen
HRP horseradish peroxidase
HSC haematopoietic stem cell
IFN interferon
IL interleukin
IP-10 IFN-γ-inducible protein 10
iPSC induced-pluripotent stem cell
iPSC-MSC induced-pluripotent stem cell-derived mesenchymal stem cell
iPSC-MSCs-CdM iPSC-MSCs-conditioned medium
JC-1 5,5‟,6,6‟-tetrachloro-1,1‟,3,3‟-tetraethylbenzimidazolylcarbocyanineiodide
KC keratinocyte-derived chemokine
KIF5 kinesin motor protein-5
LABA long-acting β2-agonist
Lm mean linear intercept
xiii
LPS lipopolysaccharide
MCP monocyte chemotactic protein
MDA malondialdehyde
MEF mouse embryonic fibroblast
MHC major histocompatibility complex
MIP macrophage inflammatory protein
Miro mitochondrial Rho GTPase
MMP matrix metalloproteinases
mMRC Modified British Medical Research Council
MPO myeloperoxidase
MSC mesenchymal stem cell
MTT methylthiazolyldiphenyl-tetrazoliumbromide
PGE2 prostaglandin E2
PLATINO Latin American Project for Investigation of Obstructive Lung Disease
PRR pattern-recognition receptor
PT permeability transition
PVDF polyvinylidene fluoride
RL pulmonary resistance
ROS reactive oxygen species
SA sham air
SABA short-acting β2 agonist
SCF stem cell factor
SCID severe combined immunodeficient
SDS sodium dodecyl sulfate
SEM standard error of mean
SFTPC surfactant protein C
SLPI secretory leukoprotease inhibitor
SOD superoxide dismutase
TGF transforming growth factor
xiv
TIMP tissue inhibitors of MMP
TL telomere length
TMB tetramethylbenzidine
TNF tumor necrosis factor
TNT tunnelling nanotube
TSG-6 tumor necrosis factor-stimulated gene-6
TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
VEGF vascular endothelial growth factor
ΔΨm mitochondrial membrane potential
1
Chapter 1. Introduction
2
1.1. Introduction to Chronic Obstructive Pulmonary Disease
(COPD)
1.1.1. Definition and diagnosis of COPD
The Global Initiative for Chronic Obstructive Lung Disease (GOLD) defines
chronic obstructive pulmonary disease (COPD) as „a common preventable and
treatable disease‟ which is „characterized by persistent airflow limitation that is
usually progressive and associated with an enhanced chronic inflammatory response
in the airways and the lung to noxious particles or gases‟ (263). The chronic
inflammation drives small airways disorder named obstructive bronchiolitis leading to
structural alterations and narrowing of small airways, and emphysema characterized
by destruction of parenchyma which causes separation of alveolar from the small
airways and loss of lung elastic recoil (263). Obstructive bronchiolitis and
emphysema together causes the persistent airway flow limitation in patients with
COPD (Figure 1.1), but the contribution from each of them varies within individual
patients (263).
COPD is diagnosed by post-bronchodilator spirometry to measure persistent
airway limitation. Spirometry measures volume of air that a patient can exhale at a
maximal force (as long and as quickly as possible) after a maximal deep inspiration
(208). The total volume of air exhalation with greatest effort of the patient is defined
as forced vital capacity (FVC) (208). The total volume of air exhalation with greatest
effort of the patient in the first second is defined as forced expiratory volume in 1 s
(FEV₁) (208). Persistent airway limitation is confirmed when the ratio of FEV₁ to
3
FVC is less than 0.7 (263) (Figure 1.2).
Figure 1.2 Measurement of airflow limitation by spirometry. Patients are asked to
exhale as long and as quickly as possible. Exhalation volume from (A) normal lungs
or (B) obstructive lungs is plotted against time. FVC: forced vital capacity; FEV1:
forced expiratory volume in the first second; Airway obstruction is defined if
FEV1/FVC is less than 0.7. The figure is reproduced based on plots by Ranu H et al,
Ulster Med J (208).
Figure 1. 1 Cause of airflow limitation in airways of patients with
COPD. Compared to (A) healthy lungs, lungs with (B) COPD demonstrate
parenchymal destruction leading to emphysema, mucosal and peribronchial
inflammation and fibrosis leading to obliterative bronchiolitis, and mucus
hypersecretion leading to luminal obstruction. As a result, patients with
COPD develop irreversible airflow limitation. Adapted from Barnes PJ, N
Engl J Med (22).
4
Further assessment of COPD includes determination of levels of symptoms,
severity of airflow limitation, exacerbation risk and comorbidities. The major
symptoms of the disease include chronic and progressive dyspnea, cough and sputum
production. The levels of symptoms can be assessed by questionnaires such as
Modified British Medical Research Council (mMRC) questionnaire on breathlessness
and COPD Assessment Test (CAT). The severity of airflow limitations is graded into
four different stages by GOLD based on the post-bronchodilator FEV₁ (Table 1.1).
The spirometric criteria for Mild, Moderate, Severe and Very Severe severities are
80%, 50% and 30% of predicted FEV₁. Exacerbations refer to short periods (at least
48 hours) of symptom worsening such as increased cough and dyspnea which is
beyond normal variations (65, 263). Exacerbations lead to reduced quality of life,
accelerated disease progression and increased risk of death (65, 71, 236).
Exacerbations usually call for changes in medication by which their severity can be
defined, including change of inhaled treatment (mild exacerbation), medical
intervention such as antibiotic and oral steroids (moderate exacerbation) and
hospitalization (severe exacerbation). A combined assessment of COPD which
integrates levels of symptoms, severity of airway limitation and exacerbation history
is suggested by GOLD. The assessment groups patients with COPD into four
sub-groups demonstrating different burden of symptom and risk of exacerbation
(Group A, low risk and less symptoms; Group B, low risk and more symptoms; Group
C, high risk and less symptoms; Group D, high risk and more symptoms). The burden
of symptoms is defined high or low by mMRC or CAT grade. The risk of
5
exacerbation is defined high if the severity of airway limitation is GOLD 3-4 or there
are two or more exacerbations in the preceding year. In addition to the combined
assessment approach, assessment of comorbidities is also important. COPD is highly
associated with comorbidities including ischaemic heart disease, diabetes, skeletal
muscle wasting, cachexia, osteoporosis, depression and lung cancer (65, 263). The
comorbidities can increase the risk of admission to hospital and death (234).
Table 1.1 GOLD Severity of airflow limitation in COPD (FEV₁/FVC<0.70)
GOLD stage Severity Post-bronchodilator FEV₁
Stage 1 Mild FEV₁≥ 80% of predicted
Stage 2 Moderate 50% ≤FEV₁< 80% of predicted
Stage 3 Severe 30% ≤FEV₁< 50% of predicted
Stage 4 Very severe FEV₁< 30% of predicted
1.1.2. Epidemiology of COPD
The prevalence of COPD varies among different reports, maybe due to different
assessing criteria and methods of survey and analysis (96). A recent meta-analysis
report estimated that the worldwide COPD cases in people aged 30 or more had
increased from 227.3 million in 1990 to 384.0 million in 2010, while the prevalence
was 10.7% in 1990 and 11.7% in 2010 (3). The actual prevalence may be higher than
the data from epidemiological studies as COPD is considered to be under-diagnosed
in general (259). It is estimated that 60-85% patients with COPD may be undiagnosed,
6
especially for the mild and moderate severities (GOLD stage 1 or 2) (114).
COPD prevalence varies among different countries and different groups of people
(e.g. gender and smoking history) (Figure 1.3). For example, the estimation of male
population with COPD of GOLD 2 severity or above was estimated as 22.2% in Cape
Town, South Africa, in comparison to 8.5% in Reykjavik, Iceland (168). Based on the
data from two large multinational studies, the Burden of Obstructive Lung Disease
(BOLD) study and the Latin American Project for Investigation of Obstructive Lung
Disease (PLATINO) study, the prevalence of COPD in different countries ranges from
3% to 23% (39, 168). The prevalence of COPD has been stable in some developed
countries, while in the United States, it is the only common cause of death that has
increased over the last 40 year (23). In developing countries such as some African
countries, the prevalence increased more dramatically (65). Variations among nations
were also observed in mortality studies. For example, the death rate of patients with
COPD was estimated to be 130.5/100,000 in China in comparison to 4.4/100,000 in
Japan, although the prevalence is similar between the two countries (168). COPD was
ranked as the 6th leading cause of death worldwide in 1990, and was predicted to be
the 3rd leading cause by 2020. A more recent study estimated COPD as 4th leading
cause of death in 2030 (172). Based on the data of 2010 Global Burden of Disease
(GBD) study, COPD led to 2.9 million deaths, accounting for about 5% of total deaths
globally (161, 181).
The economic burden associated with COPD is huge. For example, in 2003 the
United States spent US$ 32.1 billion in total on COPD, US$ 18.0 billion of which on
7
direct healthcare costs and the rest on indirect morbidity and mortality costs (168). In
2010 the number increased to US$ 49.9 billion in total and US$ 29.5 billion on direct
healthcare costs (49). In United Kingdom, in 2007 the healthcare cost of COPD is
three fold higher than asthma and the consultation rate of COPD is two to four-fold
higher than ischemic heart disease (23).
Figure 1. 3 Prevalence of COPD (GOLD stage 2 or higher) in 2007. Combined
results from BOLD study and PLATINO study are demonstrated in the figure. The
data were collected from a city or region of the country thus do not represent national
population. Adapted from Mannino DM et al, the Lancet (169).
1.1.3. Risk factors of COPD
COPD results from the interaction between gene and environmental stimuli (65).
The most common genetic risk for COPD is deficiency of α1 antitrypsin, a circulating
inhibitor of serine proteases. Mutation of genes encoding matrix metalloproteinase-12
(MMP-12) (113) and transforming growth factor β1 (47) have also been reported in
association with COPD.
8
Cigarette smoking is the most common worldwide risk factor for COPD.
Cigarette smoking is associated with an increase in prevalence of respiratory
symptoms, lung function abnormalities, annual rate of FEV1 decline, and COPD
mortality rate (136). The association between development of COPD and cigarette
smoking is dose-dependent (292), and smoking cessation reduces rates of morbidity
and mortality in comparison to continued smoking (209). Besides normal cigarette
smoking, passive exposure to cigarette smoke in the environment is also a risk factor
for COPD (73). For example, in China where 30% of the world‟s cigarettes are
consumed (277), 12.1% of male and 51.3% of female nonsmokers are exposed to
environmental smoke at home, while 26.7% of male and 26.2% of female
non-smokers are exposed to environmental smoke in the work place in 2001 (92). In
never-smokers with COPD, the passive exposure to cigarette smoke contributed to 1.9
million additional deaths in China (280). Maternal smoking during pregnancy may
also increase the risk of COPD to the fetus (245). Although cigarette smoking is
recognized as the major risk factor for COPD, COPD does not develop in some
people with the same smoking history (263). In addition, COPD may also develop in
some nonsmokers (48, 141), indicating the importance of other risk factors.
In addition to environmental smoke, other particles and gases in environment can
also contribute to development of COPD, such as occupational exposure to
hazardous dusts and fume (17) and indoor pollutant from biomass burning for family
cooking and heating (36, 75, 191, 207).
Age is commonly considered as a risk factor for COPD as the prevalence,
9
morbidity and mortality increases along with increased age (38, 126, 137). Gender
was regarded as one risk factor in the past with men presenting higher prevalence and
mortality of COPD than women (233). However, the prevalence of COPD in men and
women from high-income countries has become more similar now (39, 233).
Development of COPD can be also associated with many other risk factors
including impaired lung development, asthma, bronchial hyper-responsiveness,
asthma, infections and social status (168).
1.1.4. Pathogenesis of COPD
The most significant pathophysiological feature of COPD is airflow limitation
resulting from narrowing of the small airway compartment and destruction of
parenchyma, both leading to gas trapping and subsequently hyperinflation (186).
Other pathophysiological changes that may present in patients with COPD include gas
exchange abnormalities, mucus hypersecretion, pulmonary hypertension and other
comorbidities (263). The pathological changes in patients with COPD mainly include
infiltration of various inflammatory cells, structural changes of small airways such as
thickening of walls and mucus overproduction, and destruction of parenchyma (60,
263). The pathogenesis of COPD, including the mechanisms driving the chronic
inflammation, remodeling of small airways and parenchymal destruction is still not
fully understood (65). Current evidence indicates a complex interplay between
oxidative stress, inflammatory responses, protease and anti-protease imbalance,
ageing, and changes in apoptosis, proliferation and tissue repair process (57). A
10
simple illustration of the factors that leads to emphysema is shown in figure 1.4. The
details are described in following sections.
Figure 1. 4 Interplay of multiple events leading to emphysema. Cigarette smoke
exposure activates airway epithelial cells, which release early inflammatory cytokines
that recruit macrophages and neutrophils. They can also activate dendritic cells and
the subsequent formation of CD8+ or CD4+ T cells. Along the chronic inflammation,
various cell types are activated by inflammatory mediators and in response their
secretory function may also become activated. Their anti-oxidant system may become
defective, while cells such as neutrophils will release ROS, leading to
oxidant/anti-oxidant imbalance. The resulting oxidative stress can induce senescence
and mitochondrial dysfunction. Mitochondrial dysfunction and accelerated ageing can
further amplify the oxidative stress. Apoptosis can be induced by external cytokines
such as perforin and granzymes, as well as by cytochrome c release due to
mitochondrial damage. Both accelerated ageing and apoptosis may lead to airspace
enlargement. The immune cells and structural cells, especially neutrophils and
macrophages can release protease. When the anti-protease of the cells is not enough to
counterbalance such effect, excess protease activity can lead to destruction of alveolar
structures.
11
1.1.4.1. Oxidative stress
COPD is associated with persistent oxidative stress. Markers of oxidative stress,
such as lipid peroxidation byproducts 4-hydroxy-2-nonenal, 8-isoprostane and
malondialdehyde (MDA), are found increased in lungs of patients with COPD (135,
206). The exhaled breath or breath condensates collected from patients with COPD
also contains increased levels of various markers of oxidative stress including
hydrogen peroxide, carbon monoxide, myeloperoxidase (MPO), 8-isoprostane and
MDA (27, 176, 194, 195). Exacerbations are associated with further enhanced
oxidative stress (263).
Reactive oxygen species (ROS) are free oxygen radicals with unpaired elections
that can oxidize various molecules such as proteins, DNA and lipids (133). Typical
forms of ROS include superoxide radical (O2-), hydroxyl radical (OH
-) and hydrogen
peroxide (H2O2). Under normal conditions, the major sources of ROS are respiration
of mitochondria, xanthine oxidase activity and NADP oxidase activity (167). During
cigarette smoking, the structural and immune cells in the lung are exposed to high
levels of exogenous oxidants from inhaled cigarette smoke (CS). One puff of CS
contains about 1015
oxidants in the gas phase and 1018
oxidants in the tar phase, as
well as 3000 ppm of nitric oxide (NO) (57, 58). Exposure to CS leads to release of
inflammatory mediators and ROS from epithelial cells and immune cells, leading to
release of various inflammatory mediators and ROS (164). Although CS is an obvious
source of oxidants and indeed a major risk factor for COPD, oxidative stress is
persistent after smoking cessation in patients with COPD (160), indicating that the
12
cellular accumulation and release of ROS is an important source of oxidative stress
during the progression of COPD. Increased release of ROS was reported in alveolar
macrophages and peripheral neutrophils from patients with COPD (115, 220). The
increased production of ROS may be due to the mitochondrial ROS overproduction
and altered xanthine oxidase activity. Increased mitochondrial ROS was reported in
airway smooth muscle cells from patients with COPD (273), and products of xanthine
oxidase activity were detected in bronchoalveolar fluid (BALF) from patients with
COPD (202). Although the increased ROS may be mainly in the form of less potent
radicals such as superoxide anion and hydrogen peroxide, they can be converted to
more potent and destructive ROS such as peroxynitrite (ONOO-) by rapid reaction
between superoxide radical and NO (261), and hypohalous acids (HOCl and HOBr)
by MPO-catalyzed reactions between hydrogen peroxide and Cl- or Br
- (224). MPO
levels in neutrophils are reported to be elevated in patients with COPD (1).
ROS generation is balanced by the activity of anti-oxidant defense in the normal
body. The first anti-oxidant barrier against the noxious particles or gases is the
epithelial lining fluid containing anti-oxidants such as vitamin C, vitamin E and uric
acid (134). Reduced levels of vitamin C and vitamin E in the lung were associated
with declining lung function in patient with COPD (134). Regarding cellular
anti-oxidant activity, one of the most important reactions is the synthesis of
glutathione (GSH), an anti-oxidant that depletes oxidants via conversion to oxidized
glutathione (GSSG). Under acute oxidative stress, epithelial cells from healthy
subjects presented elevated level of glutamate-cysteine ligase which is an enzyme
13
involved in GSH synthesis (228). Such anti-oxidant defense was impaired in
bronchial epithelial cells and alveolar macrophages from patients with COPD, as
indicated by lower expression of glutamate-cysteine ligase (228). Another
anti-oxidant defense system is mediated through superoxide dismutase (SOD) which
converts superoxide radicals into hydrogen peroxide (167). Hydrogen peroxide can be
further converted to oxygen and water directly by catalase (53) or by reaction with
GSH mediated by glutathione peroxidase (GPx) (232). As mentioned earlier, under
excess ROS production, superoxide anions can react rapidly with NO to form
peroxynitrite. Peroxynitrite can inhibit SOD, thus reducing the anti-oxidant defensive
activity (261). The persistent high ROS burden and defective anti-oxidant defense
lead to oxidative stress of the cells.
1.1.4.2. Chronic airway inflammation
Chronic and persistent airway inflammation is observed in patients with COPD,
and higher grades of COPD severity is associated with higher severities of
inflammation (108). Infiltrated inflammatory cells include neutrophils, macrophages,
B-cells, lymphoid aggregates and T cells (107). In addition to the increased numbers
in the lung, the function of inflammatory cells may also change in line with the
pathogenesis of COPD (109).
The number of macrophages increases in bronchial submucosa along with the
severity of airflow limitation in patients with COPD (108). Macrophages can
contribute to oxidative stress, destruction of structural cells and further progression of
14
inflammatory responses by releasing ROS, extracellular matrix proteins, leukotrienes,
multiple cytokines, chemokines and matrix metalloproteinases (MMPs) (57). One
primary function of macrophages in normal lungs is bacterial clearance through
phagocytosis, which may be defective in patients with COPD (72). Ex vivo studies
have demonstrated a defective phagocytositic ability against bacteria commonly
detected in airways in both alveolar macrophages (29) and monocyte-derived
macrophages (250) from patients with COPD. Moreover, the secretion of
inflammatory mediators is elevated in alveolar macrophages from smokers and
ex-smokers (101).
Increased number of neutrophils in sputum has been reported to associate with
higher severity of airflow obstruction and faster decline of lung function in patients
with severe COPD (187). Neutrophils are distributed in various sites particularly in
bronchial epithelium, bronchial glands (217) and in the airway smooth muscle (20).
When recruited to the airways, neutrophils become activated and release ROS,
inflammatory cytokines, neutrophil elastase, MMPs and MPO (240). In vitro data
suggest that the phagocytic ability of neutrophils can be impaired by cigarette smoke
extract (242).
In general, infiltrating neutrophils and macrophages are considered to be the
major players in the innate immune response in COPD. However, increased numbers
of eosinophils have also been reported in sputum, BALF and the airway wall (140,
142, 156). Levels of pro-eosinophil cytokines IL-4 and IL-5 are also reported to be
increased in plasma cells of patients with COPD (298). The exact function of
15
recruited eosinophils and its contribution to the development of COPD remains
unclear (57).
There is also an adaptive immune response to the inflammatory responses in
COPD. The number of dendritic cells has been reported to increase in epithelium and
adventitia of small airways of patients with COPD compared with never-smokers and
healthy smokers and correlated with the severity of airflow limitation (66). Dendritic
cells can present both self-antigens from tissue destruction and foreign antigens to
naive T cells, and numbers of both CD4+ and CD8+ T cells have been reported to
increase in lungs of patients with COPD (175, 213, 216). CD8+ T cells from lungs of
patients with COPD have been shown to release higher levels of inflammatory
mediators including tumor necrosis factor (TNF)-α, interferon-γ and granulocyte
macrophage-colony stimulating factor (GM-CSF).
1.1.4.3. Protease and anti-protease imbalance
Parenchymal destruction is one of the most significant pathological changes in
the lungs of patients with COPD. The resulting loss of elastic recoil leads to the
airflow limitation. Destruction of connective tissue, particularly lung elastin, by
excess protease activity is believed to play an important role in this process (25).
Active neutrophils can release serine proteases including neutrophil elastase,
cathepsin G and proteinase 3 (25). Various cysteine protease cathepsin can also be
released by different cells, including cathepsin S from airway smooth muscle,
cathepsin B, K and L from alveolar macrophages and cathepsin W from CD8+ T cells
16
(25). Alveolar macrophages can provide another important protease family, MMPs,
particularly MMP-9. The alveolar macrophages from healthy smokers express more
MMP-9 than normal subject, while alveolar macrophages from patients of COPD
express an even higher level (154, 214).
The protease activities are balanced by anti-proteases in normal lungs. Serine
protease activity can be counteracted by two major serine protease inhibitors in the
lung, α1 antitrypsin and secretory leukoprotease inhibitor (SLPI). Genetic variability
of α1 antitrypsin that leads to a deficiency in circulating levels is a risk factor for
COPD. Cigarette smoking may lead to oxidized and impaired α1 antitrypsin activity,
resulting in lack of counterbalance against the activity of neutrophil elastase (46).
SLPI activity may also be reduced by oxidative stress (25). As for the other protease
family MMPs, their activity can be counteracted by tissue inhibitors of MMPs (TIMP).
Alveolar macrophages from normal subjects release increased amounts of TIMPs
after pro-inflammatory stimulation, indicating a counterbalance to the release of
MMPs. The increased release of TIMPs is reduced in the alveolar macrophages from
COPD patients, which may lead to excess activity of MMPs (214).
1.1.4.4. Apoptosis
In addition to the excess protease activity, the parenchymal destruction may also
be contributed to by increased cellular apoptosis in lung (67). Apoptosis and
proliferation are well equilibrated in healthy tissue. Increased apoptosis in endothelial
cells, alveolar epithelial cells, interstitial cells, neutrophils and lymphocytes has been
17
reported in tissue sections of the lung from patients with COPD compared to healthy
subjects (223). Another study demonstrated that lung tissue from patients with COPD
exhibited increased apoptosis of alveolar epithelial cells, endothelial cells and
mesenchymal cells in association with an increased amount of the active subunit of
caspase-3, a central mediator of apoptosis (116). Moreover, persistent apoptosis of
alveolar epithelial cells and T-lymphocytes in bronchial bushings and bronchoalveolar
lavage has been observed after smoking cessation, as indicated by comparison
between ex-smokers with COPD, current smokers with COPD and healthy
never-smokers (103). Increased apoptosis in alveolar cells in the absence of adequate
compensation by proliferation leads to the gradual destruction of alveolar walls in the
lung (67). Apoptosis of alveolar wall and endothelial cells are reported to induce
emphysema even without infiltration of inflammatory cells in an animal model (67).
The increased apoptosis in the lung may be driven by multiple mechanisms which
are currently not well understood. Oxidative stress-induced attenuation of vascular
endothelial growth factor (VEGF) activity may be a mechanism of the elevated
apoptosis in COPD. It has been reported that increased apoptosis in epithelial and
endothelial alveolar septal cells in patients with emphysema is associated with
reduced expression of VEGF and VEGF receptor 2, which may lead to the endothelial
septal death (127). The level of VEGF in induced sputum from patients with COPD
has also been reported to decrease in severe COPD, in association with increased
levels of oxidative stress (125). In animal models, apoptosis induced by inhibition in
VEGF receptor (128, 199, 255) or VEGF (249) leads to emphysema even without
18
obvious inflammation. The relationship between oxidative stress and apoptosis may
also be through mitochondrial dysfunction. Mitochondria are the major source of
cellular ROS production of cells under oxidative stress (134). Airway smooth muscle
cells (ASMCs) from patients with COPD have demonstrated increased mitochondrial
ROS compared to healthy subjects (273). In general, mitochondrial dysfunction can
be a potent driving force in inducing apoptosis through the mitochondrial pathways
(90), but more evidence is required to reveal the exact relationship between
mitochondrial damage and apoptosis in COPD. The inflammatory characteristic of
COPD may also contribute to apoptosis. The phagocytic ability of alveolar
macrophages against apoptotic airway epithelial cells is impaired in patients with
COPD (90), leading to insufficient clearance of apoptotic airway epithelial cells. The
recruitment of CD8+ cells into the lung may also induce an increase in apoptosis of
structural cells, by secretion of cytotoxic mediators such as perforins, granzymes and
TNF-α (26, 158). Indeed, increased infiltration of CD8+ T cell to alveolar walls is
associated with increased apoptosis in the lung (166).
Cell death, including apoptosis, is constantly counterbalanced by proliferation
under normal physiological conditions (67). However, whether the proliferative
activity is able to counterbalance increased apoptosis remains unclear (67). Increased
proliferative activity has been reported in the alveolar wall of patients with
emphysema (116, 287), which may indicate a compensatory mechanism to the
increased apoptosis. However, another report demonstrated similar proliferation of the
alveolar septal cells between patients with emphysema and healthy subjects with
19
increased apoptosis of alveolar epithelial cells in patients with end-stage emphysema
(44).
1.1.4.5. Ageing
Age is a risk factor for COPD. On the one hand, age is associated with longer
exposure history to noxious gas or particles, on the other hand, the process of ageing
may also contribute to the pathogenesis of COPD (119). Lung function starts to
decline approximately after the age of 30 as a result of normal ageing (198), but in
patients with COPD the decline is accelerated (79, 163). The decline in lung function
of ageing lungs may be explained by progressive distal airspace enlargement, which
leads to loss of lung elastic recoil (120). Although the airspace enlargement is not as
destructive as is the case in lungs of patients with COPD (262), the similarity of the
functional and structural changes between ageing lungs and lungs of patients with
COPD suggests a possible relationship between them. COPD is characterized by
persistent oxidative stress, while oxidative stress-induced DNA damage is believed to
play an important role in cellular senescence according to the free radical theory (99).
Senescence of cells is associated with shortening of telomere length (TL) (218).
Indeed, it has been reported that TL of circulating lymphocytes is shortened in
smokers in a dose-dependent manner, but the effect was not amplified in smokers with
COPD (178). Another study demonstrated TL shortening in circulating leucocytes
from patients with COPD compared to healthy subjects of any age range (110, 219).
TL of alveolar epithelial cells, endothelial cells (254) and fibroblasts (180) are also
shortened in patients with emphysema compared with non-emphysematous subjects.
20
Whereas oxidative stress may drive an accelerated ageing process, the ageing
process itself may also contribute to oxidative stress and inflammation, which is
evident from multiple animal studies. Acute CS exposure of mice has been reported to
induce a five-fold increase of GSH levels in BALF in two-month old mice, indicating
an anti-oxidant response. In mice of increasing age, these effects gradually diminished,
in association with increased inflammatory maker TNF-α and oxidative stress marker
8-hydroxy-2-deoxyuanosine in BALF (88). Another study suggested that older mice
had more neutrophils, inflammatory mediator keratinocyte-derived chemokine (KC, a
mouse homolog of IL-8) and MIP-2 in lungs after 9 days of cigarette smoke (177).
1.1.4.6. Bronchial epithelial cells and airway smooth muscle cells in COPD
Bronchial epithelial cells are the first cellular defensive barrier against noxious
gases and particles in lung. The epithelial injury caused by CS leads to the onset of
oxidative stress and inflammation in the lung. The direct exposure to CS induces the
intracellular release of endogenous molecules named danger-associated molecular
patterns (DAMP) which can be identified by pattern-recognition receptors (PRR) e.g.
Toll-like receptor 2 and 4 (190). This process activates epithelial cells to synthesize
and secrete early inflammatory mediators such as TNF-α, interleukin (IL)-1β and IL-8
(65). The release of these chemokine leads to recruitment of inflammatory cells such
as macrophages and neutrophils. Bronchial epithelial cells can also facilitate the onset
of the adaptive immune response in COPD. Dendritic cell migration, maturation and
activation are reported to be regulated by bronchial epithelial cells by releasing
21
various chemokines such as macrophage inflammatory protein (MIP)-3α and
chemokine ligand 20 (CCL20) (66, 201). In addition to the initiation of the
inflammatory responses, bronchial epithelial cells also contribute to the persistent
inflammation in COPD by releasing multiple cytokines and chemokines including
IL-1β, TNF-α, IL-6, IL-8 and GM-CSF. For example, epithelial cells from smokers
with COPD express higher levels of mRNA and protein of monocyte chemotactic
protein 1 (MCP-1) and IL-8 compared to the cells from smokers without COPD (64).
Bronchial epithelial cells also play an important role in the anti-oxidant system in the
lung. As mentioned in previous sections, epithelial cells from patients with COPD
demonstrated reduced ability to elevate GSH production in response to oxidative
stress (228). In addition, bronchial epithelial cells also serve as a source of the
anti-protease SLPI (25).
Patients with COPD present with an increased mass of airway smooth muscle
(ASM) in small airways (37, 59), correlating with declining lung function (57). The
thickened airway smooth muscle layer contributes to a thicker airway wall, which
plays an important role in the airflow obstruction of COPD (56). Due to its contractile
properties, ASM is the major modulator of bronchomotor tone in the airway.
Exaggerated constriction of the airways is a feature of patients with COPD, and
relaxation of contracted ASM by bronchodilators is a major therapeutic intervention
in the treatment of COPD (19). The role of ASM in the pathogenesis of COPD is not
limited to its contractile properties. Similar to epithelial cells, ASM cells (ASMCs)
also release inflammatory cytokines and chemokines including IL-6, IL-8, MCP-1,
22
MCP-2, MCP-3, growth-related oncogene (GRO)-α, IFN-γ-inducible protein (IP)-10
and GM-CSF (56). In addition, increased levels of transforming growth factor-β
(TGF-β) in patients with COPD can lead to reduced expression of catalase and
manganese-SOD in ASMCs, resulting in reduced anti-oxidant activity (174).
Unlike bronchial epithelial cells, ASMCs are usually not directly exposed to
noxious particles or gases, although this may happen when the epithelial barrier is
severely damaged by CS (102, 226). Therefore, the release of inflammatory mediators
from ASMCs is most likely not the result of a direct response to CS, but rather
induced by the effect of cytokines released by neighboring cells, e.g. epithelial cells.
As mentioned earlier, CS causes direct injury to bronchial epithelial cells leading to
release of inflammatory mediators. Amongst them, IL-1β and TNF-α can induce the
release of IL-8 from ASMCs (121), and IFN-γ and TNF-α can induce the release of
IP-10 from ASMCs (98). Moreover, bronchial epithelial cells may also contribute to
the increased TGF-β levels in COPD. An in vitro study demonstrated that epithelial
cells from smokers and patients with COPD secreted higher levels of TGF-β than the
cells from normal subjects (248). As mentioned earlier, high levels of TGF-β can
reduce the anti-oxidant activity in ASMCs. Therefore the secretory activities of
cytokines and a defective anti-oxidant system in ASMCs may partly result from an
interaction with injured epithelial cells.
1.1.4.7. Mitochondria and COPD
Mitochondria are double-membrane organelles with a unique structure and
23
important function. They are composed of an outer membrane, inner membrane
inter-membrane space and matrix (11). The inner membrane is highly folded into
cristae, and is impermeable to ions. Respiratory chain complex proteins reside on the
inner membrane where they mediate electron transport chain and the subsequent
accumulation of protons in the inter-membrane space (170). The proton gradient
between the two sides of the inner membrane leads to the flux of proton back to
matrix through adenosine triphosphate (ATP) synthase (Complex V) and drives the
synthesis of ATP (170). The electrochemical gradient across the inner membrane is
indicated by the mitochondrial membrane potential (ΔΨm) (170). The electron
leakage from oxidative phosphorylation is believed to be a major source of ROS
production in cells. It has been estimated that 0.2-2.0% of oxygen consumption by
mitochondria results in formation of superoxide radicals (149, 165). Leakage of
electrons from the electron transport chain takes place at Complex I towards the
matrix and at Complex III towards both sides of the inner membrane (97, 149, 165).
Leaked electron leads to partial reduction of oxygen. The resulting superoxide
radicals can act as precursors for many other more active ROS, or they can be
converted to hydrogen peroxide by SOD1 (superoxide dismutase 1) in the matrix or
SOD2 (superoxide dismutase 2) in the inter-membrane space (97, 149, 165).
Superoxide is believed to be short-lived while hydrogen peroxide is more stable and
permeable to other part of mitochondria and cells (149, 170). The level of hydrogen
peroxide has been estimated to be 100-fold higher than the level of superoxide
radicals in mitochondria (43). In general, mitochondria are not rich in catalase, and
24
the reduction of hydrogen peroxide relies on the reaction with GSH catalyzed by
glutathione peroxidase (149) (Figure 1.5). As the major source of ROS, mitochondria
are also a target of ROS-mediated damage. Excess mitochondrial ROS may induce
damage and mutations of mitochondrial DNA (170, 281). ROS may also induce
carbonylation and dysfunction of various mitochondrial proteins, including the
complex proteins leading to defective mitochondrial respiration, and the
mitochondrial carrier proteins regulating molecular transport across the membrane,
and the protein assembly of permeability transition (PT) pore which may trigger
signals of apoptosis (170).
ΔΨm indicates the electrochemical gradient across the inner membrane, which
results from the activity of the election transport chain (170). As ROS is a byproduct
of the electron transport chain, under the normal conditions where there is an
ROS/anti-oxidant equilibrium, ΔΨm and ROS should be positively correlated. While
this is supported by several studies (149, 165, 188), under conditions of cellular
damage ΔΨm reduction is associated with increased ROS (9, 273). Reduction of ΔΨm
may be a result of impaired proton transfer due to an interrupted electron transport
chain, or be caused by opening of PT pores resulting in increased permeability.
Mitochondria are highly dynamic organelles under constant cycles of fission and
fusion (183). Fission refers to the splitting of mitochondria leading to short and less
branched morphology while fusion is the merging of mitochondria leading to an
elongated and a more branched network (149). These morphological dynamics are
very important for normal function of the mitochondrial network in a cell.
25
Interruption of either the fusion or fission process causes dysfunction of mitochondria
(183). This might be because the exchange of mitochondrial DNA in matrix during
the fission and fusion can alleviate the effect of mutation (183).
In addition to their role in energy supply and redox regulation in the cells,
mitochondria play a central role in the regulation of apoptosis, senescence and
autophagy (5, 11, 149, 170). Excess ROS may induce mitochondrial PT pores into a
high-conductance state, leading to rising permeability for small molecules (83). This
event is reflected by loss of ΔΨm. As the inner membrane is highly folded, the fluid
influx lead to swelling of the inner membrane, resulting in further opening of
mitochondrial PT pores and sometimes rupture of outer membrane (123). During this
process cytochrome c is released from mitochondria, which triggers activation of
caspase-9 and leads to apoptosis (90). In addition to the effects on PT pores, oxidative
stress can also lead to peroxidation of cardiolipin, a lipid that immobilizes cytochrome
c in the inner membrane, which further promotes the release of cytochrome c from
mitochondria (124, 193).
There is an increasing focus on the involvement of mitochondria in the
pathogenesis of COPD (11). Most direct evidence has come from primary cells from
the patients with COPD. It has been demonstrated that primary bronchial epithelial
cells from ex-smokers with severe COPD exhibited swelling and fragmented
mitochondrial morphology which is similar to that seen in CS medium (CSM)-treated
immortalized bronchial epithelial cells (BEAS2-B) (105). Another study on ASMCs
has been reported by Wiegman and colleagues with a more comprehensive evaluation
26
of mitochondrial function (273). In this study ASMCs from patients with COPD
demonstrated elevated mitochondrial ROS levels, reduced ΔΨm, reduced amount of
Complex I, III and V compared to healthy controls. Moreover, mitochondrial
respiration was impaired in ASMCs with COPD as indicated by reduced basal and
maximum respiration levels, and respiratory reserve capacity. Healthy smokers also
demonstrated mitochondrial dysfunction compared to healthy controls, but the effects
are less pronounced compared to patients with COPD. Several other studies also
suggested mitochondria of respiratory striated muscle and skeletal muscles are
damaged in patients with COPD (5).
Mitochondrial damage is also observed in other COPD-related animal models or
CSM-treated in vitro models. It has been reported that CS exposure can impair energy
metabolism in mouse lung by switching glucose metabolism to the pentose phosphate
pathway and reducing the substrate supply to mitochondria (4). In a chronic
ozone-induced mouse model of COPD, elevation of mitochondrial ROS and reduction
of ΔΨm is observed, which can be alleviated by the mitochondria-targeted
anti-oxidant MitoQ (273). The alleviation of ozone-induced mitochondrial ROS leads
to attenuation of ozone-induced airway hyper-responsiveness (AHR) and
inflammation, indicating a potential strategy to target mitochondrial ROS for the
treatment of COPD. Another study has demonstrated that wood smoke exposure to
guinea pigs can induce impaired oxygen consumption activity and complex protein
enzyme activity (89). CSM was also reported to alter the fission-fusion activity of
mitochondria in vitro in both airway epithelial cells (105) and ASMCs (10), leading to
27
mitochondrial fragmentation and increased mitochondrial ROS.
Figure 1. 5 Generation of mitochondrial ROS from electron transport chain.
Leakage of electron from electron transport chain leads to generation of superoxide
radicals, which can be converted to hydrogen peroxide by SOD. I-V: complex
proteins; Q: coenzyme Q; Cyto c: cytochrome c; SOD: Superoxide dismutase, GPx:
Glutathione peroxidase. The figure is plotted based on Li X et al, J Hematol Oncol
(149).
1.1.5. Current therapies
For patients with COPD who are current smokers, smoking cessation is the most
effective approach that can influence the natural history of COPD. Smoking cessation
can be facilitated by nicotine replacement therapies such as nicotine gum, inhaler,
nasal spray, transdermal patch, sublingual tablet and lozenge, as well as supportive
pharmacotherapy including varenicline, bupropion and nortriptyline (263).
Pharmacologic therapies for COPD include bronchodilators, corticosteroid and
28
phosphodiesterase-4 inhibitors (263). The choice of each medications or the
combination of them is patient specific. Bronchodilators refer to the medications that
induce the dilation of bronchi and bronchioles leading to increase in FEV1 (263).
There are three classes of bronchodilators, β2-adrenorecepter agonist, anticholinergics
and methylxanthine (82). The β2-adrenoreceptor agonists are further classified as
short-acting β2 agonist (SABA) and long-acting β2-agonist (LABA), both working
through induction of cellular cyclic adenosine monophosphate (cAMP).
Bronchodilators are usually administrated through inhalation, and they are the central
medication to manage the symptoms of COPD. Combination of different classes of
bronchodilators may improve efficacy and reduce the side effects compared to a
single class. Corticosteroid treatment is mainly adopted for the severe conditions. Oral
corticosteroids aim to suppress the chronic inflammation, but they are generally
associated with high risks of adverse effects (263). Inhaled corticosteroids in
combination with bronchodilator may improve efficacy compared to single treatment,
but the long-term safety and adverse effects of inhaled corticosteroids is largely
unknown (263). Phosphodiesterase-4 inhibitors are a relatively new medication for
COPD. They are able to reduce inflammation by inhibiting cAMP breakdown, and
should be used with long-acting bronchodilators (263).
COPD is a complex, chronic disease resulting from long term interaction between
genes and environmental stimuli (65). The pathogenesis of the disease is far from
understood and the development of effective pharmacologic therapy has not been easy,
especially for the control of chronic inflammation which is corticosteroid-resistant in
29
COPD (24). To date, the pharmacologic therapy is able to reduce COPD symptoms,
exacerbations, and improve health status and exercise tolerance (263). However, none
of current treatments is able to alleviate the progressive decline of lung function in
COPD (263).
1.2. Introduction to Stem Cells
1.2.1. Definition and classification
Stem cells are cells that are able to self-renew and differentiate into other cell
types (251). In contrast, somatic cells are usually able to self-renew but cannot
differentiate, while progenitor cells are able to differentiate into somatic cells, but
with poor ability of long-term self-renewal (251). Stem cells and progenitor cells act
as sources of new somatic cells as a normal turnover or as a replenishment to
increased somatic cell death after injury (251).
Stem cells can be classified as totipotent, pluripotent and multipotent according to
their potency that is their capacity to differentiate (2, 251). Totipotent stem cells have
the highest differentiation potential with capacity to develop into new individuals
from a single cell. Although not able to form new individuals, pluripotent stem cells
are capable of differentiating into cell types of all three germ layers (ectoderm,
mesoderm and endoderm), which basically means they can generate almost any cell
types except for extra-embryonic tissues such as trophectoderm (2, 251). Multipotent
stem cells can form limited cell types, usually of one single lineage (2, 251). In
general, the potency of stem cells gradually decreases along the development process
30
(2, 251). Totipotent stem cells only exist as zygotes and cells from very early embryos,
while stem cells derived from blastocyst named embryonic stem cells (ESCs)
represent the major form of pluripotent stem cells (2, 251). Stem cells in the adult
body, or adult stem cells, are multipotent stem cells (2, 251). They are usually
distributed in specific microenvironments called niches of the parent tissues and
generate somatic cells of the same origin (2, 251). The progenitor cells have similar
differentiation features as the adult stem cells. They also reside at niches of tissues
and differentiate into specific cell types (85).
Utilising stem cells therapeutically is not a recent idea. Haematopoietic stem cells
(HSC) have been used in bone marrow transplantation for treatment of leukaemia for
more than 40 years (41). Other cells that have been tested in clinical trials include
mesenchymal stem cells (MSC) and muscle satellite stem cells (251, 269). When it
comes to stem cell-based therapy, the stem cells can be alternatively classified as
endogenous stem cells which are the adult stem cells in their niches in the body, and
exogenous stem cells which are the isolated and expanded adult stem cells ex vivo
(251).
1.2.2. Endogenous stem cells in lung
The delineation of endogenous stem cells in individual organs is not an easy task.
The most convincing approach is to examine the capacity of the cells to reconstitute
the entire organ. For example, HSCs can rebuild the entire haematopoietic system
after transplantation into irradiated animals (251). However the approach requires
31
destruction of organs which is rather difficult in most cases. An alternative approach
is to genetically trace the lineage of cells in the organs, which can provide evidence of
the differentiation of specific cell types into other cells. Intestinal stem cells (260) and
skin stem cells (34) were identified through this approach.
The lung is a complex organ with a very low turnover rate during normal
homeostasis compared with intestine or skin tissues (106). The regenerative capacity
of human lung is mostly demonstrated upon injury, such as pneumonia and severe
respiratory infections (28, 42, 131, 138). Whether there is a defective regenerative
function of endogenous lung stem cells in airway diseases is still unclear (268). In fact,
the identity and roles of stem cells and progenitor cells in the lung remains unresolved
due to the lack of markers for each cell type and lineage tracing approaches (268).
Various putative airway and alveolar epithelial stem and progenitor cells are identified
by mounting data from animal and human studies, including airway submucosal
glands, keratin-14 expressing tracheal basal cells, Clara cells in association with
neuroendocrine bodies or bronchoalveolar duct junction, and type 2 alveolar epithelial
cells (AEC2s) (85). However, there is also controversial data from other studies and
there is not yet uniform agreement on the identity of lung stem cells (268).
As COPD is associated with progressive destruction of the alveolar structure, the
identification of stem cells in alveoli, and further investigation of their role in COPD,
may provide valuable information on the disease. Alveoli are mainly composed of
type 1 alveolar epithelial cells (AEC1s) and AEC2s. AEC2s which express high levels
of surfactant protein C (SFTPC) can function as local stem cells (21). Lineage tracing
32
in mice are demonstrated that AEC2s can differentiate into AEC1s during homeostasis,
and bleomycin-induced injury promoted such activity (21). The self-renewal ability of
SFTPC+ AEC2s was also observed for at least one year. Moreover, when co-cultured
with a mesenchymal population, the SFTPC+ AEC2s can differentiate into alveolar
structures consisting both AEC2s and cells with AEC1 markers (21). In combination
with the self-renewal property in vitro, these findings demonstrated the capacity of
AEC2s for self-renewal and differentiation into other cells both in vivo and in vitro.
1.2.3. Mesenchymal stem cells (MSCs)
Mesenchymal stem cells (MSCs) are multipotent stem cells from stromal origin
(269). Originally described as a fibroblastic-like population of bone marrow (BM)
cells, MSCs now refer to similar cells found in a variety of tissues, including BM,
adipose tissue, cord blood and placenta (269). MSCs can differentiate into
chondrocyte, adipocyte, osteoblast or other lineages such as epithelial and endothelial
cells (269). The general criteria to define MSCs include the cell marker profile,
self-renewal property and the capacity of differentiating into osteoblasts, adipocytes
and chondrocytes (251). Although MSCs isolated from different tissue share common
features regarding these parameters, different gene expression, lineage tendencies and
other properties have been demonstrated amongst them (15, 132). Moreover,
individual MSC types may still present a heterogeneous group of cells of different
subtypes (200). The functional and therapeutic differences between MSCs from
different sources have been increasingly investigated (269).
33
Cells with features of MSCs have been isolated from adult mouse lungs, human
nasal mucosa, and lungs of human neonates and lung transplant recipients (269). The
functions of these cells in the lung remain unclear. They have been reported to play a
potential role in fibrotic diseases and in the bronchiolitis obliterans following lung
transplantation (14, 61, 210, 264).
MSCs express very low levels of major histocompatibility complex (MHC) class
I and class II molecules, which makes them highly immunologically-tolerant (80, 132,
144, 269). Exogenous administration of allogeneic MSCs usually lead to low levels of
immune rejections and does not require immunosuppression (251). MSCs are
commonly used to develop exogenous stem cell-based therapy for lung diseases.
There are numerous studies on this topic, which will be introduced in the later
sections.
1.2.4. Induced-pluripotent stem cells (iPSCs)
As described earlier, in nature, cells demonstrate a unidirectional reduction in
differentiation potency with development. Therefore, the access to pluripotent stem
cells had relied on the isolation of ESCs until the discovery of induced-pluripotent
stem cells (iPSCs) (247). iPSCs are pluripotent stem cells derived from somatic cells
through transcriptional factor-induced reprogramming in vitro (e.g. through induction
by Oct4, Sox2, Nanog and Lin28). Deriving iPSCs from somatic cells was first
achieved by Takahashi and Yamanaka using mouse fibroblasts in 2006 (247). Human
iPSCs were later generated also from fibroblasts in 2007 (246, 289). In theory, this
34
robust approach can generate iPSCs from any somatic cell type.
Compared to the isolation of adult stem cells from the relevant tissue, the
reprogramming of somatic cells is less restricted to the type of somatic tissue.
Moreover, the high proliferation capacity of iPSCs reduces the amount of somatic
tissue required. Therefore, iPSC provides an abundant supply of stem cells from each
particular individual. However, the therapeutic application of iPSCs is limited by
pluripotency. They are so potent that direct administration of iPSCs may lead to
formation of teratomas (122).
Although iPSCs may not be a very good candidate as a direct therapy for airway
disease, they can serve as a great source to generate other cell types that may be
effective in airway diseases. Indeed, the derivation of lung progenitor cells or
epithelial cells from iPSCs has been reported by several studies (91, 111, 179, 278,
279). AEC2 is the most common and prominent cell type derived from iPSCs, while
AEC1 can also be generated. The iPSC-derived epithelial cells can be used in ex vivo
tissue engineering of lung, providing cellular source to re-populate decellularized
whole lung scaffolds (86, 159). However, information of their effects as direct source
of cell-based therapy for airway disease is still lacking.
1.2.5. iPSC-derived MSCs
Given the numerous studies on the therapeutic potential of MSCs in airway
disease models, deriving MSCs from iPSCs provides a promising strategy. This task
was achieved by Lian and colleagues in 2010 (153) (Figure 1.6). The derivation was
35
through incubation of iPSCs in pro-MSC induction medium (detailed methodology
can be found in Chapter 2). MSC-like cells were identified as CD24- and CD105+
cells. The resulting cell culture exhibited fibroblast-like morphology which is typical
of MSCs. To acquire homogenous populations, single-cell derived colonies were
generated from these cells. In addition to the fibroblast-like morphology, the derived
cells demonstrated other features of MSC. Firstly, they presented an MSC-like surface
marker profile (CD44+, CD49a+ CD49e+, CD73+, CD105+, CD166+, CD34−,
CD45−, and CD133−). Secondly, the differentiation capacity was demonstrated by
efficient adipogenesis, osteogenesis and chondrogenesis. Lastly, they were able to
self-renew without loss of differentiation potential. In fact, they were far more
expandable than BM-MSCs, with an ability to keep their differentiation capacity up to
passage 40. As they fulfil all criteria of MSCs, these cells were named iPSC-derived
MSCs (iPSC-MSCs). Compared to iPSCs, iPSC-MSCs demonstrated reduced
expression of pluripotency-associated genes such as Oct4, Nanog and Sox2. When
subcutaneously injected into combined immunodeficient (SCID) mice, iPSC induced
teratoma while no teratoma was induced by iPSC-MSCs for up to four months.
The in vitro generation of iPSC-MSCs from iPSCs brings potential opportunities
to overcome several limitations of the adult MSCs as seen in BM-MSCs. The amount
of BM-MSCs available from BM is limited which makes ex vivo expansion important
(269). However, BM-MSCs become senescent after 20 doublings during ex vivo
expansion with loss of differentiation potential (69). Their genetically stability in vitro
also draws concerns as they may develop abnormal karyotypes during expansion
36
(148). The limited in vitro expansion capacity restricted the resource for cell therapy.
In addition, one of the future directions of MSCs-based therapy is to genetically
manipulate the cells. As MSCs can be recruited to injured tissue, by this approach
they can express and release desired molecules at the site of the injured tissue (63,
122). Such genetic manipulation also calls for high stability and expanding capacity
of the MSCs. In contrast to BM-MSCs, iPSC-MSCs demonstrated normal karyotypes
during expansion (153). They can be expanded for 120 doublings with stable surface
marker profile for MSCs (153). Another limitation of BM-MSCs is that when isolated
from aged subject or patients with ageing-related disorders, they may present reduced
survival and differentiation ability (87, 100, 267, 294). In theory, as the safety of
allogeneic MSCs has been well demonstrated, this issue can be avoided by selection
of the donor. Nevertheless, availability of personalized MSCs is appealing for future
development of MSCs-based therapy. iPSC-MSCs may hold hope as a way of
overcoming the ageing-associated impairment of BM-MSCs. When generating
iPSC-MSCs, somatic cells were firstly reprogrammed to an embryonic stem cell-like
state, which may help to avoid the problem of senescence. According to the same
study by Lian and Zhang et al., the telomerase activity of iPSC-MSCs was 10 times
higher compared to BM-MSCs (153).
As a relatively new type of MSCs, beneficial effects of iPSC-MSCs have already
been demonstrated in rodent models of limb ischemia (153), allergic airway
inflammation (244) and cardiomyopathy (293).
37
1.3. MSCs-based Therapy
1.3.1. Efficacy of MSCs in animal models
MSCs are the most studied candidates for the development of cell-based therapy
against lung diseases. Efficacy of MSCs was demonstrated in a broad spectrum of
animal models including acute lung injury and bacterial lung infection, COPD,
asthma, bronchiolitis obliterans, bronchopulmoary dysplasia, fibrosing pulmonary
injury, pulmonary hypertension, pulmonary ischemia re-perfusion injury, obstructive
sleep apnea, radiation-induced lung injury, sepsis and burns (269). The studies of
MSCs in COPD-related models are listed in Table 1.2. In general, these models can
Figure 1. 6 Change of potency: from somatic cells to iPSC-MSCs. Inducing
iPSCs from somatic cells provides great resource of stem cells. However, iPSCs are
too potent for some applications, and differentiation of them into multipotent stem
cells can generate cell types that may be more useful as treatment of diseases.
38
exhibit histological changes (primarily airspace enlargement) in the lung mimicking
the lungs of patients with COPD, which are alleviated by MSCs from various sources
including BM, adipose tissue, lung and amniotic fluid. The major approaches for
inducing emphysema in animals are by administration of elastase and by chronic
exposure to CS. Elastase can cause excess protease activity rapidly leading to direct
destruction of alveolar structures, while CS exposure can induce inflammation very
quickly but with the airspace enlargement developing much later (usually months
after exposure). However, as cigarette smoking is the major risk factor of COPD, the
CS-exposed model may be more relevant to the pathogenesis of COPD. CS-exposed
animals have been demonstrated to develop inflammation, oxidative stress and excess
protease activity both in lung and systemically (51, 52). The capacity of alleviating
CS-induced inflammation is demonstrated in MSCs from BM (94, 112), amniotic
fluid (151) and adipose tissue (222). Moreover, the MSCs from BM and adipose
tissue also inhibited CS-induced apoptosis in the lung.
39
Table 1.2 Studies of MSCs treatment in COPD-related animal models.
Studies Experimental Model
Route, and Timing of Treatment Source of MSCs Outcome Compared to Injury Effects
Potential
Mechanisms
Shigemura
2006(230)
Rat: elastase.
Direct topical application of ASC-
seeded polyglycolic acid at lung
volume reduction surgery.
Rat adipose
Plastic adherent • Improved histological repair HGF secretion
Shigemura
2006 (229)
Rat: elastase.
i.v. ASCs 1 week after elastase.
Rat adipose
Plastic adherent
• Decreased apoptosis, improved
histological repair
• Improved gas exchange and exercise
tolerance
HGF secretion
Yuhgetsu
2006 (290)
Rabbit: elastase.
i.t. MSCs 24 hours after elastase.
Rat BM - mononuclear
cells from Ficoll gradient of
total BM
• Improved histology and lung function
• Decreased BALF inflammation
• Decreased tissue MMP2 and MMP9
expression
None specified
(soluble mediators)
Zhen
2008 (295)
Rat: papain.
i.v. MSCs after papain (timing not
specified).
Rat BM
Plasticadherentmononucelar
cells from Percoll gradient
• Decreased histological injury
• Decreased alveolar cell apoptosis None specified
40
Zhen
2010 (296)
Rat: papain.
i.v. MSCs 2 hours after papain
Rat BM
Plastic adherent
mononucelar cells from
Percoll gradient
• Decreased histological injury
• Partial restoration of VEGF
expression in lung homogenates
TNF-α–stimulated
VEGF secretion by
MSCs
Katsha
2011 (130)
Mouse: i.t. elastase.
i.t. MSCs 14 day after elastase.
Mouse BM
Plastic Adherent
• Decreased histological injury
• Decreased IL-1β
• Transient increase in lung EGF, HGF,
and SLPI
None clarified
(soluble mediators)
Huh
2011 (112)
Rat: CS exposure for 6 months.
Retrobulbar MSCs at the beginning
of smoke cessation.
Rat BM
• Decreased histological injury
• Increased numbers of small vessels
and reduced RVSP
• Increased proliferation of AEC2 cells
and endothelial cells
• Decreased apoptosis
Soluble mediators
Akt signaling
suspected
41
Schweitzer
2011 (222)
Mouse: CS exposure for 4 months.
i.v. ASCs every other week for 3rd
and 4th months of CS exposure.
Human or mouse adipose
plastic adherent
• Decreased histological injury and
apoptosis (caspase activation)
• Decreased BALF inflammation
• Decreased CS-induced MAPK signal
transduction
• Decreased weight loss
• Decreased BM suppression
None specified
Immunodeficient NOD-SCID
mouse: VEGFR blockage induced
emphysema.
Human ASCs 3 days after VEGFR
blockade
Human adipose plastic
adherent
• Human ASCs decreased histological
injury and caspase 3 activation
following VEGFR blockade
• Emboli if used > 5 × 105 MSCs or if >
passage 3
None specified
Hoffman
2011 (104)
Mouse: i.t. elastase
i.v. MSCs 6-7 weeks after elastase
Mouse BM
Lung-MSCs from primary
explants cultures
• Higher retention of lung MSCs
• Decreased histological injury None specified
42
Ingenito
2012 (117)
Sheep: i.t. elastase (5
doses/20weeks)
Endoscopically transplanted in
biological delivering scaffold
Sheep lung
• Increased lung tissue mass and
perfusion
• Increased histological cellularity, cell
retention and extracellular matrix
• Improved lung mechanics
Paracrine effects.
Increased epithelial
proliferation in in
vitro co-culture.
Guan
2013 (94)
Rat: CS exposure for 11 weeks,
sacrificed after 4 weeks cessation.
i.t. MSCs on the first day of week
7
Rat BM
• Decreased histological injury
• Increased lung function
• Decreased lung apoptosis
• Decreased TNF-α, IL-1β, MCP-1,
IL-6, MMP9 and MMP12 in lung
• Increased VEGF, VEGF receptor-2
and TGFβ-1 in lung
Non specified
Song
2014 (237)
Rats: CS exposure for 7 weeks,
sacrificed after 4 weeks cessation.
i.t. MSCs at week 8
Rat BM
• Increased histological injury
• Increased lung function
• Decreased inflammatory cells in BAL
• Decreased levels of IL-1β, IL-6,
TNF-α, MDA and increased TGFβ-1 in
lung
Non specified
Tibboel
2014 (252)
Mouse: i.t. elastase
i.t. or i.v. MSCs before or after
elastase
Mouse BM
• Increased lung function only by i.v.
injection prior elastase
• No significant histological difference
Non specified
43
Li X
2014 (150)
Rat: CS exposure for 56 days
i.v. MSCs at day 29 and 43 Human iPSC-MSCs • Decreased histological injury
Mitochondrial
transfer
Li Y
2014 (151)
Rat: CS exposure for 12 weeks
plus i.t. LPS at week 4 and 8.
i.t. MSCs after CS exposureperiod
Rat amniotic fluid
• Decreased histological injury
• Increased surfactant protein A and C
• Decreased AEC2 apoptosis
Differentiation into
AEC2-like cells
Anti-apoptotic
mechanism
unknown
Gu
2015 (93)
Rat: CS exposure for 12 weeks
sacrificed after 4 weeks cessation.
i.t. MSCs started at week 7,
twice/week for 5 weeks
Rat BM
• Decreased histological injury
• Decreased inflammation
• Decreased PGE-2 and IL-6 and
increased IL-10 in BALF and serum
• Decreased COX-2 and PGE2 in lung
Inhibition of
COX-2/PGE2 in
alveolar
macrophages by p38
MAPK pathways
Abbreviations: AEC2: Type 2 Alveolar epithelial cell; ASC: adipose stem cell; BALF: bronchoalveolar lavage fluid; BM: bone marrow;
COX-2: cyclooxygenase-2; CS: cigarette smoke; EGF: epidermal growth factor; HGF: hepatocyte growth factor; IL: interleukin; iPSC-MCS:
induced pluripotent stem cell-derived mesenchymal stem cells; i.t.: intratracheal; i.v.: intravenous; MAPK: mitogen-activated protein kinase;
MCP-1: monocyte chemoattractant protein-1; MDA: malondialdehyde; MMP: matrix metalloproteinases; PGE2: prostaglandin E2; RVSP:
right ventricle systolic pressure; SLPI: secretory leukoprotease inhibitor; TGF-β: transforming growth factor-β; TNF-α: tumor necrosis factor;
VEGF(R): vascular endothelial growth factor (receptor).
The list before (including) 2012 is adapted from two reviews by Weiss et al (269, 270); studies after 2012 are described from corresponding
references.
44
1.3.2. Clinical trials
The safety and efficacy of MSCs have been tested in acute myocardial infarction
and several inflammatory and autoimmune diseases such as graft-versus-host disease
(GVHD), autoimmune diabetes and Crohn‟s disease (269). The first licensed
MSCs-based therapy has been approved in Canada, as a medication for GVHD (8,
269). The intensive clinical study in these fields showed an important fact that MSCs
may be well tolerated even by patients with very severe illness (8, 269).
The mounting evidence supporting the efficacy of MSC in COPD-related animal
models led to the initiation of the first multicenter, double-blind, placebo-controlled
phase II clinical trial of a systemic-administrated BM-MSC preparation
(PROCHYMAL, Osiris Therapeutic Inc, Columbia, MD, USA) for moderate or
severe COPD (271). 62 patients participated in the clinical trial, during which they
received 4 monthly infusions of BM-MSCs (100x106
cells/infusion) or vehicle
controls and were monitored in a subsequent 2-year period. End points included
comprehensive safety evaluation, pulmonary function testing (PFT), and
quality-of-life indicators including questionnaires, 6-minute walk test, and
assessments of systemic inflammation. The trial proved the safety of BM-MSCs in
patients with moderate or severe COPD. No pulmonary emboli formation or other
infusional toxicity was observed for every single infusion among all the 4 infusions of
all patients. No mortality or serious adverse effects were observed in the two-year
follow-up. However, the administration of BM-MSCs did not improve the lung
function of the patients compared to the placebo, as well as the quality of life
45
indicators such as 6-minute-walk evaluation and quality of life questionnaire. The
only effect observed was on the systemic C-reactive protein (CRP) levels, which may
indicate a modulation of systemic inflammation. Among the patients with high
systemic CRP levels (above 4 mg/L) at the start of the trial, infusion of BM-MSCs
significantly reduced the systemic CRP levels within the first month and the trend
persisted for 2 years although the statistical significance was lost over time.
A multicentre, open-label, dose-escalation, phase I clinical trial on treatment with
BM-MSCs in moderate to severe acute respiratory distress syndrome (ARDS) in USA
has been reported recently (276). Nine patients were included, and three dosages were
tested (106, 5x10
6, 10x10
6 cells/kg predicted body weight). The findings demonstrated
safety of the single infusion into patients with ARDS, with no adverse effects or
mortality attributable to the infusion. The trial has entered a phase II stage for
examination of efficacy. Another placebo-controlled study demonstrated safety of
adipose-derived MSCs infused in patients with ARDS (297). However, no significant
efficacy was observed in this study.
To date two non-randomized phase I clinical trials in patients with idiopathic
pulmonary fibrosis have been performed. In the first placenta-derived MSCs were
administrated intravenously (50) and in the second endobronchial infused adipose
derived stromal cells-stromal vascular fraction were used (256). Both these phase I
trials demonstrated good safety profile of MSCs in patients with idiopathic pulmonary
fibrosis.
Although the efficacy of MSCs in lung diseases has not been demonstrated in any
46
studies to date, the above studies proved strong evidence of the safety of MSCs
infusions. There are many variables regarding the potential functions of MSCs,
including dosage, preparation, and tissue source, and more investigation is required to
define its clinical role. The lack of efficacy in the clinical trial in contrast with the
numerous reports on the efficacy in animal models also calls for better pre-clinical
models and more information about the mechanisms of action of MSCs.
1.3.3. Mechanisms
1.3.3.1. Engraftment and regeneration
While intratracheal administration definitely leads to the delivery of MSCs to the
lung, systemic-administrated MSCs have also been reported to localize in the lung
vascular bed initially, and the number of localized cells can be enhanced by lung
injury (269). The retention ability of systemic-administrated MSCs in lung varies in
different studies. The effects of the MSC properties such as size, expression of
specific integrins and proteoglycan patterns on the retention ability remain unclear.
MSCs from different sources present different retention capacities. For example,
MSCs from umbilical cord blood have been reported to be more easily cleared by the
lung compared to BM-MSCs (185). In CS-exposed models, adipose stem cells have
been reported to retain in lung tissue after 7 days or 21 days (222), while another
study only detected 2-13 male BM cells per entire lung from 1 day to 1 month by
fluorescent in situ hybridization (FISH) (112). Higher retention rate of iPSC-MSCs in
the lung compared to BM-MSCs has also been reported 14 days after administration
47
(150).
The capacity of BM-MSCs and adipose-MSCs to differentiate into cells
expressing markers of alveolar or epithelial cells has been demonstrated in vitro (162,
171, 243, 282), which represents an early motivation for research on MSCs-based
therapy: to let the MSCs differentiate into progenitor or somatic cell types, replenish
the damaged cells and thus regenerate the injured tissue. Indeed, early reports
provided evidence of structural-engrafted HSCs, MSCs or endothelial progenitor cells
differentiating into airway and alveolar cells or pulmonary vascular or interstitial cells
(129, 270). However, later research suggested some major technical limitations of
these early studies and current understanding regarding the issue is that the
differentiation of MSCs into structural cells in the lung is rare and with unlikely
physiological significance (270).
Although the retention of MSCs does not lead to differentiation, it may induce
other activities. The retention is possible to modulate paracrine function of MSCs,
such as secretion of the anti-inflammatory cytokine tumor necrosis factor-stimulated
gene-6 (TSG-6) (145). The retention may also facilitate the direct contact between
MSCs and local cell types, which may induce the onset of other mechanism such as
mitochondrial transfer.
1.3.3.2. Immunoregulation
Immunoregulation is a very significant function of MSCs. As mentioned above,
treatment of the autoimmune disease GVHD using BM-MSCs has already been
48
approved for clinical use.
The role of endogenous MSCs in immunoregulation is still largely unknown.
Endogenous MSCs are found to reside perivascularly, and they may represent a
portion of the pericytes (182). At this site they can interact with HSC and may affect
immune responses. The immunoregulating capacity of exogenous MSCs has been
widely reported in vitro. They have been demonstrated to inhibit various functions of
immune cell such as dendritic cell differentiation, B cell proliferation, cytolytic
potential of natural killer cell, and proliferation and activation of T cell (33, 235, 269,
270). MSCs also facilitate the development of the M2 phenotype of macrophages,
which is believed to be anti-inflammatory (81, 266, 286). In addition, MSCs are able
to reduce the release of inflammatory mediators from these immune cells such as
IFN-γ and promote secretion of anti-inflammatory cytokines such as IL-10. The full
mechanism of the immunomodulation is unclear but the paracrine release of
immunomodulating agents including of TGF-β1, HGF, prostaglandin E2 (PGE2), IL-10
and nitric oxide from MSCs may play an important role in the process (2, 235, 270).
1.3.3.3. Mitochondrial transfer
Mitochondrial transfer was first observed in vitro in 2006 by Spees and
colleagues (238). In their study they damaged mitochondrial DNA in a lung epithelial
cell-line A549 by pre-treatment with ethidium bromide, leading to impaired aerobic
respiration and proliferation. The mutated A549 cells only survive in a permissive
medium containing uridine and pyruvate used to supplement anaerobic glycolysis.
49
However, after co-culture with BM-MSCs, these cells re-acquired the capacity to
proliferate in normal medium, indicating restoration of aerobic respiration.
Mitochondrial function was recovered as indicated by ATP levels, and mitochondrial
transfer was identified from BM-MSCs to mutated A549 cells both by DNA
measurement and microscopy. Following this study, mitochondrial transfer from
BM-MSCs to other cell types including endothelial cells, cardiomyocytes and
osteosarcoma cells in vitro has also been reported (55, 192, 204).
In 2012, Islam and colleagues reported the first in vivo study demonstrating the
occurrence and functional significance of mitochondrial transfer from exogenous
BM-MSCs to host airway epithelial cells (118). In the study acute lung injury was
induced by intratracheal infusion of lipopolysaccharide (LPS), followed by
intratracheal infusion of BM-MSCs. They found BM-MSCs attached to the alveolar
epithelia at connexin 43-containing gap junctions. Mitochondrial transfer was
observed within 24 hours from BM-MSCs to alveolar epithelial cells, which is
mediated by connexin 43-dependent generation of nanotubes and microvesicles. The
mitochondrial transfer elevated alveolar ATP concentrations. The therapeutic efficacy
of BM-MSCs on the acute lung injury, including reduction of alveolar leukocytosis
and protein leak, elevation of surfactant secretion and reduction of mortality, were
eliminated in BM-MSCs with mutated connexin 43 or dysfunctional mitochondria.
Current understanding on the mechanisms of mitochondrial transfer is still very
limited. Tunnelling nanotubes (TNTs) are identified as routes through which
mitochondria move intracellularly in most in vitro studies. TNTs were first discovered
50
in vitro by Rustom and colleagues in 2004 as thin structures connecting single cells
over long distance. TNTs are characterized with continuity of membrane connection
between the two cells as well as continued cytoskeleton in the tubes (13). F-actin is
found in most TNTs spanning along the entire length, and inhibition of F-actin
polymerization lead to blockage of TNT formation (40). Machinery of intracellular
movements of mitochondria in neuronal cells has been studied for many years. In
neuronal cells, mitochondrial Rho GTPase 1 (Miro-1, also named Rhot-1), a
calcium-sensitive adaptor protein, can bind mitochondria to kinesin motor protein-5
(KIF5) with the aid of accessory proteins Milton/TRAK (155). Kinesin can then carry
the mitochondria to move along microtubes (155). It has been proposed that this
mechanism may also mediate the intercellular movement of mitochondria along the
TNTs (Figure 1.7). A recent study demonstrated that over-expressing of Miro-1 in
BM-MSCs can promote mitochondrial transfer to epithelial cells and enhance efficacy
in vitro, while Miro-1 knock-down leads to lack of efficacy (6). Over-expression of
Miro-1 also enhanced the efficacy to attenuate rotenone-induced lung injury and
allergic airway inflammation in vivo. This finding provided a possible approach to
selectively block mitochondrial transfer, which may be very helpful for future
research about the role of mitochondrial transfer in MSCs action. Nevertheless, the
mechanisms regulating TNT formation or the Miro-1-involved movement of
mitochondria remain unclear. Further understanding on the topic may help address the
important feature of mitochondria transfer such as the direction selectivity and
enhanced activity upon injury.
51
Figure 1. 7 Proposed mechanism of mitochondrial transfer. BM-MSCs transfer
mitochondria to damaged epithelial cells through tunneling nanotubes (TNTs). Miro-1
loads mitochondria on KIF5 with aid of accessory protein Milton/Trak. KIF-5 moves
along microtubes in the TNTs and thus transfers mitochondria between cells. The
image showing the enlarged region is adapted from Sheng ZH et al, Nat Rev Neurosci
(227).
52
1.4. Aims of Study
The overall hypothesis for this thesis is that iPSC-MSCs are able to alleviate
oxidative stress (CS or ozone)-induced lung damage (histological changes,
hyper-responsiveness, inflammation, apoptosis and mitochondrial dysfunction) in
vivo as well as mitochondrial dysfunction and apoptosis in bronchial epithelial cells
and ASMCs in vitro, through multiple mechanisms including paracrine effect and
mitochondrial transfer.
Based on previous sections, the background of the study can be summarized as
follows:
1. COPD is a chronic and complex disease. It is a rising cause of death associated
with huge social and economic burden. There are no current medications that can
slow the progression of the disease. Cigarette smoking is the major cause of
COPD. Age is also an important risk factor.
2. Mitochondrial dysfunction may play an important role in the pathogenesis of
COPD. Targeting mitochondria may be a novel strategy to develop new treatment
for the disease.
3. Efficacy of MSCs isolated from BM, adipose tissue and cord blood on
CS-induced emphysema models has been demonstrated. BM-MSCs have been
proven to be safe in clinical trials but without efficacy in COPD patients.
4. iPSC-MSCs have several advantages over BM-MSCs including more abundant
resource, high capacity of expanding without loss of differentiation potency and
53
potential to overcome ageing-associated impairment.
5. The mechanisms of action of MSCs are not fully understood. Paracrine effects and
mitochondrial transfer represents two possible mechanisms.
Based on the above knowledge, my Ph.D study focuses on the effects of
iPSC-MSCs on airway cells in a COPD or oxidative stress-related context. The aims
of the study include:
1. To compare effects of iPSC-MSCs and BM-MSCs on CS-induced damage in
the rat lung in vivo and in human bronchial epithelial cells in vitro with an
emphasis on apoptosis. Both mitochondrial transfer and paracrine effects
were examined.
2. To study the capacity of iPSC-MSCs to alleviate mitochondrial dysfunction in
CS-treated human ASMCs in vitro and to examine the relevant contribution of
mitochondrial transfer and paracrine effects to the efficacy.
3. To investigate the capacity of iPSC-MSCs to alleviate oxidative
stress-induced AHR, lung inflammation, mitochondrial dysfunction and
apoptosis in an acute ozone-exposed mouse model.
These studies should provide evidence to demonstrate the potential of
iPSC-MSCs as a candidate for cell-based therapy for COPD.
54
Chapter 2 Material and Methods
55
2.1 Materials
2.1.1 Reagents and chemicals
Chemicals, reagents and recombinant proteins were supplied by Thermo Fisher
Scientific (Carlsbad, CA, USA), Sigma-Aldrich (St. Louis, MO, USA), Amresco
(Solon, OH, USA), Bio-Rad Laboratories (Richmond, CA, USA), Cayman Chemical
(Ann Arbor, MI, USA), Life Technologies (Eugene, OR, USA) and Roche (Mannheim,
Germany). Cell culture reagents were purchased from Invitrogen (Thermo Fisher
Scientific) and Sigma-Aldrich. Cigarettes including Camel (11 mg TAR and 0.8 mg
nicotine, R.J. Reynolds, Winston-Salem, NC, USA) or Marlboro Red (10 mg TAR
and 0.8 mg nicotine, Philip Morris International, New York City, NY, United States)
were obtained from commercial retailers.
2.1.2 Experimental animals
For the in vivo study performed in Hong Kong, the procedures of the project and
the personnel involved were under licenses from Department of Health, Hong Kong
SAR and approved by the Committee on the Use of Live Animals in Teaching and
Research (CULATR) of the University of Hong Kong. Male Sprague-Dawley (SD)
rats (weighted 170-200 g) were purchased from and kept in the Laboratory Animal
Unit, the University of Hong Kong.
For the in vivo study performed at Imperial College London, the procedures of
the project and the personnel involved were under a Project License and Personal
Licenses from the British Home Office, United Kingdom, under the Animals
56
(Scientific Procedures) Act 1986. Male C57BL/6 mice (6 weeks old) were purchased
from Harlan UK Ltd (Wyton, UK) and kept in Central Biomedical Services, Imperial
College London.
The living environment of all animals was monitored and controlled with respect
to temperature, humidity, and day/night cycle. The animals‟ general conditions were
monitored daily.
2.1.3 Human iPSC-MSCs
The iPSC-MSCs were kindly provided by Dr. Lian Q and Professor Tse HF from
the Cardiology Division, Department of Medicine, LKS Faculty of Medicine, HKU.
The protocol to generate iPSC-MSCs includes two steps: inducing iPSCs from
somatic cells, and generating iPSC-MSCs from the iPSCs. Firstly, iMR90 fibroblast
cells (CCL-186, American Type Culture Collection, Manassas, VA, USA) were
transduced with human OCT4, SOX2, NANOG and LIN28 genes through lentiviral
vectors (Plasmid 16577-80, Addgene, Cambridge, MA, USA) overnight. The
transduction gene mixture was then replaced with human embryonic stem cell culture
medium and cells transferred to inactivated mouse embryonic fibroblast (MEF) feeder.
20 days later, cell colonies with human embryonic stem cell morphology were
identified as iPSC-MSCs and propagated. The resulting iPSC lines were differentiated
into iPSC-MSCs as previously described (153). iPSCs were harvested and placed on a
gelatinized 10-cm dish containing knockout Dulbecco‟s modified Eagle medium
(DMEM) (Invitrogen) supplemented with 10% serum replacement medium
57
(Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF) (Invitrogen), 10 ng/mL
platelet-derived growth factor AB (Peprotech, Rocky Hill, NH, USA), and 10 ng/mL
epidermal growth factor (EGF) (Peprotech) to induce differentiation. Cells were left
to differentiate for 7 days after which they were harvested and incubated with
CD24-phycoerythrin and CD105-FITC (BD PharMingen, San Diego, CA, USA). The
CD24-CD105
+ population were separated by fluorescence-activated cell sorting
(FACS). The CD24-CD105
+ cells were diluted and seeded in 96-well plates to select
wells containing a single cell. Upon reaching 70% confluence, the single cell-derived
clones were serially sub-cultured to confluence in a 175-cm2 tissue culture flask. They
were then trypinised, aliquoted and frozen as stocks in liquid nitrogen for future
experiments.
2.1.4 Human bone marrow – derived MSCs (BM-MSCs)
Human BM-MSCs were obtained from a commercial supplier (Cat. No. PT-2501,
Cambrex Bioscience, Rockland, ME, USA). Both iPSC-MSCs and BM-MSCs were
cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum
(Invitrogen), 5 ng/mL bFGF (R&D Systems, Minneapolis, MN, USA), 10 ng/mL EGF
(R&D Systems), 55 μM 2-mercaptoethanol (Invitrogen), 20 units/ml penicillin and 20
μg/ml streptomycin.
2.1.5 BEAS-2B cells
The BEAS-2B cells were virus-transformed immortalized human bronchial
58
epithelial cells from autopsy of non-cancerous individuals. The BEAS-2B cells in this
study were from a commercial supplier (American Type Culture Collection, Rockville,
MD, USA). They were cultured in keratinocyte serum free media K-SFM (Invitrogen)
supplemented with 5 ng/ml EGF and 50 mg/ml bovine pituitary extract. Passages
42-50 were used for experiments.
2.1.6 Primary human airway smooth muscle cells (ASMCs)
ASMCs were isolated from biopsies of bronchi or tracheas of healthy transplant
donor lungs as previously described (174). Briefly, biopsies were cut into small pieces
(<1 mm2) and placed in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 20
units/ml penicillin, 20 μg/ml streptomycin and 2.5 μg/ml amphotericin B. ASMCs
were confirmed by the characteristic “hill and valley” morphology under light
microscopy. Cells were sub-cultured to reach confluence in 175-cm2 tissue culture
flasks and then frozen as stock. Passages 3 to 7 were used for experiments. When
serum-free medium was required in the experiments, it was prepared as phenol-free
DMEM supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, 1:100
non-essential amino acids, 0.1% BSA, 20 units/ml penicillin, 20 μg/ml streptomycin
and 2.5 μg/ml amphotericin B.
All of the above cell types were maintained in a humidified incubator of 5% CO2
and 37C and fed with medium every 2-3 days.
59
2.2 Methods
2.2.1 MSCs treatment to cigarette smoke-exposed rats
Male Sprague-Dawley rats were exposed to 4% (v/v) CS for one hour daily for 56
days (Figure 2.1) using a same protocol as described in a previous study (52). Briefly,
a maximum of 8 rats were placed into a ventilated 20 liter-chamber. 800 ml of CS was
injected into the chamber through a syringe to raise the initial CS concentration to
4%.The filters of the cigarettes were removed beforehand. The chamber was then
ventilated with a steady inflow of 4% CS at 1 L/min – a gas mixture of 100% CS (40
ml/min) and normal air (960 ml/min) from two separate peristaltic pumps (Masterflex,
Cole-Parmer Instruments Co, Niles, IL, USA) (Figure 2.2). The control group was
exposed to sham air (SA) using the same protocol. The exposure was performed in a
chemical fume hood at the same time of each day. At day 29 and day 43, CS-exposed
rats were intravenously injected with plain PBS, 3x106 human BM-MSCs or 3x10
6
human iPSC-MSCs into tail vein (Figure 2.1). The SA group was injected with PBS.
Daily check of general conditions showed no obvious adverse reactions towards the
injections.
Rats were sacrificed 24 hours after the last CS or air exposure by intraperitoneal
injection of over-dosed pentobarbitone (100 mg/kg body weight). The larger lobe of
left lungs was firstly inflated with 1 ml formalin and then fixed, dehydrated and
paraffin embedded. The paraffin block was cut into 5 μm sections. The rest of the lung
tissue was frozen and stored at -80℃.
60
Figure 2. 1 Schematic diagram of BM-/iPSC-MSCs treatments in a cigarette
smoke (CS)-exposed rat model. Male Sprague-Dawley rats were exposed to 4% CS
for 1 hour daily for 56 days. At Days 29 and 43, 3x106
human iPSC-MSCs (n = 8) or
BM-MSCs (n = 8) were injected intravenously through tail veins. A sham air (SA)
group was included in which the rats were exposed to fresh air. At Day 57, the rats
were sacrificed, and the larger lobe of the left lungs were fixed and sectioned for
histological studies.
Air Exposure (1 hour/day, 56 days)
PBS
4% CS Exposure (1 hour/day, 56 days)
4% CS Exposure (1 hour/day, 56 days)
4% CS Exposure (1 hour/day, 56 days)
PBS
Sacrifice
PBS
PBS
BM-MSCs
Day 29 Day 43 Day 57
BM-MSCs
iPSC-MSCs
iPSC-MSCs
Sacrifice
Sacrifice
Sacrifice
SA
CS
iPSC-MSCs
BM-MSCs
Male SD rats
(170-200 g)
61
Figure 2. 2 The set-up of the ventilated smoking chamber. In the right chamber, the
fresh air and CS were mixed in ratio of 96:4 to generate constant flow of 4% CS at
1000 ml/min. In the left chamber, only fresh air was pumped. The whole set-up was
placed in a chemical fume hood.
2.2.2 Histology and morphometric analysis of rat lungs
The paraffin sections were always deparaffinised and dehydrated before staining.
The slides were incubated at 40℃ in an oven for one hour to melt the paraffin,
followed by incubation twice in xylene for 15 minutes. A serial rehydration was then
applied by incubation in 100%, 95%, 75% ethanol and double distilled water (ddH2O)
successively, each for 10 minutes, twice.
Mean linear intercept (Lm) was measured to evaluate airspace enlargement based
on a modified method (62). Briefly, the paraffin sections from the largest lobes of rat
62
lungs were rehydrated and stained with hematoxylin and eosin. The slides were then
mounted in a xylene-based mounting medium, observed using a brightfield
microscope at 10x magnification and analysed using AxioVision (Zeiss, Germany). A
100 μm-length interval was inserted in the field of a randomly selected region of the
lung sections. The interval passed through several alveoli, resulting in multiple
alveolar intercepts. Lm was calculated as 100 μm divided by the number of alveolar
intercepts. 10 fields per section, 8 rats per group were analysed.
Masson's trichrome staining was performed to measure the pulmonary fibrosis
according to the standard protocol. The pulmonary fibrosis was assessed by
semi-quantitative Ashcroft score, which gave each field a score ranging from 0
(normal lung), 1 (minimal fibrous thickening of alveolar or bronchiolar walls), 3
(moderate thickening of walls without obvious damage to lung architecture), 5
(increased fibrosis with definite damage to lung structure and formation of fibrous
bands or small fibrous masses), 7 (severe distortion of structure and large fibrous
areas) to 8 (total fibrous obliteration of the field) (12). Scores from 5 fields per section,
total 6 rats, were assessed under 20x magnification.
2.2.3 iPSC-MSCs treatment to ozone-exposed mice
Male C57BL/6 mice were put into a chamber ventilated with gas mixture of
normal air and ozone produced by an ozone generator (model 500 Ozoniser, Sander,
Wuppertal, Germany). The concentration of ozone in the chamber was monitored by
an ozone detector and maintained at 3 ppm by tuning the ozone generator (Figure 2.3).
63
Control groups were exposed to normal air. The exposure lasted for 3 hours.
iPSC-MSCs or PBS were administrated to mice by intravenous injection through
tail vein. The administration was performed either 24 hours prior or 6 hours after the
exposure (Figure 2.4). There were 5 groups of mice in total: Air, Air with iPSC-MSCs
administrated 24 hours prior (-24 hr), Ozone, Ozone with iPSC-MSCs administrated
24 hours prior-exposure (-24 hr) and Ozone with iPSC-MSCs administrated 6 hour
post-exposure (+6 hr). The Mice from all groups were anaesthetised for AHR
measurements 21 hours after the exposure. They were sacrificed immediately after the
AHR measurements by intraperitoneal injection of over-dosed pentobarbitone (100
mg/kg body weight). Bronchoalveolar lavage (BAL) was collected before the
collection of the lungs. The right lobes were frozen for mitochondria extraction while
left lobes were inflated with and fixed in paraformaldehyde. The larger lobe of the
fixed lungs were later embedded into paraffin blocks and sectioned into 5
μm-sections.
Figure 2. 3 The set-up of the ventilated chamber for ozone exposure. Ozone
generated from an ozone generator and normal air from an air pump was ventilated
into the chamber. The ozone concentration was constantly monitored by an ozone
detector and maintained at 3 ppm by tuning the ozone generator.
64
Figure 2. 4 Schematic diagram of iPSC-MSCs treatments in an ozone-exposed
mouse model. Male C57BL/6 mice were exposed to ozone (3 ppm) for 3 hours.
Control groups were exposed to fresh air. 106
iPSC-MSCs were intravenously injected
into the tail vein either 24 hours prior to the ozone exposure, or 6 hours after the
exposure. Airway hyper-responsiveness (AHR) was measured 21 hours after the
exposure. The mice were then sacrificed and BAL and lung tissues collected.
65
2.2.4 Measurement of airways hyper-responsiveness (AHR) of mice
The AHR was measured as previously described (272). Before the measurement
of AHR, mice were anaesthetised by intraperitoneal injection of
ketamine/xylazine/saline mixture. The trachea was then opened and inserted with a
catheter through which the mouse was ventilated (MiniVent type 845, Hugo Sach
Electronic, Germany) at 250 breaths/minute and a tidal volume of 250 μl. The animal
was continuously monitored in a whole body plethysmograph with a
pneumotachograph connected to a transducer (EMMS, Hants, UK). Another
transducer was connected to an esophageal catheter to evaluate the intrapleural
pressure. The transpulmonary pressure was assessed based on the pressure
measurements from the two transducers. Increasing concentrations (4-256 mg/ml) of
acetylcholine in PBS were then administered into airway via a nebulizer (Aeroneb
Lab Micropump Nebulizer, EMMS) and the pulmonary resistance (RL) was recorded
for 3 minute periods during each dose. Baseline RL was defined as the RL with
nebulised PBS. A concentration-response curve was plotted for each animal and the
concentration of acetylcholine that induced 100% elevation of RL from baseline was
derived (PC100). The value of -log PC100 was taken as a measurement of AHR.
2.2.5 Measurement of cells and cytokines in BAL of mice
Bronchoalveolar lavage (BAL) was collected by rinsing the lungs three times
with 0.8 ml cold PBS through an endotracheal tube. After centrifugation at 3000 rpm,
4°C for 5 min, the supernatant was collected as BAL fluid (BALF) while the cell
66
pellets were resuspended in 200 μl PBS and counted on a haemocytometer for total
cell number. BAL cell samples were centrifuged again onto glass slides by a cytospin
device at 30g for 6 minutes (Shandon Cytospin 4; Thermo Electron Corporation,
Waltham, MA, US). The resulting cell slides were air-dried overnight and stained
using Diff-Quick kit (Reagena, Toivala, Finland) according to manufacturer‟s
instructions. Differential count of white blood cells (neutrophil, macrophage,
lymphocyte and eosinophil) was performed under an optical microscope (Olympus
BH2, Olympus).
The cytokine levels in BALF of mice were determined using a MAGPIX
Luminex multiplexing analyser (Merck Millipore, Darmstadt, Germany), which
simultaneously measured levels of multiple analytes, including MCP-1, Eotaxin, IL-6,
IL-5 and macrophage inflammatory proteins (MIP)-1α.
2.2.6 Isolation of intact mitochondria from mouse lungs
Intact mitochondria were isolated from mouse lungs using a Mitochondria
Isolation Kit for Tissue (Thermo Fisher Scientific) and Dounce tissue grinder set
(Sigma) following the manufacturer‟s instructions. In brief, approximately 20 mg of
lung tissue was washed with PBS supplemented with EDTA-free protease inhibitors
(Roche). The tissue was homogenized in corresponding reagent with 5 strokes of
Dounce A followed by 20 strokes of Dounce B. The homogenates were centrifuged
firstly at 700xg for 10 minutes at 4°C, to sediment large organelle fractions containing
nucleus, plasma membrane, endoplasmic reticulum, Golgi and so on. The
67
supernatants were centrifuged again at 12,000xg for 5 minutes. The supernatants and
pellets were collected as cytosolic fractions and mitochondrial fractions, respectively.
2.2.7 Generation of cigarette smoke medium (CSM)
CSM was prepared as previously described (143). Briefly, cigarette smoke from
two filter-removed cigarettes was bubbled into 20 ml of serum free medium. The
medium was then filtered through a 0.22 μm filter. The optical density (OD) at
wavelength 320 nm was measured.
For the study with BEAS-2B cells which was performed at Hong Kong university,
the cigarettes smoke was from Camel cigarettes (11 mg TAR and 0.8 mg nicotine),
and pumped into serum free medium manually by syringe. 100% CSM was generated
by adjusting the OD at 320 nm with serum free medium to 3.5.
For the study of ASMCs which was performed at Imperial College London, the
cigarette smoke was from Marlboro Red cigarettes (10 mg TAR and 0.8 mg nicotine)
and pumped into serum free medium through a peristaltic pump, which resulted in a
lower OD read-out than the manual method. Thus the 100% CSM was generated by
adjusting the OD at 320 nm with serum free medium to 1.1. The advantage of
peristaltic pump is a very consistent output for each batch of CSM.
After preparation, the 100% CSM was aliquoted and stored at -80℃.
2.2.8 Co-culture of BEAS-2B cells with MSCs.
BEAS-2B cells were pre-stained with CellTrace Violet (Invitrogen) which
68
fluorescently labelled the cells for several generations while iPSC-MSCs or
BM-MSCs were pre-stained with MitoTracker Green (Invitrogen), which specifically
stained the mitochondria in the cells. In brief, BEAS-2B cells were harvested, counted,
incubated with 5 µM Celltrace Violet for 20 minutes, while MSCs were harvested,
counted and incubated with 200 nM Mitotracker Green for 30 minutes. They were
centrifuged at 1000 rpm for 5 min and incubated in complete medium for 10 min after
the staining to wash away the remaining dye. After another centrifugation, they were
re-suspended in 1:1 mixed medium and co-cultured at a 1:1 ratio in a 24-well plate.
Approximately 5 hours later, when the cells attached to the plates, 100% CSM was
added into the medium to make the final concentration 2%. After 24 hours, cells were
visualized by motorized inverted microscope (IX81-ZDC2; Olympus, Shinjuku,
Tokyo City, Tokyo, Japan) or analyzed by flow cytometry with a BD FACS Aria I
Cell Sorter (BD Biosciences, Oxford, UK).
2.2.9 Determination of ATP content in BEAS-2B cells following co-culture
BEAS-2B cells in the co-culture were sorted as CellTrace positive cells by a BD
FACS Aria I Cell Sorter. ATP was extracted with boiling distilled water according to a
one-step method previously described (284). The ATP content was measured by a
luciferase-catalysed luminescent assay using an ATP Determination Kit (Invitrogen)
according to the manufacturer‟s instruction.
69
2.2.10 BM-MSC and iPSC-MSC conditioned medium treatment of CSM-treated
BEAS-2B cells
The conditioned medium (CdM) was prepared as previously described (294).
Briefly, BM-MSCs and iPSC-MSCs were re-fed with DMEM without serum and
supplements. After 24 hr, the medium was collected and concentrated via
centrifugation through ultrafiltration conical tubes (Amicon Ultra-15 with membranes
selective for 5 kDa). The final concentration was adjusted to 20 times that of the
collected CdM.
Alternatively, to investigate the effects of stem cell factor (SCF), SCF was
depleted from iPSC-MSCs-CdM as previously described (139). Briefly, 0.5 μg of
anti-SCF (AF-255-NA; R&D) or normal human IgG control antibody (1-001-A; R&D)
were mixed with protein G-agarose beads in PBS at 4°C for 1 hour with intermittent
shaking. After centrifugation, beads were washed three times and used for
immunedepletion of SCF. iPSC-MSCs-CdM was incubated with protein G-agarose
beads immobilized with anti-SCF antibodies or human IgG control antibody for 1
hour at 4°C. The beads with immune complexes attached were precipitated by
centrifugation. The supernatants collected were either SCF-depleted
iPSC-MSCs-CdM or control iPSC-MSCs-CdM.
The BEAS-2B cells were cultured on top of coverslips in 24-well plates. The
medium was replaced by keratinocyte medium with no supplements 24 hr before the
treatment. They were then treated with 2% (v/v) CSM. BM-MSCs-CdM or
iPSC-MSCs-CdM containing 3 μg of total protein was added at the same time. After
70
24 hours, the supernatant was removed and the cells fixed for immunohistochemical
tests or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
(TUNEL) assay. To investigate role of SCF, the cells were treated with either
SCF-depleted iPSC-MSCs-CdM or recombinant SCF (255-SC, R&D) with 2% CSM.
2.2.11 Immunohistochemistry
To detect expression and localization of various proteins in lung sections and cell
culture, standard immunohistochemistry procedures were applied. Briefly, lung
sections were serially rehydrated and antigen retrieved by boiling for 5 minutes at
120C in Sodium Citrate Buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0).
They were then blocked by 5% bovine serum albumin (BSA) for 30 minutes at room
temperature followed by overnight incubation in primary antibodies at 4C. The
primary antibodies included human nuclear antigen (HNA) (MAB1281, Chemicon),
human/rat clara cell secretory protein 10 (CC10) (SC-9772, Santa Cruz, CA, USA),
human/rat/mouse Ki-67 (ab15580, Abcam, Cambridge, UK), human/rat Complex I
(SC-271387, Santa Cruz), human COX4 (SC-133478, Santa Cruz), rat/mouse CD68
(SC-7084, Santa Cruz), mouse/rat/human C-kit (SC-1494, Santa Cruz). After 3 washs
with PBS, the sections were incubated in secondary antibodies including horseradish
peroxidase-conjugated or fluorescent-conjugated anti-mouse IgG, anti-rat IgG and
anti-rabbit IgG for 1 hour at room temperature. For HRP-conjugated secondary
antibodies, the targets were recognized by an intense brown colour after developing
with 3,3'-diaminobenzidine (DAB) substrate solution. For fluorescent-conjugated
71
secondary antibodies, the sections were mounted with mounting medium (ProLong
Gold antifade reagent with DAPI, Invitrogen) containing
4,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. At least five fields per
section were randomly captured under a conventional fluorescent microscope
(IX81-ZDC2; Olympus) at the desired magnification and analyzed using AxioVision
(Zeiss, Oberkochen, Germany).
For cell culture, the cells were seeded and treated on coverslips in 24-well dishes.
They were fixed with 4% paraformaldehyde in PBS for 15 min, blocked with 5%
BSA for 30 min and permeabilized with 0.1% Triton-100 in PBS for 3 min, all at
room temperature. The cells were incubated in primary antibodies including C-kit
(SC-1494, Santa Cruz) and cytochrome c (SC-13156, Santa Cruz) at room
temperature for 1 hr and then in fluorescent-conjugated secondary antibodies. The
coverslips were taken out from the wells, flipped and put on a drop of mounting
medium containing DAPI on glass slides. Images of 5 fields for each slide were
captured randomly by a motorized inverted microscope (Olympus) and analyzed
using AxioVision (Zeiss). The ratio of positively stained cells in the total cell
population was calculated as the rate of cytochrome c translocation, apoptosis or
proliferation.
2.2.12 Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
(TUNEL) assay
TUNEL assay detects cell apoptosis by labelling the terminal end of nucleic acids
72
which increase in abundance in cells only if the DNA becomes fragmented. Apoptosis
of BEAS-2B cells and mouse lung sections were determined via TUNEL assay using
the In Situ Cell Death Detection Kit, POD (Roche Applied Science, Mannheim,
Germany).
For analysis of the lung sections, they were rehydrated as per the
immunohistochemistry protocol, and heated in citrate buffer in a microwave oven for
5 minutes. They were then washed with PBS and incubated with TUNEL reaction
mixture for 1 hour. Nucleus of apoptotic cells were stained green by the reaction.
For the BEAS-2B cells, they were fixed with 4% paraformaldehyde in PBS,
blocked with 3% H2O2 in methanol, permeabilized with 0.1% Triton-100 in 0.1%
sodium citrate and washed with PBS. TUNEL reaction mixture was then applied for a
one-hour incubation at 37 ℃.
All samples were mounted in mounting medium containing DAPI. Images of 5
fields for each slide were captured randomly by a motorized inverted microscope
(Olympus) and analysed using AxioVision (Zeiss).
2.2.13 Total protein extraction from cell culture
To extract total protein from cultured cells, cells in 6-well plates were first
washed with ice-cold PBS and then frozen at -80℃. The cells were then scraped with
approximately 50 μl/well T-PER Cell Protein Extraction Reagent (Thermo Fisher
Scientific) supplemented with protease/phosphatase inhibitor cocktail (Thermo Fisher
Scientific) and 100 mM PMSF. The cell/reagent mixture were vortexed, put on ice for
73
30 min and centrifuged at 16000xg at 4℃ for 30 minutes. The supernatant was
collected as cell lysates and stored at -80℃. Protein concentrations were determined
by Bradford protein assay using bovine BSA as standard.
2.2.14 Western blot
Cell lysates containing the comparable amounts of total protein (20-40 μg) were
denatured and reduced by boiling at 95℃ for 5 minutes in Laemmli buffer with
sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. The proteins in the samples
were separated by size by running through an SDS–polyacrylamide gel. They were
transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories)
which was then blocked in 5% skimmed milk in Tris-buffered saline (pH 7.4) with 0.1%
Tween-20. The membranes were incubated with diluted anti-C-kit (1:500) (SC-1494,
Santa Cruz) or anti-β-actin (1:5000) antibodies at 4°C overnight, washed three times,
and then incubated with horseradish peroxide-conjugated anti-goat antibody (1:2000)
or anti-mouse antibody (1:2000) (Dako, Danmark) for 1 h at room temperature. After
three further washes and a one-minute incubation with enhanced chemiluminescence
(ECL), the membranes were exposed to x-ray films (Fujifilm, Tokyo, Japan) on which
the target proteins were developed as dark bands.
2.2.15 Enzyme-linked immunosorbent assay (ELISA)
The concentration of SCF in CdM was measured by Human SCF DuoSet ELISA
Kit (R&D) according to manufacturer‟s instructions. In general, samples and serially
74
diluted standards were added into 96-well plates coated with capture antibody. After
adequate washing, the captured target proteins were further incubated with detecting
antibody and streptavidin conjugated with horseradish peroxidase (HRP),
consecutively. HRP catalysed a colorimetric reaction of tetramethylbenzidine (TMB)
substrates. The reaction was stopped by adding 2 N H2SO4, and the OD was measured
at wavelength 450/570 nm in a microplate reader (FLUOstar Optima, BMG Labtech).
A standard curve was generated according to OD values of the standards, and SCF
levels of CdM were calculated from the standard curve.
2.2.16 Treatment of CSM or H2O2 to ASMCs or iPSC-MSCs
ASMCs were cultured in 6-well plates or 96-well plates. On reaching 90%
confluence, the medium was replaced with serum-free medium for 24 hours. The cells
were then treated with different concentrations of H2O2 (50, 100 and 200 μM) or CSM
(10, 25, 50 and 75%) for 2, 4 and 24 hours. A cessation group was also included, in
which after 4 hours of treatment H2O2 or CSM was replaced with fresh serum-free
medium for the next 20 hours. The iPSC-MSCs were similarly treated for 4 hours and
24 hours. The cell viability, mitochondria membrane potential and mitochondrial ROS
levels were measured by methylthiazolyldiphenyl-tetrazoliumbromide (MTT) assay,
JC-1 (5,5‟,6,6‟-tetrachloro-1,1‟,3,3‟-tetraethylbenzimidazolylcarbocyanineiodide) and
MitoSOX staining, respectively.
75
2.2.17 Cell viability assay
MTT assay is a commonly used cell viability assay. In brief, MTT
(Sigma-Aldrich) was dissolved in 37 ℃ serum free medium at 1 mg/ml. ASMCs or
iPSC-MSCs cultured in 96-well plates were incubated with 100 μl MTT solution at
37 ℃ after treatments. After 15 min, the cells were checked every 5 minutes using
microscope to observe formation of purple color within. When the color is visible but
not too intense, the MTT solution was replaced with 50 μl DMSO. After shaking for 5
minutes, the OD at wavelength 550 nm was measured.
2.2.18 Measurement of mitochondrial ROS
Mitochondrial ROS levels were assessed by staining with MitoSOX Red
Mitochondrial Superoxide Indicator (Invitrogen). 50 μg MitoSOX powder was
dissolved in 13 μl DMSO to make a 5 mM stock solution and further diluted to 5 μM
with relevant buffers to make working solutions.
For the in vitro study of ASMCs, cells in 6-well plates were washed with
modified Hank‟s Balanced Salt Solution (HBSS) (containing Ca/Mg) (Sigma-Aldrich),
and incubated with 5 μM MitoSOX diluted in modified HBSS for 30 minutes. The
cells were then washed with HBSS (Sigma-Aldrich) and incubated with 500 µl/well
accutase solution (Sigma-Aldrich) for up to 5 min at 37 °C and 5% CO2. One ml/well
DMEM with 4 mM L-glutamine and 10% FBS was then added to the detached cells.
After centrifugation and resuspension in HBSS, the intensity of red fluorescence was
measured at 510/580 nm by a FACSCanto II flow cytometer (BD Biosciences).
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For the in vivo study of ozone-exposed mice, the intact mitochondria were
isolated from lungs, and then incubated with 5 μM MitoSOX in 96-well plates for 30
minutes at 37°C. The intensity of red fluorescence was measured at 510/580 nm using
a fluorescence plate reader.
2.2.19 Mitochondrial membrane potential
Mitochondrial membrane potential (ΔΨm) was determined by staining with JC-1
dye (Invitrogen). The dye exists as monomers with green fluorescence, but when
they enter live mitochondria they form J-aggregates which exhibit red fluorescence.
The accumulation of the dye in mitochondria is potential-dependent, making the ratio
of red to green fluorescence as an indicator of ΔΨm. ASMCs or intact mitochondria
from mouse lungs were incubated with 2 µM of JC-1 for 30 minutes at 37°C. The
fluorescent signal was determined either using a FACSCanto II flow cytometer (BD
Biosciences) for the living cells, or a fluorescence plate reader (Synergy HT Biotek,
Winooski, VT, USA) for the isolated mouse mitochondria. The green fluorescence
from JC-1 monomers was measured at excitation/emission ratios of 485/535 nm and
the red fluorescence from J-aggregates at 560/595 nm.
2.2.20 Annexin V staining
The apoptosis of ASMCs/iPSC-MSCs co-culture was detected by Annexin V
staining through flow cytometry. Annexin V detects apoptosis by binding to the
externalized phosphatidylserine in apoptotic cells. Briefly, cells were harvested and
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resuspended at a density of 106 cells/ml in assay buffer. 5 μl of FITC-conjugated
Annexin V (Invitrogen) was added into 100 μl of cell suspension. After 15
minutes-incubation at room temperature, the intensity of Annexin V was measured by
a FACSCanto II flow cytometer (BD Biosciences).
2.2.21 Direct co-culture of ASMCs and iPSC-MSCs
To study the effects of direct interaction between ASMCs and iPSC-MSCs on
mitochondrial ROS, ΔΨm and apoptosis, ASMCs and iPSC-MSCs were co-cultured
in using two different procedures, one as a prophylactic study and the other as
therapeutic (Figure 2.5).
For the prophylactic study, the ASMCs and iPSC-MSCs were cultured in T150
flasks separately before the experiments. The ASMCs were trypsinized, resuspended
and counted. They were washed with HBSS by centrifugation and resuspension,
followed by 20 minutes-incubation with CellTrace Violet (1:1000 in HBSS, 1 ml/106
cells). After centrifugation, they were incubated with complete medium for 10
minutes to wash and neutralize the dye. Meanwhile, the iPSC-MSCs were trypsinized,
resuspended and counted. The two cell types were mixed and seeded in 6-well plates
at a density of 105 ASMCs plus 1.5x10
5 iPSC-MSCs per well. Single cell culture
groups were included as comparison, in which only 105 ASMCs or 1.5x10
5
iPSC-MSCs were seeded into each well. After 20 hours, the medium was replaced
with 10% or 25% CSM for 4 hours. The mitochondrial ROS, ΔΨm and apoptosis
were then assessed by MitoSOX, JC-1 and Annexin V staining through flow
78
cytometry.
For the therapeutic study, 105 ASMCs were first seeded in each well of 6-well
plates. 20 hours later, they were incubated with CellTrace Violet for 20 minutes
followed by treatment with 10% or 25% CSM for 4 hours. The CSM were then
removed and 1.5x105
iPSC-MSCs were added into each well. After another 24 hours,
the mitochondrial ROS, mitochondrial membrane potential and apoptosis were
measured through flow cytometry.
2.2.22 Detection of mitochondrial transfer in co-culture of ASMCs and
iPSC-MSCs
To detect mitochondrial transfer in the co-culture of ASMCs and iPSC-MSCs,
ASMCs pre-stained with CellTrace Violet and iPSC-MSCs pre-stained with
MitoTracker Red (for fluorescent microscope) or MitoTracker Green (for flow
cytometry) were mixed and seeded into 6-well plates at a density of 105 ASMCs plus
1.5x105
iPSC-MSCs per well. 20 hours later, they were treated with 10% or 25%
CSM and analyzed by flow cytometry. The percentage of ASMCs that were
MitoTracker positive was subsequently calculated. For fluorescent microscopy, the
cells were fixed in 3.7% formaldehyde (Sigma-Aldrich) for 10 minutes, permeabilized
in 0.1% Triton X-100 (Sigma-Aldrich) for 5 minutes and stained by Alexa Fluor 488
phalloidin (Invitrogen) in PBS with 1% BSA. Phalloidin is a probe that labels
filamentous actin selectively. The cells were then visualized by a motorized inverted
fluorescent microscope.
79
A
B
Figure 2. 5 Protocols of direct co-culture of iPSC-MSCs and ASMCs. (A)
Prophylactic co-culture with iPSC-MSCs. 1.5x 105 ASMCs were pre-stained with
CellTrace Violet and seeded in 6-well plates either with or without 105
iPSC-MSCs. 20 hours later they were treated with CSM for 4 hours. MitoSOX ,
JC-1 and Annexin V staining were applied to measure mitochondrial ROS, ΔΨm
and apoptosis. (B) Therapeutic co-culture with iPSC-MSCs. 1.5x 105 ASMCs were
labeled with CellTrace Violet and treated with CSM for 4 hours. CSM was
removed and either 105 iPSC-MSCs or serum free medium was added. After 20
hours of co-culture, the mitochondrial ROS and membrane potential were
measured.
80
2.2.23 iPSC-MSCs-conditioned medium treatment to ASMCs
The iPSC-MSCs-CdM was prepared as described above. The ASMCs were
pretreated with 20-fold diluted iPSC-MSCs-CdM for 4 hours. The CSM were then
added on top of the iPSC-MSCs-CdM to make 10% or 25% CSM. Serum free
medium was added to the control group or 10% CSM group to keep the total volume
of each well the same. 4 hours later, the mitochondrial ROS, ΔΨm and apoptosis were
measured.
2.2.24 Transwell co-culture between ASMCs and iPSC-MSCs
To study the effects of paracrine crosstalk between ASMCs and iPSC-MSCs, they
were co-cultured in a transwell co-culture system. In brief, 105 ASMCs were seeded
in 6-well plates while 1.5x105
iPSC-MSCs were seeded in a cell-culture insert with
pores sized 0.4 μm (Falcon, Corning, NY, USA). They were incubated overnight and
then combined. Single-culture groups were included with empty inserts or empty
wells. After 20 hours‟ co-culture, they were treated with 10% or 25% CSM for 4
hours, and assessed for the mitochondrial ROS, ΔΨm and apoptosis.
2.2.25 Statistical analysis
Numerical data are presented as mean±standard error of mean (SEM). The
statistical analysis was performed using software Prism 5.0 (Graphpad, San Diego,
CA, USA). Differences between groups were evaluated by student t-test or one-way
ANOVA followed by Newman-Keuls test when appropriate. Significant difference
was defined as when p-value < 0.05. The number of animals required for the study
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was determined by power calculations (78) using data from previous studies
conducted in the department. In particular, for CS-exposed rats, the Lm were
31.28±1.75 μm vs. 19.79±0.81 μm for CS and SA groups (n=9) in a previous report
(52). Based on this change, the maximal number of animals to attain statistical
significance of p<0.01 with a 95% probability equated to 6. Considering possible loss
of animals during experimental period, n=8 was chosen for the rat experiments. For
the ozone-exposed mice, -logPC100 was reported to be 1.059±0.127 vs. 2.088±0.136
for ozone and air groups (n=8) respectively (275). Based on these data, the maximal
number of animals to attain statistical significance of p<0.01 with a 95% probability
equated to 5. n=5-6 was chosen for the mouse study.
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Chapter 3. iPSC-MSCs Attenuate CS-induced
Damage in Airway Epithelial Cells through
Mitochondrial Transfer and Paracrine Effect
83
3.1. Introduction
Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary
disease (COPD) (23, 39). In the first part of this study a rat model of chronic exposure
to CS was used to study the effects of BM-MSCs and iPSC-MSCs on CS-induced
alterations of lung regarding emphysema, fibrosis, inflammation, apoptosis and
proliferation. Male SD rats were exposed to 4% CS for 1 hour/day for 56 consecutive
days. 3x106 of BM-MSCs or iPSC-MSCs were intravenously administrated at day 29
and 43. A control group exposed to sham air (SA) was included.
Despite numerous reports of MSCs‟ effectiveness in diverse disease models, the
mechanism of MSCs‟ action remains unresolved (270). A major breakthrough was the
report of mitochondrial transfer from BM-MSCs to pulmonary alveoli which
protected mice against acute lung injury (118). In this study I hypothesized that a
similar phenomenon also existed in the CS-exposed model. The results demonstrated
mitochondrial transfer in vivo from BM-MSCs or iPSC-MSCs to the local cells of
CS-exposed lung tissue. The in vivo findings were followed by in vitro studies on
such activity in a co-culture of MSCs and bronchial epithelial cells (BEAS-2B)
treated with CSM. The mitochondrial transfer was determined through fluorescent
microscope and flow cytometry, and the effect on ATP depletion was also examined.
Although mitochondrial transfer may be a potent way to rescue damaged cells,
such action requires direct cell contact and is therefore restricted to small regions
around the MSCs. On the other hand, MSCs have been reported to induce paracrine
effects via release of various immunomodulators, such as TGF-β1, hepatic growth
84
factor, prostaglandin E2, interleukin (IL)-10 and nitric oxide (2, 235, 270). The
paracrine effect can be effective in a large radius from MSCs as it does not rely on
direct cell-cell contact. Therefore I further investigated the effects of the paracrine
factors released from MSCs in an in vitro model using bronchial epithelial cells. In
particular, the role of stem cell factor (SCF) secreted from MSCs, a growth factor that
can mediate cell survival, migration, and proliferation was examined (146). BEAS-2B
cells were treated with conditioned medium (CdM) from BM-MSCs or iPSC-MSCs to
test a general paracrine effect on apoptosis and proliferation. SCF was then depleted
from CdM for a more specific investigation on the role of SCF in the CdM.
The aims of the study include:
1. To examine the effect of iPSC-MSCs on CS-induced lung damage in a chronic
CS-exposed rat model compared with BM-MSCs.
2. To determine whether there is mitochondrial transfer from iPSC-MSCs to
CS-damaged airway epithelial cells both in vivo and in vitro.
3. To investigate the involvement of paracrine regulation on the protective effects of
iPSC-MSCs on CSM-induced apoptosis in bronchial epithelial cells by using
conditioned medium (CdM), and to specifically determine the role of a potential
functional molecule, namely stem cell factor (SCF).
85
3.2. Effects of iPSC-MSCs on CS-induced lung damage in
rats.
3.2.1. iPSC-MSCs attenuated CS-induced airspace enlargement, fibrosis and
inflammation in rat lung
Emphysema is one of the major characteristics of COPD. In this study, airspace
enlargement in the CS-exposed rat lung was assessed by histological examination
after haematoxylin and eosin staining. CS exposure caused obvious alveolar
destruction (Figure 3.1A), leading to a much longer mean linear intercept (Lm) (60 ±
4.6 μm) compared to the SA group (31.0±2.3 μm, p<0.001) (Figure 3.1B). The
airspace enlargement was attenuated by the BM-MSCs treatment (Figure 3.1A), with
a significantly reduced Lm compared with the CS group (48.0±4.0 μm, p<0.01)
(Figure 3.1B). Treatment with iPSC-MSCs also attenuated alveolar wall destruction
(Figure 3.1A), leading to significant reduction in Lm compared to the CS group
(40.0±2 μm, p<0.05). Moreover, the treatment with iPSC-MSCs was more effective
than BM-MSCs, as indicated by a shorter Lm value (p < 0.05) (Figure 3.1B).
CS also induced airway fibrosis compared to the SA group, as shown by the
Masson's Trichrome staining (Figure 3.2) (5±0.13% vs. 0.98±0.09%, p < 0.05).
Fibrosis was reduced by both BM-MSCs (3.85±0.17%, p < 0.05) and iPSC-MSCs
treatment (2.37±0.12%, p < 0.05), and iPSC-MSCs demonstrated superior effect
compared to BM-MSCs (p < 0.05) (Figure 3.2).
The inflammation induced by CS in lung tissues was illustrated by
immunohistochemistry staining against CD68, a transmembrane glycoprotein highly
86
expressed by monocytes and tissue macrophages (26) (Figure 3.3A). CS exposure
significantly elevated the number of CD68-positive cells compared to SA group
(21.3±1.9 vs. 2.0±0.41%; p<0.01) (Figure 3.3B). Both treatments of iPSC-MSCs and
BM-MSCs significantly reduced the number of CD68-positive cells compared to the
CS group (4.3±0.8% for iPSC-MSCs and 12.3±1.3% for BM-MSCs respectively;
p<0.01) with a superior effect seen iPSC-MSCs (p<0.01) (Figure 3.3B).
87
Figure 3.1 Human iPSC-MSCs and BM-MSCs alleviated CS-induced airspace
enlargement in rats. (A) Histological assessment of lung sections stained with
haematoxylin and eosin at 10x magnification. Scale bar = 100 μm. (B) Morphometric
analysis of mean linear intercept (Lm) (n=8). *p<0.05 compared with SA group;
#p<0.05 compared with CS group;
†p<0.05 compared with BM-MSCs group.
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Figure 3.2 BM-MSCs and iPSC-MSCs attenuated CS-induced lung fibrosis in
rats. (A) Masson's trichrome staining of lung sections. Blue staining indicates site of
fibrosis. 40x magnification. Scale bar = 50 μm. (B) Percentage of bronchioles with
fibrosis (n=8). *p<0.05 compared to SA group; #p<0.05 compared to CS group;
†p<0.05 compared to BM-MSCs group.
89
Figure 3.3 BM-MSCs and iPSC-MSCs ameliorated CS-induced inflammation in
rat lungs. (A) Illustration of monocytes/macrophages as CD68 positive cells with
brown staining. (B) Number of CD68 positive cells in lung sections (n=6-8).
**p<0.01 compared to SA group; ##
p<0.01 compared to CS group; ††
p<0.01 compared
to BM-MSCs group.
90
3.2.2. iPSC-MSCs attenuated CS-induced apoptosis/proliferation imbalance in
rat lung epithelium.
To illustrate apoptosis in the epithelium, lung sections were co-stained with the
TUNEL assay which labels apoptotic cells, and Clara cell 10-kDa protein (CC-10)
which is a marker of epithelium (Figure 3.4A). CS exposure induced increased
apoptosis in lung epithelium (5.2±0.7% vs. 0.4±0.2%; p<0.01), which was attenuated
by both BM-MSCs (3.4±0.5%; p<0.01 in comparison to CS group) and iPSC-MSCs
(2.0±0.6%; p<0.01 in comparison to CS group) (Figure 3.4B). The treatment of
iPSC-MSCs was more effective than BM-MSCs group (p<0.01) (Figure 3.4B).
CS exposure also led to reduction of proliferation in the epithelium, which was
shown by co-staining against CC-10 and Ki-67, a marker of cell proliferation (221)
(Figure 3.4A). CS exposure significantly reduced the number of proliferating cells in
the epithelium (5.5±0.5% vs. 25.8±2.8%; p<0.01), which was improved by the
treatment with either BM-MSCs (11.2±0.8%; p<0.01 in comparison to CS group) or
iPSC-MSCs (15.2±2.3%; p<0.01 in comparison to CS group) (Figure 3.4B). The
iPSC-MSCs group demonstrated higher proliferation activity than the BM-MSCs
group (p<0.01) (Figure 3.4B).
91
3.2.3. Mitochondrial transfer from MSCs to adjacent lung cells
The epithelium in the rat lung was stained with CC-10, and mitochondria in both
MSCs and host cells were labeled with an antibody against human/rat Complex I.
Meanwhile, human mitochondria were detected by staining using a human specific
Cox-4 antibody (Figure 3.5A&B). For both BM-MSCs and iPSC-MSCs treatments,
human mitochondria were detected inside some of the CC-10-expressing cells
(Figure 3.5A&B), indicating events of mitochondrial adoption by the bronchial
epithelium from the foreign human MSCs. With the same number of cells injected,
rats treated with iPSC-MSCs demonstrated a higher number of cells receiving
MSC-derived mitochondria than those treated with BM-MSCs (number/40x field:
8.4 ± 1.6 vs. 4.6 ± 0.9, p< 0.05; Figure 3.5C).
92
Figure 3.4 BM-MSCs and iPSC-MSCs attenuated CS-induced increase in
apoptosis and reduction of proliferation in rat lung epithelium. (A) Apoptotic
cells in airway epithelium. Red (CC-10): airway epithelium; green (TUNEL):
apoptotic cells; blue (DAPI): nuclei. (B) Proliferative cells in airway epithelium.
Green (CC-10): airway epithelium, Red (Ki-67): proliferative cells; Blue (DAPI):
nuclei. (C) Number of apoptotic cells in airway epithelium (n=6-8). (D) Number of
proliferating cells in airway epithelium. **p<0.01 compared to SA; ##
p<0.01
compared to CS; ††
p<0.01 compared to BM-MSCs.
93
Figure 3.5 Mitochondria transfer from MSCs to lung epithelium in rats. Lung
sections treated with (A) BM-MSCs and (B) iPSC-MSCs were co-stained against
CC-10 (red) marking lung epithelium, human/rat Complex I (green) labeling
human/rat mitochondria and human Cox-4 (violet) detecting human mitochondria
specifically. Co-localization of three signals indicates receiving of human
mitochondria by lung epithelium of rats. Scale bar = 50 μm. (C) Number of cells
receiving foreign mitochondria in 40x fields. *p<0.05 compared between iPSC-MSCs
and BM-MSCs groups.
94
3.3. Mitochondrial Transfer from MSCs to BEAS-2B cells in
vitro
3.3.1. Mitochondrial transfer from MSCs to BEAS-2B cells
Mitochondrial transfer was further investigated in an in vitro co-culture model in
the context of CSM treatment. According to a previous report, treatment of 2% CSM
for 24 hours induces a significant inflammatory response and oxidative stress without
loss of viability in human bronchial epithelial BEAS-2B cells (143). This condition
was adopted in the co-culture study. BEAS-2B cells were pre-labeled with CellTrace
Violet while mitochondria of BM-MSCs or iPSC-MSCs were pre-labeled with
MitoTracker Green. BEAS-2B cells were then co-cultured with either BM-MSCs or
iPSC-MSCs in a 1:1 ratio and treated with 2% CSM for 24 hours. The
MitoTracker-labeled mitochondria which originated from the MSCs were observed in
neighboring BEAS-2B cells, indicating that mitochondria transferred from MSCs to
BEAS-2B cells within 24 hours (Figure 3.6A&B). Moreover, formation of tunneling
nanotube (TNT)-like structures between the MSCs and BEAS-2B cells was identified
after 24 hours (Figure 3.6A&B). The formation of TNTs was reported to be blocked
by Cytochalasin B at nanomolar concentrations without affecting endocytosis and
phagocytosis (40). In this study, when 300 nM of cytochalasin B was added to the
co-culture together with the CSM, the formation of TNTs was highly inhibited and
few MSCs-derived mitochondria were observed in BEAS-2B cells (Figure 3.6 C&D),
indicating that that mitochondrial transfer may be mediated through TNTs.
95
Figure 3.6 Mitochondrial transfer from BM-MSCs and iPSC-MSCs to BEAS-2B
cells in co-culture. (A) BM-MSCs or (B) iPSC-MSCs pre-stained with MitoTracker
Green and BEAS-2B cells pre-labeled by CellTrace Violet were co-cultured in a 1:1
ratio and treated with 2% CSM for 24 hours. The observation of MitoTracker Green
in the BEAS-2B cells (Blue) indicated the adoption of MSCs-derived mitochondria by
BEAS-2B cells. Connection between the cells through TNT-like structures was
observed. Treatment of Cytochalasin B suppressed the formation of TNT-like
structures and mitochondrial transfer in both (C) BM-MSCs and (D) iPSC-MSCs
group. Scale bar = 20 μm. B2B: BEAS-2B cells; DIC: differential interference
contrast. A representative image from three independent experiments is shown.
96
3.3.2. CSM promoted mitochondrial transfer from MSCs to BEAS-2B cells
In addition to fluorescent microscopy, mitochondrial transfer in the co-culture
was further evidenced by flow cytometry at different time-points. The percentage of
CellTrace/MitoTracker double positive cells in the whole CellTrace positive
population was defined as the transfer rate (Figure 3.7A). There were three findings
from this experiment. First, the transfer rate gradually increased along the co-culture
period in both BM-MSCs and iPSC-MSCs groups. Second, after 24 hours, the
iPSC-MSCs group demonstrated a higher transfer rate compared with the BM-MSCs
group (38.8±2% vs. 27.8±1.6%, p<0.05), suggesting a higher potential for
mitochondrial transfer between iPSC-MSCs and BEAS-2B cells. Lastly, at the 24
hour time-point, CSM treatment significantly increased the transfer rate compared to
the untreated group, both for the BM-MSCs co-culture (27.8±1.6% vs. 14.4±1%, p<
0.05) and iPSC-MSCs co-culture (38.8±2% vs. 18±1.9%, p<0.05), suggesting that
mitochondrial transfer is promoted in the presence of CSM.
3.3.3. Co-culture with iPSC-MSCs elevated ATP levels in BEAS-2B cells
Given the observation of mitochondrial transfer in the co-culture, the effect of
co-culture with BM-MSCs and iPSC-MSCs on intracellular ATP levels in BEAS-2B
cells was investigated. BEAS-2B cells were identified from the co-culture followed
by determination of intracellular ATP levels. The 2% CSM treatment for 24 hours
significantly reduced the intracellular ATP level (percentage of control: 76.5 ±3.5% vs.
100 ±10.1%, p<0.05) (Figure 3.8). The drop of ATP level was restored by co-culture
97
with iPSC-MSCs (percentage of control: 97.8 ±9.1% vs. 76.5 ±3.5% for iPSC-MSCs
and CSM group respectively, p<0.05), but not the BM-MSCs (percentage of control:
80.7 ± 7.1%) (Figure 3.8).
Figure 3.7 CSM promoted mitochondrial transfer. (A) Representative flow
cytometry outcome of single culture of MSCs (left), BEAS-2B cells (middle) and
co-culture (right). BM-MSCs or iPSC-MSCs pre-labeled with MitoTracker Green and
BEAS-2B cells pre-labeled with CellTrace Violet were co-cultured at a 1:1 ratio in
the presence of 2% CSM. (B) Effects of time, CSM and cell type on mitochondrial
transfer rate (n=3).Transfer rate was defined as the percentage of BEAS-2B cells
containing MSCs-derived mitochondria in the total BEAS-2B population. W/O stands
for CSM untreated group while other groups were treated with 2% CSM for 24 hours.
*p<0.05 represents statistical difference as indicated.
98
Figure 3.8 iPSC-MSCs restored intracellular ATP levels of BEAS-2B cells.
BEAS-2B cells were co-cultured with MSCs with 2% CSM for 24 hours before sorted
out through flow cytometry and determination of intracellular ATP levels (n=3).
*p<0.05 compared to control group; #p<0.05 compared to CSM group;
†p<0.05
compared to BM-MSCs group.
3.4. iPSC-MSCs Attenuated CSM-induced Apoptosis and
Proliferation Imbalance in BEAS-2B Cells by Paracrine
Effect
3.4.1. iPSC-MSCs-CdM attenuates CSM-induced apoptosis in BEAS-2B cells
Conditioned medium (CdM) from BM-MSCs and iPSC-MSCs contains paracrine
factors released from the MSCs accumulated over 24 hours. Thus the effects of
MSCs-CdM on BEAS-2B cells were investigated in order to study the paracrine effect.
BEAS-2B cells were treated with 2% CSM and CdM for 24 hr. The release of
cytochrome c from mitochondria and its translocation to nucleus (Figure 3.9A) is a
major event in triggering the onset of apoptosis (184, 211). CSM elevated the
percentage of cells with cytochrome c translocation compared to the untreated group
99
(73.4±6.8%vs. 1.88±1%; p<0.01), which was attenuated by both BM-MSCs-CdM
(41.2±6.3%; p<0.01 in comparison to CSM group) and iPSC-MSCs-CdM (16.6±5%;
p<0.01 in comparison to CSM group) (Figure 3.9D). iPSC-MSCs-CdM exhibited a
superior effect compared with to BM-MSCs-CdM (p<0.01) (Fig. 3.9D). In addition,
cellular apoptosis was identified by TUNEL staining (Figure 3.9B). CSM elevated the
percentage of apoptotic cells (16.2±2% vs. 1.4±0.4%; p<0.01), which was attenuated
by both BM-MSCs-CdM (9.3±0.7%; p<0.01 in comparison to CSM group) and
iPSC-MSCs-CdM (5.5±0.5%; p<0.01 in comparison to CSM group) (Figure 3.9E).
There were fewer apoptotic cells in the iPSC-MSCs-CdM group compared to
BM-MSCs-CdM group (p<0.01), suggesting a better anti-apoptotic activity of
iPSC-MSCs-CdM.
While apoptosis was enhanced, the percentage of proliferating cells was reduced
by the 2% CSM (3.4±0.5% vs. 19.2±0.8%; p<0.01) as determined by staining against
Ki-67 (Figure 3.9C&F). This reduction was improved by both BM-MSCs-CdM
(8.2±0.6%; p<0.01 in comparison to CSM group) and iPSC-MSCs-CdM (13.4±0.5%;
p<0.01 in comparison to CSM group) (Figure 3.9F), in which iPSC-MSCs-CdM
demonstrated a more potent effect (p<0.1) (Figure 3.9F), indicating a higher ability to
promote cell proliferation.
100
Figure 3.9 CdM from BM-MSCs and iPSC-MSCs ameliorated CSM-induced
apoptosis/proliferation imbalance in BEAS-2B cells. BEAS-2B cells were treated
with 2% CSM and BM-MSCs-CdM or iPSC-MSCs-CdM for 24hr. (A) Cytochrome c
translocation in BEAS-2B cells. Green: cytochrome c; blue: DAPI. Cells with
cytochrome c translocation to nuclei are marked by red arrows. (B) Apoptotic
BEAS-2B cells as stained by TUNEL. Green: TUNEL; blue: DAPI. (C) Proliferative
BEAS-2B cells as marked by Ki-67. Red: Ki-67; blue: DAPI. (D) Percentage of
BEAS-2B cells with cytochrome c translocation (n=3). (E) Percentage of apoptotic
BEAS-2B cells (n=3). (F) Percentage of proliferative BEAS-2B cells. **p<0.01
compared to control group; ##
p<0.01 compared to CSM group; ††
p<0.01 compared to
BM-MSCs-CdM group.
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3.4.2. Effects of iPSC-MSCs-CdM on apoptosis and proliferation of BEAS-2B
cells were SCF-dependent
Given the superior effects of iPSC-MSCs-CdM over BM-MSCs-CdM regarding
apoptosis and proliferation, the active components responsible for such effects were
further investigated. In particular, the role of SCF was examined in my model.
First of all, the levels of SCF in CdM were measured, showing that
iPSC-MSCs-CdM contains a much higher level of SCF than BM-MSCs-CdM
(518±57 vs. 113±13 pg/mg protein; p<0.01) (Figure 3.10A). The expression of SCF
receptor, C-kit, by BEAS-2B cells was confirmed by both immunohistochemistry
staining and Western blotting (Figure 3.10B) which showed that the level of C-kit was
not changed by CSM treatment (Figure 3.10B).
To investigate the role of SCF, SCF-deprived iPSC-MSCs-CdM was generated by
incubation with immobilized anti-SCF antibody. Effects of iPSC-MSCs-CdM,
SCF-deprived iPSC-MSCs-CdM and recombinant SCF were evaluated on BEAS-2B
cells treated with 2% CSM for 24 hours. All three treatments attenuated CSM-induced
morphological change (Figure 3.11A), cytochrome c translocation (22.0±2.1%,
42.4±2.5% and 41.8±1.9% respectively, p<0.01 in comparison to CSM group,
74.8±2.9%) (Figure 3.11BE), apoptosis (5.6±0.5%, 10.4±0.5% and 10.2±1.0%
respectively, p<0.01 in comparison to CSM group, 20±1.7%) (Figure 3.11CF) and
reduction of proliferation (15.4±0.5%, 11.0±0.5% and 11.0±0.9% respectively, p<0.01
in comparison to the CSM group, 5.8±0.4%) (Figure 3.11DG). Compared to the
normal iPSC-MSCs-CdM treatment, depletion of SCF in the CdM resulted in
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significantly weakened activity, as shown by more cytochrome c translocation
(p<0.01), more apoptosis (p<0.01) and less proliferation (p<0.01). This finding, in
combination with the result that recombinant SCF was able to induce considerable
effects by itself, indicated that SCF contributed to the activity of iPSC-MSCs-CdM.
Similarly, the recombinant SCF were less effective than iPSC-MSCs-CdM regarding
cytochrome c translocation (p<0.01), apoptosis (p<0.01) and proliferation (p<0.01).
Given that the deprived iPSC-MSCs-CdM still retained some capacity to restore all
three parameters, this finding indicated that in addition to SCF, there were other
components promoting similar effects. In summary, the data suggested that the action
of iPSC-MSCs-CdM to alleviate CSM-induced apoptosis/ proliferation imbalance was
partly through SCF.
103
Figure 3.10 Secretion of SCF by BM-MSCs and iPSC-MSCs and expression of
C-kit by BEAS-2B cells. (A) SCF levels in CdM from BM-MSCs or iPSC-MSCs as
measured by ELISA (n=3). (B) Expression of C-kit in BEAS-2B cells. C-kit
expression was identified by immunohistostaining (i) with or (ii) without 2% CSM
treatment for 24 hours, and by (iii) Western blotting. Red: C-kit; blue: DAPI.
**p<0.01 compared to BM-MSCs-CdM.
A
104
Figure 3.11 The anti-apoptotic and pro-proliferation activity of
iPSC-MSCs-CdM was partly through SCF. Immobilized anti-SCF antibody was
used to scavenge SCF from iPSC-MSCs-CdM. BEAS-2B cells were treated with 2%
CSM and iPSC-MSCs-CdM for 24 hours before fixation and TUNEL staining or
immunostaining against cytochrome c and Ki-67 (n=3). (A) The morphological
change of the BEAS-2B cells. DIC: differential interference contrast. (B) Cytochrome
c translocation in BEAS-2B cells. Green: cytochrome c; blue: DAPI. Cells with
cytochrome c translocation to nuclei are marked by red arrows. (C) Apoptotic
BEAS-2B cells as stained by TUNEL. Green: TUNEL; blue: DAPI. (D) Proliferative
BEAS-2B cells as marked by Ki-67. Red: Ki-67; blue: DAPI. (E) The percentage of
BEAS-2B cells with cytochrome c translocation to nuclei (n=3). (F) The apoptotic
rate of BEAS-2B cells (n=3). (G) Percentage of proliferative BEAS-2B cells (n=3).
**p<0.01 compared to control group; ##
p<0.01 compared to CSM group; ††
p<0.01
compared to normal iPSC-MSCs-CdM group.
105
3.5. Discussion
This study elucidated that iPSC-MSCs are more effective than BM-MSCs in
attenuating CS-induced alterations in rat lungs. The higher efficacy may be explained
by a higher activity of mitochondrial transfer, which was evidenced in vivo and
further confirmed by in vitro co-culture study. Meanwhile, the higher efficacy may
also be attributed to more potent paracrine effects, partly through SCF.
MSCs isolated from BM (94, 112), amniotic fluid (151) and adipose tissue (222)
have been reported to attenuate CS-induced emphysema in murine models. These
MSCs are isolated directly from adult tissue, hence relevant patient tissues such as
bone marrow and adipose tissue need to be obtained to collect them. The technique to
differentiate iPSCs into MSCs provides a different source of MSCs, which has several
advantages over classic MSCs. Firstly, large quantities of iPSC-MSCs can be
generated from a small number of somatic cells which can be of a number cell types
such as fibroblasts. In other words, the supply of iPSC-MSCs is unlimited and
personalised. Secondly, iPSC-MSCs have a strong capacity of proliferation and
differentiation, with differentiation potential maintained for over 120 population
doublings (152), while normal MSCs may reach senescence after 20 doublings (69).
Thirdly, impaired survival and differentiation ability may be associated with MSCs
isolated from aged subject or patients with ageing-related disorders (87, 100, 267,
294). However, iPSC-MSCs may hold hope to overcome these issues because when
deriving iPSC-MSCs, somatic cells are firstly reprogrammed to an embryonic stem
cell-like state, which may provide the cells with a rejuvenated phenotype. Therefore
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iPSC-MSCs are valuable candidates for the development of cell therapy against
COPD whose prevalence, morbidity, and mortality are all highly associated with age
(38, 126, 137). Regarding airway disease models, iPSC-MSCs have been reported to
attenuate allergic airway inflammation in mice (244).
The first part of this study evaluated a general effect of iPSC-MSCs on
CS-induced lung alterations in rats. The parameters included emphysema, fibrosis,
inflammation, apoptosis and proliferation. My results show that, iPSC-MSCs
demonstrated a better effect regarding each of the parameters in comparison to those
elicited in response to treatment with BM-MSCs. In fact, as far as iPSC-MSCs can
produce comparable effects to BM-MSCs, due to iPSC-MSCs‟ advantages regarding
the resource and ageing issue, they would at the outset be a better candidate than the
BM-MSCs. The superior efficacy over BM-MSCs further defined iPSC-MSCs as a
promising candidate. Moreover, for the BM-MSCs, a placebo-controlled, randomized
clinical trial in COPD subjects has already been reported (271). The trial observed no
toxicity caused by the treatment. However, there were also no effects regarding both
pulmonary function and indicators of quality-of-life. It did reveal some potential
anti-inflammatory activity though, as the treatment decreased C-reactive protein (CRP)
levels at an early stage. There were some limitations of the study including the
determination of doses, but it still pointed out to the possibility that BM-MSCs might
not work in the clinical setting, despite the clear efficacy demonstrated in animal
models. From our study, iPSC-MSCs are much more effective than BM-MSCs in the
rat model. Therefore the possible failure of BM-MSCs in the clinical context will not
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necessarily indicate a failure of iPSC-MSCs. Instead, it will call for improved MSCs
therapies, which makes study of iPSC-MSCs even more worthwhile and important.
However, on the other hand, the clinical study also revealed the difference between
animal models and diseased subjects, thus the effects of iPSC-MSCs in the clinical
context are not predictable.
Despite numerous reports of MSCs‟ effectiveness in diverse disease models, the
mechanism of MSCs‟ action remains unresolved (270). A major breakthrough is the
report of that in vivo mitochondrial transfer from BM-MSCs into pulmonary alveoli
protect the lung against lipopolysaccharide-induced acute lung injury (118),
illustrating a new mechanism of MSCs‟ action. However, mitochondrial transfer in the
context of CS-induced lung damage was unknown. This study demonstrated that
BM-MSCs and iPSC-MSCs could effectively transfer mitochondria to CS-damaged
lung epithelial cells, similar to sending in a “Trojan horse”. This is one of the very
early demonstrations of such activity following the first report in the acute lung injury
model mentioned above. Although the signaling that drives the MSCs to transfer
mitochondria for rescue of lung epithelial cell was unknown, in vitro formation of
TNTs was observed between MSCs and bronchial epithelial cells. The interruption of
TNT formation by cytochalasin B almost completely blocked mitochondrial transfer,
suggesting that mitochondria might be transferred via TNTs in vitro. Such
TNT-mediated mitochondrial transfer between MSCs and damaged lung epithelial
cells might be an important mechanism through which the exhausted epithelial
cellular bioenergetics can be restored and damaged lung epithelium be rejuvenated.
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Bronchial epithelial cells were chosen for the in vitro study because they act as
the first barrier against CS exposure. The inflammation and oxidative stress in COPD
was initiated by the bronchial epithelial cells‟ response to CS-exposure such as
expression of cell injury–associated endogenous molecules and secretion of
inflammatory mediators including TNF-α and IL-8 (190). Mitochondria are mostly
known as the powerhouse of the cell. In patients with COPD, decreased energy
metabolism is associated with declining lung function (285). Given the observation of
mitochondrial transfer from MSCs to bronchial epithelial cells, the effect of MSCs on
the cellular bioenergetics was studied by measuring the ATP level. The CSM
treatment reduced ATP levels of bronchial epithelial cells, which was alleviated by
co-culture with iPSC-MSCs, indicating improvement in bioenergetics. Such activity
was coupled with CSM-induced enhancement of mitochondrial transfer from
iPSC-MSCs to bronchial epithelial cells, which may be the source of the restored
bioenergetics. Moreover, iPSC-MSCs have demonstrated a more active role in
mitochondrial transfer in both in vivo and in vitro experiments, which may contribute
to the higher efficacy of iPSC-MSCs in attenuating CS-induced lung alterations.
The limitation of mitochondrial transfer is its working radius. Relying on direct
contact between iPSC-MSCs and pulmonary cells, the primary effects of
mitochondrial transfer might be only effective within a limited distance from the
iPSC-MSCs. Although the retention of the iPSC-MSCs were identified even two
weeks after injection based on our previous study (150), it is unlikely that all the cells
in the lungs would have the opportunity to directly adopt mitochondria from them.
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The full activity of the MSCs may be better explained with a complementary
mechanism that overcomes the restriction imposed by the distance between cells,
namely, the paracrine effect. The paracrine effect of MSCs has been documented in
various studies, with different active factors identified, such as vascular endothelial
growth factor, TGF-β1, hepatic growth factor, bFGFr and platelet derived growth
factor (2, 235, 270). In this study the potential of paracrine effects was demonstrated
by treating bronchial epithelial cells with CdM in vitro. The treatment of CSM led to
increased apoptosis and reduced proliferation in bronchial epithelial cells in vitro,
resulting in an imbalance between apoptosis and proliferation. CdM restored the
imbalance by reducing the apoptotic rate and promoting proliferation. The
iPSC-MSCs-CdM was superior than BM-MSC-CdM regarding all the observed
effects. From the in vivo data discussed earlier, iPSC-MSCs have shown superior
effects than BM-MSCs in attenuating apoptosis and promoting proliferation in rat
lung epithelium. The in vivo and in vitro findings together suggested a better
anti-apoptotic and pro-proliferation capacity of iPSC-MSCs through paracrine action.
There are two reasons for studying the effects on apoptosis. Firstly, most studies
on paracrine effects have focused on the immunoregulatory role (e.g. suppression of
inflammation) of MSCs, which is therefore well demonstrated by previous reports (2,
235, 270). In contrast, the modulation of apoptosis by the CdM is less well
investigated. Secondly, there is mounting data suggesting that the gradual alveolar
destruction in COPD pathogenesis may be partly driven by imbalance between
apoptosis and proliferation (67), although classically it is explained by the imbalance
110
of protease and anti-protease activity (68, 76, 77, 189). Apoptosis and proliferation are
well equilibrated in healthy tissue. The induction of apoptosis by CS has been
reported in various cell types of lung, such as endothelial cells, alveolar epithelial
cells, fibroblasts and immune cells (16, 103, 116, 127, 166, 223). Excess apoptosis in
alveolar cells without compensation from proliferation led to the gradual destruction
of alveolar walls in lung (67). Apoptosis of alveolar wall and endothelial cells was
reported to induce emphysema even without infiltration of inflammatory cells in an
animal model (67). Therefore the anti-apoptotic and pro-proliferation potential might
identify a key capacity of iPSC-MSCs as a promising candidate for cell therapy
development against COPD.
Given the effects of CdM on apoptosis and inflammation, the active component
of CdM was the next research question. SCF, a dimeric protein, triggers
homodimerization and autophosphorylation of its receptor c-Kit upon binding, which
further activates diverse pathways involving cell survival, migration, and proliferation
(146). In addition to its ability to induce proliferation, SCF/c-Kit may also affect
apoptosis through down-stream activation of the serine/threonine kinase Akt (35). Akt
subsequently phosphorylates and inhibits pro-apoptotic protein Bad which regulates
the cytochrome c release from the mitochondria through Bcl-2/Bax signalling (35,
265, 283). Despite of a lack of understanding regarding the mechanism, the SCF/c-Kit
signalling has been proved essential to maintain normal alveolar structure in mouse
lung. Deficiency in SCF/c-Kit signalling was reported to cause spontaneous alveolar
destruction, increased ex vivo lung compliance and enlarged residual volume in mice
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(157). Given its important role in proliferation/apoptosis regulation and maintaining
alveolar structure, this study targeted SCF as a potential active component in CdM.
SCF alone was able to attenuate CSM-induced cytochrome c translocation, apoptosis
and reduction in proliferation, indicating its anti-apoptotic and pro-proliferative
activity. Meanwhile, SCF depletion led to a weakened ability of iPSC-MSCs-CdM to
attenuate CSM-induced cytochrome c translocation, apoptosis and reduction in
proliferation, indicating that SCF played an important role in the action of
iPSC-MSCs-CdM. However, the data also showed that SCF alone was less effective
than iPSC-MSCs-CdM, and that SCF depletion only weakened but did not eliminate
the efficacy of iPSC-MSCs-CdM, suggesting that the action of iPSC-MSC-CdM was
only partly dependent on SCF. Moreover, iPSC-MSCs secreted much higher levels of
SCF than BM-MSCs, which may explain its superior anti-apoptotic and
pro-proliferation effects through paracrine action.
From the above discussion, this study has demonstrated two possible mechanisms
through which the iPSC-MSCs protect lung epithelial cells against CS-induced
damage. However, the relative importance of each mechanism was not evaluated in
the study. As mitochondrial transfer was limited by distance, the total therapeutic
effects of the treatment strongly rely on the number of MSCs retained in the lung
tissue. However, the ability of the MSCs to be retained in the lung varies between
different reports. From our previous study in the CS-exposed model, clusters of MSCs
were identified even two weeks after injection. Similarly, adipose stem cells have
been reported to be retained in lung tissue in CS-exposed model after 7 days or 21
112
days (222). However, in another study using BM cells in CS-exposed female rats,
fluorescent in situ hybridization (FISH) only found 2-13 male BM cells per entire
lung from 1 day to 1 month (112). The factors that determine this retention ability
remain unknown, and such factors would also be key to give mitochondrial transfer
higher importance in therapeutic action of MSCs. The paracrine effect, on the other
hand, may be more effective when the retained MSCs are less abundant, as the effects
might be initiated by low level secretion of relevant paracrine factors but amplified by
signalling pathways in the responding cells. The MSCs in the circulation might also
possess systemic effects through release of molecules into the circulation. However,
regarding acute responses, the direct delivery of mitochondria seemed to be a more
straightforward and immediate rescue, which may be why the mitochondrial transfer
has been identified as such an important mechanism in the context of acute lung
injury models where bioenergetics is rapidly impaired.
There are nonetheless several limitations of this study, and more future research
questions can be raised from current findings.
First of all, for the general advantages discussed earlier, although the iPSC-MSCs
may be able to overcome the age-related impairment of normal MSCs, its phenotype
when derived from patients with genetic mutations/inherited diseases remains unclear.
About 1-3% of COPD patients are associated with a genetic alteration which leads to
deficiency of serine protease α-1 antitrypsin (241). There are also other genes
suspected to be associated, such as TGF-β1 (47).
The mitochondrial transfer was proposed as an important mechanism, but the
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TNT may also facilitate the transfer of other organelles, proteins and compounds. For
example, whether the ATP was directly transferred from MSCs to bronchial epithelial
cells was not determined. In addition, other mechanisms for the transfer of material
between cells may also play important roles, such as gap junction and microvesicles.
Lastly, despite the identification of mitochondrial transfer via TNTs in vitro, the route
of mitochondrial transfer in vivo was not demonstrated and requires further
investigation.
Regarding the paracrine effects, the CdM was collected from normal culture of
BM-MSCs or iPSC-MSCs without any interactions with BEAS-2B cells. However, it
is possible that CSM can induce BEAS-2B cells to release cytokines, which may act
on MSCs and modulate secretory functions of MSCs. A trans-well co-culture with
both cells in the same system may overcome this limitation in vitro.
SCF has been identified to contribute to the anti-apoptosis and pro-proliferation
action of iPSC-MSCs-CdM, but the depletion of SCF did not eliminate the effects.
Therefore more active molecules remain to be determined.
In summary, this study demonstrated that iPSC-MSCs attenuate CS-induced lung
damage and observed evidence of mitochondrial transfer from iPSC-MSCs to lung
epithelial cells. The higher activity to transfer mitochondria to CS-damaged bronchial
epithelial cells in vitro and in vivo, in line with the higher anti-apoptosis and
pro-proliferation capacity by paracrine effect partly due to more abundant SCF
secretion, together explained the superior effects of iPSC-MSCs over BM-MSCs. This
114
study defines iPSC-MSCs as a promising candidate for the development of cellular
therapy against COPD.
115
Chapter 4. iPSC-MSCs attenuated
CSM-induced Mitochondrial Dysfunction in
Human Airway Smooth Muscle Cells
116
4.1. Introduction
There is an increasing interest in the role of mitochondria in the pathogenesis of
airway diseases. Mitochondria, most well-known as the powerhouse of the cells, also
play crucial roles in other cellular functions in addition to bioenergetic regulation,
such as calcium metabolism, reactive oxygen species (ROS) modulation and onset of
apoptosis (45, 90, 95, 212). As the site of oxidative phosphorylation, mitochondria are
an important intracellular source of ROS. Defective oxidative phosphorylation may
lead to overwhelming generation of ROS in mitochondria. Increased external and
internal ROS production, without sufficient protection from anti-oxidants, leads to
cellular oxidative stress, which is a key player in the pathogenesis of COPD (57). The
effectiveness of MSCs has been demonstrated in multiple airway disease models
including emphysema and allergic airway inflammation, but the underlining
mechanisms remain unresolved (270). Given that mitochondria are a potential
therapeutic target for airway diseases, investigating the effects of MSCs on oxidative
stress-induced mitochondrial damage in airway cells may be of value. In this study I
hypothesized that interaction with iPSC-MSCs can attenuate cigarette smoke medium
(CSM)-induced mitochondrial damage in ASMCs.
Recently, mitochondrial dysfunction has been reported in airway smooth muscle
cells (ASMCs) isolated from patients with COPD (273), indicating that mitochondria
may be a promising therapeutic target for COPD. Airway smooth muscle plays an
important role in airway remodelling and inflammation (56). In addition to their
contractile properties, ASMCs are also involved in airway inflammation by releasing
117
pro-inflammatory cytokines (10). The first part of this chapter aims to evaluate
changes in mitochondrial function in normal ASMCs in response to oxidative stress.
Cigarette smoke (CS), the primary cause of COPD, is a significant source of oxidants.
Cigarette smoke extract was reported to alter the morphology of mitochondria in vitro
in both airway epithelial cells (105) and ASMCs (10), leading to mitochondrial
fragmentation. Therefore CSM was used in this study to induce mitochondrial damage.
In addition, H2O2 was used as a direct source of oxidants. Mitochondrial ROS levels
and ΔΨm were examined to evaluate changes in mitochondrial function.
Mitochondria play a key role in the regulation of cell fate. The depolarization of
mitochondria is regarded as an early event triggering mitochondrial apoptosis (90).
Therefore, in the second part of this chapter I investigated the effects of co-culture
with iPSC-MSCs on CSM-induced mitochondrial dysfunction and apoptosis in
ASMCs.
Paracrine regulation by MSCs is a widely reported mechanism through which
MSCs can suppress inflammation (2, 235, 270) or attenuate apoptosis, as suggested in
the previous chapter. The third part of this chapter aims to determine whether the
effects of iPSC-MSCs on CSM-induced mitochondrial alterations were through
secretion of paracrine factors. To investigate this, the effects of
iPSC-MSCs-conditioned medium (iPSC-MSCs-CdM) on CSM-induced
mitochondrial alterations were firstly examined. The contents of the
iPSC-MSCs-CdM represented the paracrine factors secreted by iPSC-MSCs under
normal conditions. However, in a real interaction, ASMCs may affect the iPSC-MSCs
118
and modulate the latter‟s synthetic and secretory functions. CSM may also affect the
paracrine effects of iPSC-MSCs as well as the interaction between iPSC-MSCs and
ASMCs. Therefore a study using a trans-well co-culture system was carried out in
which the two cell types are separated by a membrane that is permeable to the
paracrine factors but prevents direct cell-cell contact.
Another recently reported mechanism of the action of MSCs is through
mitochondrial transfer from MSCs to recipient cells, which was firstly described in
vivo in an acute lung injury rodent model (118). In the previous chapter, mitochondrial
transfer from iPSC-MSCs to airway epithelial cells was observed in CS-exposed rat
lungs and CSM-treated bronchial epithelial cells. However, the effect of
mitochondrial transfer on markers of mitochondrial damage was not studied. In the
final part of this chapter, I studied mitochondrial transfer between ASMCs and
iPSC-MSCs using fluorescent microscopy and flow cytometry.
The aims of this chapter were:
1. To evaluate oxidative stress-induced mitochondrial dysfunction in human primary
ASMCs
2. To examine the capacity of iPSC-MSCs to reduce CSM-induced mitochondrial
dysfunction and apoptosis in ASMCs
3. To evaluate the relative contributions of paracrine effects and cell-cell contact in
the protective effects of iPSC-MSC‟s on mitochondrial dysfunction and apoptosis
in ASMCs
4. To investigate mitochondrial transfer from iPSC-MSCs to ASMCs
119
4.2. Oxidative Stress-induced Mitochondrial Dysfunction in
ASMCs and iPSC-MSCs
4.2.1. Oxidative stress reduced the viability of ASMCs
Normal ASMCs isolated from bronchi or tracheas of healthy transplant donor
lungs were treated with H2O2 (50, 100 and 200 μM) or CSM (10, 25, 50 and 75%) for
2, 4 and 24 hours. In the 4/20 hour group cells were stimulated for 4 hours and then
incubated with serum-free medium for 20 hours. Cell viability was measured by MTT
assay. H2O2 led to a concentration-dependent reduction in viability at each time point
(Maximum reduction at 200 μM, fold-change vs. controls: 2 hours, 0.70±0.05,
p<0.001; 4 hours, 0.62 ±0.02, p<0.001; 24 hours, 0.62 ±0.06, p<0.01; 4/24 hours, 0.59
±0.06, p<0.001) (Figure 4.1A). A similar effect was observed at all time points.
CSM also caused loss of viability in a concentration-dependent manner at 2 hours,
4 hours and 24 hours (Maximum effects at 75% CSM, fold-change vs. controls: 2
hours, 0.88±0.04, p<0.01; 4 hours, 0.81±0.07, p<0.01; 24 hours, 0.57±0.13, p<0.05)
(Figure 4.1B). However, no significant reduction in viability was observed in the 4/20
hours group (fold to control, 75% CSM: 0.93±0.15). Moreover, the 75% CSM
treatment led to a significantly lower viability at 24 hours compared with the viability
at 2 hours (p<0.05) or 4 hours (p<0.05), indicating that the effect of 75% CSM was
time-dependent.
120
MTT assay is based on NAD(P)H-dependent reductive reactions catalyzed by
oxidoreductases in cells (30-32). Mitochondria are one of the major carriers of such
enzymes, thus the reduction of MTT readout may be a reflection of mitochondrial
dysfunction.
2hr 4hr 24hr 4/20hr0.0
0.5
1.0
1.5
*
*** ***
* *
*****
Control
50 M
100 M
200 M
[H2O2]
Cell Viability (n=5)
Cel
l via
bili
ty
(Fo
ld c
han
ge
to c
on
tro
l)
A
2hr 4hr 24hr 4/20hr0.0
0.5
1.0
1.5
** ***
*
Control
10%
25%
50%
75%
CSM%
#
#
#
Cell Viability (n=4)
Cel
l via
bili
ty
(Fo
ld c
han
ge
to c
on
tro
l)
B
Figure 4. 1 Effects of H2O2 and CSM on the viability of ASMCs. Viability of
ASMC was measured by MTT assay after treatment with (A) H2O2 (50, 100 and
200 μM) or (B) CSM (10, 25, 50 and 75%) for 2 hours, 4 hours or 24 hours. In the
4/20 hr group ASMCs were stimulated for 4 hours and then incubated with
serum-free medium for 20 hours. *p<0.05, **p<0.01 and ***p<0.001 compare
corresponding groups with relevant control groups; #p<0.05 compares groups as
indicated.
121
4.2.2. Oxidative stress induced mitochondrial ROS in ASMCs
Mitochondrial ROS levels were measured by staining with MitoSOX, a dye that
targets the mitochondria of live cells and emits red fluorescence when oxidized by
superoxide. H2O2 treatment increased the mitochondrial ROS levels in a
concentration-dependent manner at 2 hours (Maximum induction at 200 μM, fold to
control, 1.61±0.16, p<0.05 vs. control) and 4 hours (Maximum induction at 200 μM,
fold to control, 1.75±0.15, p<0.01vs. control). After 24 hours only 200 μM H2O2
increased mitochondrial ROS levels (fold to controls: 24 hours, 1.6±0.25, p<0.05 vs.
control) (Figure 4.2A). Treatment for 24 hours did not show a more potent induction
of mitochondrial ROS than the 2 hour or 4 hour treatments. The induction of
mitochondrial ROS persisted up to 20 hours after the removal of H2O2 (Maximum
induction at 200 μM, fold to control, 1.56±0.22, p<0.01 vs. control).
Mitochondrial ROS was highly induced by CSM treatment in a
concentration-dependent manner (Max induction at 75% CSM, fold to controls: 2
hours, 8.54±1.99, p<0.001; 4 hours, 8.70±1.17, p<0.001; 24 hours, 11.98±2.85,
p<0.001; p-value compared to control of each time point) (Figure 4.2B). The increase
in mitochondrial ROS persisted up to 20 hour after the removal of CSM (Max
induction at 75% CSM, 8.74±2.87, p<0.001).
122
A
B
2hr 4hr 24hr 4/20hr0.0
0.5
1.0
1.5
2.0
2.5
** *** **
[H2O2]
Ctrl
50M
100M
200M
#
Mito-ROS (n=5)
Mito-R
OS
(fold
chan
ge
to c
ontr
ol)
2hr 4hr 24hr 4/20hr0
5
10
15
20
*
*** ***
***
***
** ***
*
*
Control
10%
25%
50%
75%
CSM%
Mito-ROS (n=5)
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
Figure 4. 2 H2O2 and CSM induced mitochondrial ROS in ASMCs.
Mitochondrial ROS in ASMCS was measured by staining with MitoSOX after
treatment with (A) H2O2 (50, 100 and 200 μM) or (B) CSM (10, 25, 50 and 75%)
for 2, 4 or 24 hours. In the 4/20 hr group ASMCs were stimulated for 4 hours and
then incubated with serum-free medium for 20 hours. *p<0.05, **p<0.01 and
***p<0.001 compare corresponding groups with relevant control groups;
#p<0.05 compares groups as indicated.
123
4.2.3. Oxidative stress induced reduction of ΔΨm in ASMCs
Maintenance of the mitochondrial membrane potential (ΔΨm) is very important
for the normal function of mitochondria. Mitochondrial depolarization can lead to an
interruption in the action of the respiratory chain and triggers apoptosis (90). In this
study ΔΨm was measured by staining with JC-1. A decreased ratio of red/green
fluorescence indicates loss of ΔΨm.
The ΔΨm was reduced by H2O2 treatment in a concentration-dependent manner at
each time point (Maximum effects at 200 μM, fold to controls: 2 hours, 0.88±0.02,
p<0.05; 4 hours, 0.89±0.01, p<0.05; 24 hours, 0.70±0.10, p<0.01; p-value compared
to control of each time point) (Figure 4.3A). The reduction of ΔΨm was observed
even 20 hours after the removal of H2O2 (Maximum effects at 200 μM, fold to
controls: 0.64±0.09, p<0.01).
There was a trend showing a decrease in ΔΨm after 2 hours treatment with CSM,
which did not reach statistical significance. Significant reduction in ΔΨm was
observed in a concentration-dependent manner at the 4 hour and 24 hour time points
(Maximum effects at 75% CSM, fold to controls: 4 hours, 0.62±0.04, p<0.001; 24
hours, 0.39±0.07, p<0.001) (Figure 4.3B). Moreover, 75% CSM treatment led to
significantly lower ΔΨm at 24 hours compared to the 2 hours (p<0.001) and 4 hours
(p<0.001) time points. The reduction of ΔΨm was still evident even 20 hours after the
removal of CSM (Maximum effects at 75% CSM, fold to controls: 0.67±0.07,
p<0.01)
124
2hr 4hr 24hr 4hr/20hr0.0
0.5
1.0
1.5
********
***
**
Control
10%
25%
50%
75%
CSM%
## #
#########
Mitochondria membrane potential (n=5)
m
(Fo
ld c
ha
ng
e t
o c
on
tro
l)
2hr 4hr 24hr 4hr/20hr0.0
0.5
1.0
1.5
* ****
**
Ctrl
50 M
100 M
200 M
[H2O2]
Mitochondria membrane potential (n=6)
m
(Fo
ld c
han
ge t
o c
on
tro
l)
B
A
Figure 4. 3 H2O2 and CSM reduced ΔΨm in ASMCs. ΔΨm in ASMCS was
measured by staining with JC-1 after treatment with (A) H2O2 (50, 100 and 200
μM) or (B) CSM (10, 25, 50 and 75%) for 2, 4 or 24 hours. In the 4/20 hr group
ASMCs were stimulated for 4 hours and then incubated with serum-free medium
for 20 hours. *p<0.05, **p<0.01 and ***p<0.001 compare corresponding groups
with relevant control groups; #p<0.05, ##p<0.01, ###p<0.001 compare groups as
indicated.
125
4.2.4. Oxidative stress reduced cell viability of iPSC-MSCs
To examine the effects of oxidative stress on iPSC-MSCs, iPSC-MSCs were
treated with H2O2 (50, 100 and 200 μM) or CSM (10, 25, 50 and 75%) for 4 and 24
hours. The effects were compared with those observed in ASMCs.
H2O2 reduced the viability of iPSC-MSCs in a concentration-dependent manner
after 4 and 24 hours (Max effect at 200 μM, fold to controls: 4 hours, 0.43 ±0.14,
p<0.05; 24 hours, 0.44 ±0.05, p<0.001; p-value compared to control of each time
point), in line with the effects seen in ASMCs (Figure 4.4A).
CSM also reduced the viability of iPSC-MSCs in a concentration-dependent
manner after 4 and 24 hours. At 4 hours, significant reduction was observed with 50%
CSM (fold to controls: 0.60 ±0.12, p<0.05 vs. control) and 75% CSM (fold to controls:
0.49 ±0.09, p<0.01 vs. controls; Figure 4.4B). At 24 hours, significant reduction was
observed with 25% CSM (fold to controls: 0.76 ±0.09, p<0.05 vs. control), 50% CSM
(0.18 ±0.02, p<0.001 vs. control) and 75% CSM (0.16±0.02, p<0.001 vs. control).
The viability of iPSC-MSCs was significantly lower than ASMCs (p<0.05 at each
treatment and time point), indicating that iPSC-MSCs are more sensitive to high
concentrations of CSM than ASMCs with respect to their viability.
126
4hr H2O2 24hr H2O2
4hr CSM 24hr CSM
Figure 4. 4 Effects of H2O2 and CSM on viability of iPSC-MSCs. Changes in the
viability of iPSC-MSCs were measured by MTT assay after treatment with (A) H2O2
or (B) CSM for 4 hours and 24 hours. Changes in the viability of ASMCs were shown
for comparison. *p<0.05, **p<0.01 and ***p<0.001 compare corresponding groups
with relevant control groups; #p<0.05 compares groups as indicated.
0.0
0.5
1.0
1.5
[H2O2]Ctrl 50 M 100 M 200 M
ASMC (n=6) iPSC-MSC (n=3)
*
****
Cell v
iab
ilit
y
(Fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5
[H2O2]Ctrl 50 M 100 M 200 M
ASMC (n=6) iPSC-MSC (n=3)
*****
Cell v
iab
ilit
y
(Fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5
CSM%
*
******
*
# ##
Ctrl 10% 25% 50% 75%
ASMC (n=4) iPSC-MSC (n=3)
Cell v
iab
ilit
y
(Fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5
CSM%
***
* **
# #
Ctrl 10% 25% 50% 75%
ASMC (n=4) iPSC-MSC (n=3)
Cell v
iab
ilit
y
(Fo
ld c
han
ge t
o c
on
tro
l)
A
B
127
4.2.5. Oxidative stress induced mitochondrial ROS in iPSC-MSCs
H2O2 increased the mitochondrial ROS levels in iPSC-MSCs in a
concentration-dependent manner at both 4 and 24 hours (Maximum effect at 200 μM,
fold to controls: 4 hours, 4.12±1.73, p<0.05; 24 hours, 4.46±0.81, p<0.05; p-value
compared to control of each time point) (Figure 4.5A). The increase in mitochondrial
ROS levels induced by CSM (200 μM) after 24 hours was greater in iPSC-MSCs
compared to ASMCs (p<0.01.)
CSM also induced mitochondrial ROS in iPSC-MSCs, in a
concentration-dependent manner after 4 and 24 hours (Maximum effect at 75% CSM,
fold to controls: 4 hours, 6.36±1.94, p<0.05; 24 hours, 10.33±3.29, p<0.001; p-value
compared to control of each time point) (Figure 4.5B). No significant difference was
observed between the response of iPSC-MSCs and ASMCs.
128
Figure 4. 5 Effects of H2O2 and CSM on mitochondrial ROS of iPSC-MSCs.
Mitochondrial ROS levels of iPSC-MSCs were measured by MitoSOX staining after
treatment with (A) H2O2 or (B) CSM for 4 and 24 hours. Changes in the mitochondrial
ROS levels of ASMCs were shown for comparison. *p<0.05, **p<0.01 and
***p<0.001 compare corresponding groups with relevant control groups; ##p<0.01
compares groups as indicated.
0
2
4
6
8
[H2O2]Ctrl 50 M 100 M 200 M
ASMC (n=5) iPSC-MSC (n=4)
** **
*
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
0
2
4
6
[H2O2]Ctrl 50 M 100 M 200 M
ASMC (n=5) iPSC-MSC (n=4)
*
*
##
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
0
5
10
15
ASMC (n=5) iPSC-MSC (n=4)
CSM%
*
*
***
***
*
Ctrl 10% 25% 50% 75%
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
0
5
10
15
20
ASMC (n=5) iPSC-MSC (n=4)
CSM%
****
*
Ctrl 10% 25% 50% 75%
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
A
B
4hr H2O2 24hr H2O2
4hr CSM 24hr CSM
129
4.2.6. Oxidative stress induced reduction of ΔΨm in iPSC-MSCs
H2O2 reduced the ΔΨm in iPSC-MSCs in a concentration-dependent manner at
both 4 hours and 24 hours (Maximum effect at 200 μM, fold to controls: 4 hours,
0.80±0.09, p<0.05; 24 hours, 0.41±0.09, p<0.001; p-value compared to control of
each time point) (Figure 4.6A). The reduction in ΔΨm by H2O2 (200 μM) was
stronger in iPSC-MSCs compared to ASMCs but it did not reach statistical
significance.
CSM also reduced the ΔΨm in iPSC-MSCs in a concentration-dependent manner
at both 4 and 24 hours (Maximum effect at 75% CSM, fold to controls: 4 hours,
0.23±0.06, p<0.001; 24 hours, 0.21±0.01, p<0.001; p-value compared to control of
each time point) (Figure 4.6B). The reduction in ΔΨm was significantly stronger in
iPSC-MSCs compared with ASMCs after treatment with 50% and 75% CSM for 4
hours or with any concentration of CSM for 24 hours.
130
Figure 4. 6 Effects of H2O2 and CSM on ΔΨm of iPSC-MSCs. The ΔΨm of
iPSC-MSCs was measured by JC-1 assay after treatment with H2O2 (A) or CSM (B)
for 4 hours and 24 hours. Changes in the ΔΨm of ASMCs were shown for
comparison. *p<0.05, **p<0.01 and ***p<0.001 compare corresponding groups
with relevant control groups; #p<0.05, ##p<0.01 compare groups as indicated.
A 4hr H2O2 24hr H2O2
0.0
0.5
1.0
1.5
[H2O2]Ctrl 50 M 100 M 200 M
ASMC (n=6) iPSC-MSC (n=5)
*
***
**
m
(Fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5
ASMC (n=5) iPSC-MSC (n=5)
CSM%
*******
**
******
# ##
Ctrl 10% 25% 50% 75%
m
(Fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5
ASMC (n=5) iPSC-MSC (n=5)
CSM%
***
*****
***
***
***
# ## # #
Ctrl 10% 25% 50% 75%
m
(Fo
ld c
han
ge t
o c
on
tro
l)4hr CSM 24hr CSM
B
0.0
0.5
1.0
1.5
[H2O2]Ctrl 50 M 100 M 200 M
ASMC (n=6) iPSC-MSC (n=5)
** *
m
(Fo
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ge
to
co
ntr
ol)
131
4.3. Direct Co-culture with iPSC-MSCs Attenuated
CSM-induced Mitochondrial Dysfunction and Apoptosis in
ASMCs
In order to determine whether iPSC-MSCs are capable of preventing
CSM-induced mitochondrial dysfunction in ASMCs, in a prophylactic protocol
iPSC-MSCs were directly co-cultured with CellTrace-labeled ASMCs for 20 hours
and then treated with CSM (10 and 25%) for 4 hours. As a control, single cultures of
ASMCs were stained with Cell Trace, incubated for 20 hrs and then treated with CSM
for 4 hours (Figure 2.5 A). In a therapeutic protocol, CellTrace-labeled ASMCs were
treated with CSM (10 and 25%) for 4 hours before iPSC-MSCs were added to the
culture and incubated for a further 20 hours. As a control, single cultures of ASMCs
were stained with Cell Trace, treated with CSM for 4 hours and then incubated in the
absence of stimulation for 20 hrs (Figure 2.5B).
4.3.1. iPSC-MSCs prevented and reversed CSM-induced mitochondrial ROS in
ASMCs
The two cell types in the co-cultures were distinguished by the CellTrace staining
using flow cytometry (Figure 4.7A). The mitochondrial ROS levels were measured in
the gated ASMC (CellTrace-positive) population and compared with the ASMCs in
the single-culture (Figure 4.7B). 10% CSM significantly increased the levels of
mitochondrial ROS in ASMCs in the single culture (fold to control: 3.26±0.33,
p<0.01 in comparison to control), which was prevented by co-culture with
132
iPSC-MSCs (fold to control: 2.12±0.17, p<0.05 in comparison to single-culture). 25%
CSM further increased the level of mitochondrial ROS in single-culture compared to
10% CSM (fold to control: 6.71±0.75, p<0.001), which was also reduced by
iPSC-MSCs (fold to control: 4.01±0.44, p<0.05 in comparison to single-culture).
However, a concentration-dependent increase of mitochondrial ROS levels of ASMCs
in the co-culture was still observed, indicating the effects of CSM were only
alleviated but not eliminated.
In addition, a therapeutic protocol was also performed, in which the CSM was
removed from the ASMCs and replaced by either serum free medium or iPSC-MSCs
(Figure 4.7C). Under these conditions only 25% CSM led to a significant increase in
mitochondrial ROS (fold to control: 3.42±0.46, p<0.01 in comparison to control). A
small but significant reduction in mitochondrial ROS was observed in the co-culture
group compared to the single culture group treated with 25% CSM (fold to control:
2.70±0.48, p<0.05), indicating therapeutic recovery from CSM-induced mitochondrial
ROS by iPSC-MSCs. Again the concentration-dependent elevation of mitochondrial
ROS was still evident among the co-culture groups, indicating the effects of CSM
were not eliminated.
133
0
2
4
6
8
*
*
Control 10% CSM 25% CSM
*****
***
****
***
ASMC only
ASMC+iPSC-MSC
MitoROS of ASMCs (n=7)
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
B
A
C
Figure 4. 7 Effects of direct co-culture with iPSC-MSCs on mitochondrial
ROS in ASMCs. (A) Flow cytometry histograms showing CellTrace positive
and negative populations corresponding to ASMCs and iPSC-MSCs,
respectively. (B) Prophylactic effects of iPSC-MSCs on CSM-induced
mitochondrial ROS in ASMCs. iPSC-MSCs were co-cultured with
CellTrace-labeled ASMCs for 20 hours and then treated with CSM (10 and
25%) for 4 hours. Single ASMCs cultures were used as controls. Mitochondrial
ROS levels were determined using MitoSOX staining. (C) Therapeutic effects
of iPSC-MSCs on CSM-induced mitochondrial ROS in ASMCs.
CellTrace-labeled ASMCs were treated with CSM (10 and 25%) for 4 hours.
CSM was then removed and iPSC-MSCs were loaded. Single ASMCs cultures
were used as controls. *p<0.05, **p<0.01, ***p<0.001 compare groups as
indicated.
0
1
2
3
4
Control 10% CSM 25% CSM
**** *
***
ASMC only
ASMC+iPSC-MSC
MitoROS of ASMCs (n=6)
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
134
4.3.2. iPSC-MSCs prevented but did not reverse CSM-induced reduction in ΔΨm
in ASMCs
In the prophylactic protocol, CSM induced reduction of ΔΨm at both 10% (fold
to control: 0.80±0.04, p<0.01) and 25% concentration (fold to control: 0.52±0.07,
p<0.001). 25% CSM led to lower ΔΨm than 10% CSM (p<0.001). Co-culture with
iPSC-MSCs improved the ΔΨm under both 10% CSM treatment (fold to control:
0.95±0.04, p<0.01) and 25% CSM treatment (fold to control: 0.78±0.10, p=0.059)
(Figure 4.8A). CSM still led to a trend showing a reduction in ΔΨm in ASMCs in the
co-culture, but the difference was not statistically-significant.
In the therapeutic protocol, after recovery for 20 hours, single-cultures of ASMCs
still showed significantly reduced ΔΨm in both 10% CSM-treated group (fold to
control: 0.84±0.07, p<0.01) and 25% CSM-treated group (fold to control: 0.75±0.59,
p<0.001) (Figure 4.8B). There is a trend showing an increase in mitochondrial
potential in the co-culture groups compared to the single culture at both 10% (fold to
control: 0.94±0.04) and 25% CSM treatments (fold to control: 0.87±0.11), which did
not reach statistical significance.
135
0.0
0.5
1.0
1.5
Control 10% CSM 25% CSM
******** p=0.059
**
ASMC only
ASMC+iPSC-MSC
Mitochondrial Membrane Potential (n=5)
m
(Fo
ld c
han
ge t
o c
on
tro
l)
A
0.0
0.5
1.0
1.5
Control 10% CSM 25% CSM
*
* ASMC only
ASMC+iPSC-MSC
Mitochondrial Membrane Potential (n=3)
m
(Fo
ld c
han
ge t
o c
on
tro
l)
B
Figure 4. 8 Effects of direct co-culture with iPSC-MSCs on ΔΨm in ASMCs.
(A) Prophylactic effects of iPSC-MSCs on CSM-induced reduction in ΔΨm in
ASMCs. iPSC-MSCs were co-cultured with CellTrace-labeled ASMCs for 20
hours and then treated with CSM (10 and 25%) for 4 hours. Single ASMCs
cultures were used as controls. ΔΨm was determined using JC-1 staining. (B)
Therapeutic effects of iPSC-MSCs on CSM-induced reduction in ΔΨm in ASMCs.
CellTrace-labeled ASMCs were treated with CSM (10 and 25%) for 4 hours. CSM
was then removed and iPSC-MSCs were loaded. Single ASMCs cultures were used
as controls. *p<0.05, **p<0.01, ***p<0.001 compares groups as indicated.
136
4.3.3. iPSC-MSCs prevented CSM-induced apoptosis of ASMCs
Based on the observations of a protective role of iPSC-MSCs against
CSM-induced mitochondrial dysfunction in ASMCs, further investigations were
carried out on whether such mitochondrial improvement led to improved function of
the cells. In particular apoptosis, as a possible outcome of severe mitochondrial
damage, was investigated in the co-culture using the prophylactic protocol (Figure
4.9). Results from Annexin V staining confirmed induction of apoptosis by CSM in
single-cultured ASMCs at 25% CSM (46.6±1.3% vs. 17.4±2.9%, p<0.001) and 50%
CSM (86.8±3.8% vs. 17.4±2.9%, p<0.001). In the absence of stimulation, the
iPSC-MSCs-treated ASMCs demonstrated higher percentage of apoptosis than those
in the single-culture (30.7±1.4% vs. 17.4±2.9%, p<0.05), which indicates that
introducing iPSC-MSCs to the system may induce stress to the ASMCs at baseline.
However, iPSC-MSCs led to a significant reduction in apoptosis induced by 25%
(34.7±1.5% vs. 46.6±1.3%, p<0.05) and 50% CSM (49.5±4.2% vs. 86.8±3.8%,
p<0.001), indicating that interaction with iPSC-MSCs can protect ASMCs from
CSM-induced mitochondrial dysfunction and apoptosis.
137
Figure 4. 9 Prophylactic effects of iPSC-MSCs on CSM-induced apoptosis
in ASMCs. iPSC-MSCs were co-cultured with CellTrace-labeled ASMCs for
20 hours and then treated with CSM (10 and 25%) for 4 hours. Single ASMCs
cultures were used as controls. Apoptosis was detected by Annexin V staining.
*p<0.05, **p<0.01, ***p<0.001 compare groups as indicated.
0
20
40
60
80
100 ASMC only
ASMC+iPSC-MSC
Control 10%CSM 25%CSM 50%CSM
***
*
******
*****
***
***
*
Apoptotic rate in ASMCs (n=5)
An
nexin
V p
osit
ive c
ells (
%)
138
4.4. Paracrine Effects are only Partly Involved in the
Protective Effects of iPSC-MSCs on CSM-induced
Mitochondrial Dysfunction and Apoptosis in ASMCs
4.4.1. Effects of conditioned medium from iPSC-MSCs on CSM-induced
mitochondrial dysfunction in ASMCs
In order to examine the role of paracrine effects in the protective effects of
iPSC-MSCs on CSM-induced mitochondrial damage in ASMCs, the effects of
conditioned medium from iPSC-MSCs (iPSC-MSCs-CdM) on CSM-induced
mitochondrial damage in ASMCs were investigated. CdM was a concentrate of all
paracrine factors released over a 24 hour period from confluent cultures of
iPSC-MSCs. The ASMCs were pre-treated with iPSC-MSCs-CdM for 4 hours
followed by CSM treatment for a further 4 hours in the presence of CdM. The
evaluation of mitochondrial ROS revealed very similar results with the direct
co-culture system (Figure 4.10A). CSM treatments increased mitochondrial ROS in a
concentration-dependent manner in both untreated ASMCs (fold to controls: 10%
CSM, 2.96±0.34, p<0.05; 25% CSM, 7.76±0.06, p<0.001; p-value compared to
relevant controls) and ASMCs treated with iPSC-MSCs-CdM (fold to controls: 10%
CSM, 2.56±0.29, p<0.01; 25% CSM, 5.81±0.49, p<0.001; p-value compared to
relevant controls). However, compared to untreated ASMCs, ASMCs treated with
iPSC-MSCs-CdM demonstrated significantly lower levels of mitochondrial ROS in
both 10% CSM group (p<0.05) and 25% CSM group (p<0.05).
The reduction of mitochondrial ROS by iPSC-MSCs-CdM was not associated
139
with prevention of ΔΨm reduction. CSM treatments reduced ΔΨm in the untreated
ASMCs (fold to controls: 10% CSM, 0.84±0.03, p<0.01; 25% CSM, 0.59±0.05,
p<0.001; p-value compared to relevant controls) as well as in the ASMCs treated with
iPSC-MSCs-CdM (fold to controls: 10% CSM, 0.87±0.02, p<0.05; 25% CSM,
0.59±0.05, p<0.001; p-value compared to relevant controls; Figure 4.10B). The effect
of CSM on ΔΨm was similar in untreated and iPSC-MSCs-CdM-treated ASMCs.
These findings suggest that paracrine factors are only partly involved in the capacity
of iPSC-MSCs to protect ASMCs from CSM-induced mitochondrial damage
4.4.2. Effect of iPSC-MSCs-CdM on CSM-induce apoptosis of ASMCs
The effect of iPSC-MSCs-CdM on CSM-induced ASMC apoptosis was also
determined. CSM increased the percentage of apoptotic ASMCs in a
concentration-dependent manner (Maximum induction by 50% CSM: 64.8±9.2% vs.
12.6±1.4%, p<0.001; p-value compared to control). Within the iPSC-MSCs-CdM
treated groups, a similar induction was observed (Maximum induction by 50% CSM:
67.8±9.0% vs. 13.8±1.3%, p<0.001; p-value compared to control) (Figure 4.11).
Treatment with iPSC-MSCs-CdM did not affect the baseline or CSM-induced
apoptosis.
140
0
2
4
6
8
10Without CdM
With CdM
******
*****
***
Control 10% CSM 25% CSM
*
*
*
Mitochondrial-ROS (n=6)
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5Without CdM
With CdM
Control 10% CSM 25% CSM
******
****
***
**
Mitochondrial Membrane Potential (n=4)
m
(Fo
ld c
han
ge t
o c
on
tro
l)
B
A
Figure 4. 10 Effects of iPSC-MSCs-CdM on mitochondrial ROS and
ΔΨm in ASMCs. ASMCs were pre-treated with iPSC-MSCs-CdM for 4
hours before stimulating with CSM for 4 hours. (A) Mitochondrial ROS
was determined by MitoSOX staining and (B) ΔΨm by JC-1 staining.
*p<0.05, **p<0.01, ***p<0.001 compare groups as indicated.
141
0
20
40
60
80
100Without CdM
With CdM
Control 10%CSM 25%CSM 50%CSM
******
*****
**
******
*****
**
Apoptotic rate of ASMCs (n=4)
An
nexin
V p
osit
ive c
ells (
%)
Figure 4. 11 Effects of iPSC-MSCs-CdM on CSM-induced apoptosis in
ASMCs. ASMCs were pre-treated with iPSC-MSCs-CdM for 4 hours before
stimulating with CSM for 4 hours. Apoptosis was determined by Annexin V
staining. **p<0.01, ***p<0.001 compare groups as indicated.
142
4.4.3. Effects of trans-well co-culture with iPSC-MSCs on CSM-induced
mitochondrial alterations in ASMCs
To better replicate the crosstalk between the ASMCs and iPSC-MSCs through
paracrine effects, trans-well co-culture was performed using cell culture inserts with
0.4 μm pores. ASMCs in 6-well plates were incubated with either blank inserts or
inserts containing iPSC-MSCs for 20 hours before being treated with CSM (10 or
25%) for 4 hours (Figure 4.12A). Mitochondrial ROS levels were increased by 10%
CSM (fold to control: 3.55±0.59, p<0.01) and 25% CSM (fold to control: 8.35±0.83,
p<0.001) in the ASMCs treated with blank inserts. In the trans-well co-culture group,
mitochondrial ROS levels in ASMCs were also elevated by 10% CSM (fold to control:
3.12±0.57, p<0.05) and 25% CSM (fold to control: 7.15±0.84, p<0.001), but the
mitochondrial ROS induced by 25% CSM was significant reduced compared to the
blank insert group (p<0.05) (Figure 4.12B).
Significant reduction of ΔΨm was induced by 25% CSM at both blank insert
group (fold to control: 0.39±0.06, p<0.01) and iPSC-MSCs-treated group (fold to
control: 0.49±0.12, p<0.01). No significant difference was observed between the
iPSC-MSCs treatment and blank insert (Figure 4.12C). These results are in line with
the findings from the iPSC-MSCs-CdM experiment.
143
A
Figure 4. 12 Effects of co-culture with iPSC-MSCs using the trans-well
system on mitochondrial ROS and ΔΨm in ASMCs. (A) Illustration of the
trans-well setup. Inserts with or without iPSC-MSCs were placed into the wells of
6-well tissue culture plates, containing ASMCs, for 20 hours. The whole system
was treated with CSM for 4 hours. In the ASMCs mitochondrial ROS was
determined by (B) MitoSOX staining and (C) ΔΨm by JC-1 staining. *p<0.05,
**p<0.01, ***p<0.001 compare groups as indicated.
0
2
4
6
8
10
Ctrl 10%CSM 25%CSM
****
*****
*******
Blank Insert
iPSC-MSCs
Mitochondrial-ROS (n=4)
Mit
o-R
OS
(fo
ld c
han
ge t
o c
on
tro
l)
0.0
0.5
1.0
1.5
Ctrl 10%CSM 25%CSM
Blank Insert
iPSC-MSCs
****
Mitochondrial Membrane Potential (n=4)
m
(Fo
ld c
han
ge t
o c
on
tro
l)
C
B
144
4.4.4. Effect of trans-well co-culture with iPSC-MSCs on CSM-induced apoptosis
in ASMCs
The effect of co-culture using a trans-well system on ASMC apoptosis was also
examined. 25% CSM induced a significant increase in ASMC apoptosis in both the
blank insert group (48.6±10.9% vs. 11.7±2.6%, p<0.05) and the iPSC-MSC-treated
group (54.6±8.3% vs. 16.7±3.3%, p<0.01) (Figure 4.13). No difference was observed
in the iPSC-MSCs-treated group in comparison with the blank group.
0
20
40
60
80
Ctrl 10%CSM 25%CSM
iPSC-MSCs
Blank Insert
***
Apoptotic rate of ASMCs (n=4)
An
nexin
V p
osit
ive c
ells (
%)
Figure 4. 13 Effects of trans-well co-culture with iPSC-MSCs on apoptosis in
ASMCs. Inserts with or without iPSC-MSCs were placed into the wells of 6-well
tissue culture plates, containing ASMCs, for 20 hours. The whole system was treated
with CSM for 4 hours. In the ASMCs apoptosis was determined by Annexin V
staining.*p<0.05, **p<0.01 compares corresponding groups with relevant controls.
145
4.5. Mitochondrial Transfer from iPSC-MSCs to ASMCs
4.5.1. Mitochondrial transfer from iPSC-MSCs to ASMCs
A possible mechanism underlining the protective effects of iPSC-MSCs against
CSM-induced mitochondrial dysfunction in ASMCs is mitochondrial transfer. To
investigate whether mitochondrial transfer from iPSC-MSCs to ASMCs takes place,
ASMCs were pre-stained with CellTrace Violet while iPSC-MSCs were pre-stained
with MitoTracker Red, a mitochondrial-targeted fluorescent dye. They were
co-cultured for 20 hours followed by 4 hours stimulation with 25% CSM. The cells
were fixed and stained with phalloidin which selectively labels F-actin. Using
fluorescent microscopy, iPSC-MSCs-derived MitoTracker-labeled mitochondria were
observed in CellTrace-labeled ASMCs, indicating transfer of mitochondria from
iPSC-MSCs to ASMCs (Figure 4.14). In addition, tunneling nanotube (TNT)-like
structures, containing iPSC-MSC mitochondria, were observed connecting
iPSC-MSCs and ASMCs. Actin filaments were identified in the TNTs by phalloidin
staining confirming a connection between the cytoskeleton systems of iPSC-MSCs
and ASMCs, which may provide a path through which the mitochondria are
transferred.
4.5.2. CSM increased mitochondrial transfer from iPSC-MSCs to ASMCs
Mitochondrial transfer was quantified by determining the percentage of
MitoTracker-positive cells in the ASMC population using flow cytometry.
Mitochondrial transfer was detected after co-culture for 24 hours without CSM
146
stimulation or after stimulation with 10% or 25% CSM during the last 4 hours of the
co-culture period. 10% CSM enhanced mitochondrial transfer, as shown by a
significant elevation of the transfer rate (48.9±6.8% vs. 18.8±4.1%, p<0.05). 25%
CSM further increased the transfer rate compared to 10% CSM (75.5±8.5% vs.
48.9±6.8%, p<0.05), suggesting that CSM promoted mitochondrial transfer from
iPSC-MSCs to ASMCs in a concentration-dependent manner (Figure 4.15).
147
Figure 4. 14 Mitochondrial transfer from iPSC-MSCs to ASMCs. ASMCs were
pre-stained with CellTrace and iPSC-MSCs with MitoTracker. Cells were co-cultured
for 20 hours and then treated with 25% CSM for 4 hours. Phalloidin staining was
applied after fixation. The presence of MitoTracker-labeled mitochondria was
observed in CellTrace-labeled cells, indicating transfer of iPSC-MSC-derived
mitochondria to ASMCs. Tunneling nanotube–like structures between iPSC-MSCs
and ASMCs can be observed. Scale bar=25 μm.
148
Ctrl 10% CSM 25% CSM0
20
40
60
80
100 ****
*
Percentage of ASMCs that recievedMSCs-derived mitochondria (n=3)
Mit
oT
racker+
AS
MC
s (
%)
Figure 4. 15 Effect of CSM on the rate of mitochondrial transfer from
iPSC-MSCs to ASMCs. ASMCs were pre-stained with CellTrace and
iPSC-MSCs with MitoTracker. Cells were co-cultured for 20 hours and then
treated with 25% CSM for 4 hours. (A) Mitochondrial transfer from
iPSC-MSCs (MitoTracker-positive) to ASMCs (CellTrace-positive) was
determined by flow cytometry. The ASMC population is indicated by the gate
in the plots of co-culture. (B) The rate of mitochondrial transfer was evaluated
by the percentage of MitoTracker-positive ASMCs in total ASMC population.
*p<0.05, ***p<0.001 compare groups as indicated.
A
B
149
4.6. Discussion
This study demonstrated increased mitochondrial ROS and reduced ΔΨm in
healthy ASMCs, in response to H2O2 or CSM. Direct contact with iPSC-MSCs
protected the ASMCs from CSM-induced mitochondrial ROS and reduction of ΔΨm,
which may explain the attenuation of CSM-induced apoptosis. When the interaction
between the two cell types was restricted to paracrine effects, only CSM-induced
mitochondrial ROS but not CSM-induced ΔΨm loss and apoptosis in ASMCs were
ameliorated, indicating that the paracrine effects can only partly explain the protective
effects of iPSC-MSCs. Under direct interaction, mitochondria were transferred from
iPSC-MSCs to ASMCs, which was enhanced by CSM treatment. Formation of TNTs
between iPSC-MSCs and ASMCs was observed in the co-culture. The prophylactic
effects of iPSC-MSCs on CSM-induced mitochondrial damage in ASMCs were lost
when the co-culture was carried out using a trans-well system. As the trans-well
inserts would block nanotube formation, whilst allowing only paracrine
communication, this suggests an important role of mitochondrial transfer in the
protective effects of iPSC-MSCs. These findings highlight that the full protective
capacity of iPSC-MSCs may rely on direct contact with target cells through which
mitochondrial transfer can take place. This study suggested that iPSC-MSCs may be
promising candidates for cell-based treatments targeting mitochondrial damage in
patients with COPD.
Targeting mitochondrial damage provides a new promising strategy for
developing treatments for COPD. Mitochondrial dysfunction was recently
150
demonstrated in ASMCs from patients with COPD, as characterized by reduced ATP
levels, mitochondrial complex protein expression and ΔΨm, as well as increased
mitochondrial ROS (273). The study also demonstrated mitochondrial damage, as
reflected by the same parameters, in an ozone-induced mouse model of airway
inflammation and hyper-responsiveness. Mitochondrial damage as well as airway
inflammation and hyper-responsiveness were attenuated by the mitochondrial-
targeted antioxidant MitoQ (273). In an earlier report, swelling and fragmented
mitochondria with depleted cristae were observed in airway epithelial cells from
patients with stage 4 COPD (105). In the present study, oxidative stress-induced
mitochondrial damage was identified by increased mitochondrial ROS and loss of
ΔΨm in ASMCs, which provided a model in which to study the effects of
iPSC-MSCs.
CS, the major cause of COPD, contains numerous oxidants. One puff of cigarette
smoke contains approximately 1015
oxidants in the gas phase and 1018
oxidants in the
tar phase, together with 3000 ppm of nitrite oxide (57, 58). Acute exposure to CS is
reported to impair energy metabolism and mitochondrial protein expression in mouse
lungs, leading to switch of glucose metabolism to pentose phosphate pathway and
reduction of substrate supply to mitochondrial respiration (4). Cigarette smoke
medium was reported to alter the morphology of mitochondria in vitro, in both airway
epithelial cells (105) and ASMCs (10), inducing mitochondrial fragmentation in both
cell types and mitochondrial ROS elevation in ASMCs. However, the effect on ΔΨm
was not examined in these studies. In the present study CSM was demonstrated to
151
induce mitochondrial ROS, ΔΨm loss and apoptosis in normal ASMCs from healthy
subjects. Although inhalation of CS primarily affects the epithelium, CS-derived
chemicals could also affect airway smooth muscle directly via the circulation (205) or
indirectly through inducing the production of epithelial cell-derived inflammatory
mediators such as IL-1β, IFN-γ and TNF-α which affect ASMC function (70, 231).
Moreover, chronic exposure to CS can lead to a defective epithelial barrier function
(102, 226), which may result in a direct exposure of CS to ASMCs. Hence the in vitro
model of CSM-induced ASMC dysfunction is physiologically relevant. On the other
hand, it is to my interest to establish a model of oxidative stress-induced
mitochondrial damage, through which mitochondrial-targeted therapeutic approaches
can be tested. The concentrations of CSM chosen for the co-culture experiments were
based on the findings of concentration response experiments performed on single
cultures of ASMCs, as it would be impossible to determine physiologically-relevant
concentrations of cigarette smoke constituents. CSM-induced apoptosis was observed
in association with mitochondrial dysfunction. Induction of apoptosis by CS has also
been demonstrated in other cell types in the lung including endothelial cells, alveolar
epithelial cells, fibroblasts and immune cells (16, 103, 116, 127, 166, 223). The
anti-apoptotic role of iPSC-MSCs demonstrated in the ASMC model may also be
important in other cell types in the lung.
MSCs have been reported to attenuate lung emphysema and allergic airway
inflammation in murine models (94, 150, 151, 244). Although MSCs are considered
as putative cell-based therapies for COPD, their potential mechanisms of their action
152
remain unclear. It is possible that their effects are at least partly mediated by
improving mitochondrial function in COPD lungs. Indeed, MSCs have been shown to
rescue defective mitochondria and impaired bioenergetics of mitochondria in an
animal model of lipopolysaccharide-induced acute lung injury (118), as well as in an
in vitro model of mitochondrial DNA mutation (238). In my study, the effects of
iPSC-MSCs on CSM-induced mitochondrial damage in ASMCs were examined.
Using a prophylactic protocol, I have shown that interaction with iPSC-MSCs
prevents CSM-induced mitochondrial ROS and reduction in ΔΨm in ASMCs.
CSM-induced apoptosis in ASMCs was also attenuated by iPSC-MSCs, which may
be a result of improved mitochondrial function. Using the therapeutic protocol, I
observed significant attenuation of CSM-induced mitochondrial ROS but not reversal
of CSM-induced ΔΨm reduction. The therapeutic effects of iPSC-MSCs appear to be
less pronounced, possibly due to the partial recovery of mitochondrial function in
absence of CSM stimulation, or the progression of mitochondrial damage to an
irreversible stage. Therefore the prophylactic protocol was chosen for later
experiments. These findings support the hypothesis that iPSC-MSCs can attenuate
CSM-induced mitochondrial damage, suggesting their potential capacity to repair
mitochondrial dysfunction in patients with COPD.
MSCs have been reported to induce paracrine effects by releasing
immunoregulatory cytokines such as VEGF, TGF-β1, HGF, bFGF and platelet derived
growth factor (2, 235, 270). In the previous chapter iPSC-MSCs-CdM was found to
attenuate CSM-induced apoptosis in bronchial epithelial cells in vitro. However, in
153
the present study, iPSC-MSCs-CdM was only able to ameliorate CSM-induced
mitochondrial ROS in ASMCs without any effectiveness on ΔΨm or apoptosis. As
iPSC-MSCs were not in close proximity to ASMCs or exposed to CSM treatment
before the collection of CdM, the lack of effect may indicate that the protective effects
of iPSC-MSCs on ASMCs may be induced by the CSM or mediators produced by the
ASMCs themselves. However, in a trans-well co-culture system where there is
paracrine crosstalk between the two cell types, just as in the direct co-culture system,
the iPSC-MSCs prevented CSM-induced mitochondrial ROS production but not the
reduction of ΔΨm or apoptosis in ASMCs.
Therefore, the protective role of iPSC-MSCs against CSM-induced mitochondrial
depolarization and apoptosis in ASMCs seems to be dependent on the direct contact
between the two cell types, which may also involve mitochondrial transfer.
Mitochondrial transfer was firstly identified in co-cultures of BM-MSCs and lung
epithelial cells with defective mitochondria (238). Since then, mitochondrial transfer
from BM-MSCs to epithelial cells, endothelial cells, cardiomyocytes and
osteosarcoma cells has been demonstrated (55, 192, 204, 238). Mitochondrial transfer
from MSCs to epithelial cells was also observed in vivo in rodent models of acute
lung injury (118) and CS-induced emphysema (150). In this study mitochondrial
transfer from iPSC-MSCs to ASMCs was demonstrated. Moreover, CSM significantly
enhanced the transfer in a concentration-dependent manner. A previous study reported
that CS exposure can lead to a reduction in the supply of substrates to the electron
transport chain, leading to increased mitochondrial workload to maintain normal
154
energy supply (4). The transfer of healthy mitochondria may alleviate such burden
and thus relieve the mitochondrial stress as indicated by the reversal of mitochondrial
depolarization. In this study the formation of TNTs connecting the cytoskeleton
systems of the two cells were evident by staining the actin filaments. TNTs are highly
sensitive nanotubular structures between cells which can facilitate the selective
transfer of membrane vesicles and organelles (215). TNTs have been reported to
mediate mitochondrial transfer from BM-MSCs to epithelial cells, from endothelial
cells to cancer cells and from vascular smooth muscle cells to MSCs (196, 238, 258).
ASMCs are also reported to form TNTs with CD4+ T cells in vitro, leading to
enhanced T cell survival (7). The formation of TNTs between iPSC-MSCs and
ASMCs may provide a path through which mitochondria can be transferred, but the
mechanisms underlining the regulation of TNT formation by the two cell types or by
CSM remain unknown. The mechanism by which cells regulate mitochondrial
transfer through the TNTs is also unclear. A recent study demonstrated that
mitochondria are carried by a mitochondrial Rho-GTPase, Miro1, which can move
along the actin filament in the TNTs between two cell types (6). Possible mechanisms
of regulation of Miro1 by CS should be further investigated in the future.
Despite the evidence indicating iPSC-MSC-mediated protection of ASMCs from
CSM-induced mitochondrial damage, some of my findings need to be considered
carefully. Firstly, although MSCs have been reported to rescue injured cells in various
models of inflammation and oxidative stress, their own response to oxidative stress is
often neglected. In this study I have demonstrated that iPSC-MSCs are not more
155
resistant to oxidative stress than ASMCs. Therefore caution is required in the
development of MSC-based therapies as MSCs may also show mitochondrial damage
under oxidative stress. Secondly, in the absence of CSM treatment, the co-culture with
iPSC-MSCs increased the apoptosis of ASMCs suggesting that iPSC-MSCs may
induce stress in the target cells at baseline. Such data imply that the safety of
iPSC-MSCs should be carefully examined if they are to be used therapeutically in
man.
There are several limitations to this study and therefore further work is required
to better understand the protective effects of iPSC-MSCs and the mechanisms
mediating these effects. Firstly, the ASMCs used in my study were isolated from
healthy subjects. A better understanding of the therapeutic potential of iPSC-MSCs in
COPD would result from a study of ASMCs from COPD patients, in which
mitochondria are known to be already impaired (273). Secondly, although the
paracrine effects were not shown to mediate the full protective effect of iPSC-MSCs,
protection from CSM-induced mitochondrial ROS was still evident. The molecules
mediating these effects require further investigation. Thirdly, the formation of TNTs
may also facilitate the exchange of other cellular content, which may also protect the
ASMCs from mitochondrial damage. Lastly, the mechanism of CSM‟s effect on
promotion of mitochondrial transfer need to be further addressed.
In summary, this study demonstrated that the capacity of iPSC-MSCs to attenuate
CSM-induced mitochondrial damage in ASMCs relies highly on direct cell-cell
contact. Mitochondrial transfer through the TNTs may play an important role in this
156
process. I elucidated the potential of iPSC-MSCs to target mitochondrial dysfunction
in patients with COPD, thus further highlighting iPSC-MSCs as a promising
candidate for development of cell-based therapies in COPD.
157
Chapter 5. Effects of iPSC-MSCs on
Ozone-induced Airway
Hyper-responsiveness, Lung Inflammation,
Apoptosis and Mitochondrial Dysfunction
in Mice
158
5.1. Introduction
Ozone (O3) is a potent oxidizing pollutant. High concentration of ozone in the
environment have been reported to be associated with worsening of symptoms in
patients with COPD and asthma (18, 74, 84, 173, 197, 239, 257). In mouse models,
exposure to ozone is reported to induce airway oxidative stress, airway
hyper-responsiveness (AHR) and inflammation (54, 253, 272, 274, 275). Repeated
exposure to ozone can destroy alveolar structures in mice, finally leading to
emphysema (203, 253).
Beneficial effects of iPSC-MSCs have been reported in rodent models of allergic
airway inflammation and CS-exposed emphysema (150, 244). However, their effects
on AHR have not been investigated yet. In this study, a previously reported mouse
model of acute ozone-exposure was used (274, 275) in order to examine the effects of
iPSC-MSCs on oxidative stress-induced AHR and airway inflammation.
Although in the last chapter the capacity of iPSC-MSCs to prevent oxidative
stress-induced mitochondrial dysfunction in ASMCs was demonstrated in vitro, the
capacity of iPSC-MSCs to modulate oxidative stress-induced mitochondrial
dysfunction in the lung in vivo remains unknown. Mitochondrial dysfunction in lung
tissue has been documented in the ozone-exposed mouse model (273). Therefore the
other aim of the study was to examine the effects of iPSC-MSCs on oxidative
stress-induced mitochondrial dysfunction in the ozone-exposed mouse model. The
hypothesis of the study is that intravenous administration of iPSC-MSCs can
prophylactically prevent and/or therapeutically reverse ozone-induced AHR, airway
159
inflammation, apoptosis and mitochondrial dysfunction in the lung.
The aims of this chapter were:
1. To evaluate the effects of iPSC-MSCs on AHR and lung inflammation in an acute
ozone-exposed mouse model.
2. To examine effects of iPSC-MSCs on mitochondrial dysfunction and apoptosis in
the lung, in an acute ozone-exposed mouse model.
5.2. Results
5.2.1. iPSC-MSCs prevented ozone-induced AHR
Male C57BL/6 mice were exposed to normal air or ozone (3 ppm) for 3 hours.
1x106 iPSC-MSCs or PBS were administrated to mice through intravenous injection
either 24 hours prior or 6 hours after the exposure. There were 5 groups of mice in
total: the Air/saline (n=5), the Air with iPSC-MSCs administrated 24 hours
prior-exposure (Air/-24hr) (n=5), the Ozone/saline (n=6), the Ozone with iPSC-MSCs
administrated 24 hours prior-exposure (Ozone/-24hr) (n=6) and the Ozone with
iPSC-MSCs administrated 6 hour post-exposure (Ozone/+6hr) (n=6).
Pulmonary resistance (RL) to increasing concentration of acetylcholine was
measured 24 hours after exposure (Figure 5.1A). The injection of iPSC-MSCs 24
hours prior to air exposure did not show significant effect on AHR compared to the
Air/saline group, as indicated by -log PC100 (2.01±0.06 vs. 2.15±0.06, p>0.05) Figure
5.1B). Ozone/saline group demonstrated significantly increased AHR compared with
Air/saline group, as indicated by significantly reduced -log PC100 (1.68±0.06 vs.
160
2.15±0.06, p<0.01). The treatment of iPSC-MSCs 24 hours prior-exposure
significantly reduced ozone-induced AHR as compared with Ozone/saline group (-log
PC100: 1.99±0.08 vs.1.68±0.06, p<0.05). The Ozone/+6hr group, on the other hand,
did not exhibit a difference in AHR compared to the Ozone group (-log PC100:
1.75±0.07 vs.1.68±0.06, p>0.05). The data suggest that intravenously injected
iPSC-MSCs can prevent rather than reverse ozone-induced AHR in mice.
0.0 0.5 1.0 1.5 2.0 2.50
50
100
150
200
air -24hr
ozone
ozone -24hr
air
ozone +6hr
Log[ACh] (mg/ml)
Ch
an
ge in
RL(%
)
A
air air ozone ozone ozone0
1
2
3
salinesaline MSCs
-24hrMSCs
+6hr
MSCs
-24hr
**
B
†
-lo
gP
C1
00
Figure 5. 1 Effect of iPSC-MSCs on ozone-induced AHR. Mice were exposed to
ozone (3 ppm) or air for 3 hours. iPSC-MSCs were intravenously injected 24 hrs
before or 6 hours post exposure. (A) RL in response to increasing concentration of
acetylcholine. (B) -log PC100 derived from plot. **p<0.01 compared to air/saline; †p<0.05 compared to ozone/saline.
161
5.2.2. Effect of iPSC-MSCs on ozone-induced infiltration of inflammatory cells in
the lung
The total cell number in bronchoalveolar lavage (BAL) was determined as
indication of inflammatory cell infiltration in the lung (Figure 5.2A). The
Ozone/saline group demonstrated significantly higher total cell numbers in the BAL
compared to the Air/saline group (51.5±6.7x105 vs. 13.8±4.8x10
5, p<0.01). The
Ozone/-24hr group also demonstrated higher total cell numbers in the BAL compared
to Air/-24hr group (31.9±4.3x105 vs. 12.4±2.1x10
5, p<0.01). However, the total cell
numbers in Ozone/-24hr group was significantly lower compared to Ozone/saline
group (p<0.05). The Ozone/+6hr group did not show any significant difference in
total cell numbers compared to the Ozone/Saline group.
Diff-Quik staining was performed on the BAL cells after cytospin. The numbers
of neutrophils, macrophages, eosinophils and lymphocytes were determined. In the
saline groups, ozone exposure significantly increased the neutrophil number in the
BAL compared to air exposure (20.6±3.5x105 vs. 0.44±0.20x10
5, p<0.05). Treatment
with iPSC-MSCs 24 hours before the ozone exposure significantly reduced the
neutrophil number compared to the ozone/saline group (7.6±1.3x105 vs. 20.6±3.5x10
5,
p<0.01), but the level was still higher than the air/-24hr group (7.6±1.3x105 vs.
0.26±0.07x105, p<0.05). The Ozone/+6hr group did not show any significant
difference compared to Ozone/Saline group. Macrophage number was also elevated in
Ozone/saline group compared to Air/saline (21.6±1.7x105 vs. 1.58±0.67x10
5, p<0.01).
There was a trend of decrease in Ozone/-24hr compared to Ozone/saline but not
162
reaching significance (14.6±2.8x105 vs. 21.6±1.7x10
5). The Ozone/+6hr group did not
show trend of difference compared to Ozone/Saline group (21.2±3.1x105 vs.
21.6±1.7x105). Similarly, eosinophil number in the BAL was increased in
Ozone/saline group compared with Air/saline (2.58±0.63x105 vs. 0.16±0.09x10
5,
p<0.01). There was a trend of decreased eosinophil number in Ozone/-24hr group
compared with Ozone/saline though not reaching significance (0.98±0.27x105 vs.
2.58±0.63x105). Similar trend existed in Ozone/+6hr group as compared with
Ozone/Saline group, also not reaching significance (1.31±0.52x105 vs.
2.58±0.63x105). As for number of lymphocytes, the induction by ozone is significant
(5.76±1.21x105 vs. 1.06±0.41x10
5, p<0.05). There were trends of decrease in both
Ozone/-24hr (4.44±0.86x105) and Ozone/+6hr groups (4.13±1.21x10
5), neither of
which reached significance.
The results suggested that iPSC-MSCs were able to prevent but not reverse
ozone-induced recruitment of inflammatory cells into mouse lung.
163
air air ozone ozone ozone0
20
40
60
80
**##
**
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
†
To
tal C
ells
(cells
x 1
05)
air air ozone ozone ozone0
10
20
30
40
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
*
#
**
††
Neu
tro
ph
ils (
cells x
10
5)
air air ozone ozone ozone0
10
20
30
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
**##
**
Macro
ph
ag
es (
cells x
10
5)
air air ozone ozone ozone0
2
4
6
8
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
*##
Lym
ph
ocyte
s (
cells x
10
5)
air air ozone ozone ozone0
1
2
3
4
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
*
#
Eo
sin
op
hils
(cells
x 1
05)
A
B
Figure 5. 2 Effects of iPSC-MSCs on total and differential cell counts in BAL
from ozone-exposed mice. BAL was collected 24 hours after ozone exposure. (A)
The number of total cells was determined. (B) Differential counts of cells in BAL.
BAL cells were subjected to a cytospin protocol and stained by Diff-Quik.
Neutrophils, macrophages, eosinophils and lymphocytes were identified, based on
their color and morphology, and counted. *p<0.05, **p<0.01 compared to air/saline;
#p<0.05, ##p<0.01 compared to air/-24hr, †p<0.05,
††p<0.01 compared to
ozone/saline.
164
5.2.3. Effect of iPSC-MSCs on cytokine contents of BALF
The BAL fluid (BALF) was collected as the non-cellular compartment of the
BAL. Levels of the inflammatory mediators MCP-1, eotaxin, IL-6, IL-5 and MIP-1α
in BALF were measured by MAGPIX Luminex multiplexing analyzer. Compared
with the Air/saline group, ozone exposure significantly elevated the levels of MCP-1
(181.5±13.3 pg/ml vs. 151.9±1.6 pg/ml, p<0.05), eotaxin (48.6±2.2 pg/ml vs.
30.2±2.7 pg/ml, p<0.01), IL-6 (76.1±9.8 pg/ml vs. 37.4±2.7 pg/ml, p<0.01), IL-5
(11.9±2.6 pg/ml vs. 1.9±0.07 pg/ml, p<0.05) and MIP-1α (9.2±1.6 pg/ml vs. 2.2±0.1
pg/ml, p<0.05). Prophylactic administration of iPSC-MSCs significantly reduced IL-6
(45.1±3.2 pg/ml, p<0.05) and IL-5 (4.66±0.60 pg/ml, p<0.01) levels compared with
the Ozone/saline group. Trends of reduction in eotaxin (40.8±2.3 pg/ml, p=0.053) and
MIP-1α (5.4±0.8 pg/ml, p=0.055) in the BALF were also demonstrated in the
Ozone/-24hr group compared with Ozone/saline group. Therapeutic treatment with
iPSC-MSCs reversed the ozone-induced increase in eotaxin (36.8±2.5 pg/ml, p<0.01),
IL-6 (47.8±3.6 pg/ml, p<0.05), IL-5 (4.6±0.9 pg/ml, p<0.05) and MIP-1α (4.8±0.6
pg/ml, p<0.05) in the BALF. Both prophylactic and therapeutic treatments induced a
trend of reduction in MCP-1 levels which did not reach statistical significance
(Ozone/-24hr, 160.0±1.0 pg/ml; Ozone/+6hr, 157.9±2.8 pg/ml).
The results indicated that iPSC-MCSs prevented and reversed ozone-induced
elevation in inflammatory mediator levels in BALF, including eotaxin, IL-6, IL-5 and
MIP-1α.
165
air air ozone ozone ozone0
50
100
150
200
250
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
*M
CP
-1 (
pg
/ml)
air air ozone ozone ozone0
20
40
60
80
100
†**
p=0.053
†
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
Eo
taxin
(p
g/m
l)
air air ozone ozone ozone0
50
100
150
200
†
**†
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
IL-6
(p
g/m
l)
air air ozone ozone ozone0
10
20
30
†
*
##
†
*†
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
IL-5
(p
g/m
l)
air air ozone ozone ozone0
10
20
30
40
*# †
*
p=0.055
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
MIP
-1
(p
g/m
l)
Figure 5. 3 Effects of iPSC-MSCs on inflammatory mediator levels in BALF after
ozone exposure. Levels of MCP-1, eotaxin, IL-6, IL-5 and MIP-1α in BALF were
measured using a MAGPIX Luminex multiplexing analyser. *p<0.05, **p<0.01
compares to air/saline; #p<0.05, ##p<0.01 compared to air/-24hr, †p<0.05,
††p<0.01
compares to ozone/saline.
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5.2.4. Effects of iPSC-MSCs on apoptosis and proliferation in ozone-exposed
lung tissue
Apoptosis in lung sections was detected using TUNEL staining (Figure 5.4A).
Apoptotic cells were labeled green and the nuclei were labeled blue. The Ozone/saline
group demonstrated significantly increased number of apoptotic cells in lung sections
in comparison with the air/saline group (number/20x field: 21.7±2.9 vs. 3.7±0.4,
p<0.05) (Figure 5.4B). Both Ozone/-24hr and Ozone/+6hr groups showed
significantly reduced number of apoptotic cells compared with Ozone/saline group
(number/20x field: 9.0±1.2, p<0.01 and 9.3±1.3, p<0.01 respectively) (Figure 5.4B).
Proliferative cells in lung sections were identified by immunofluorescent staining
against Ki-67, a marker of proliferation (Figure 5.5A). Ozone exposure significantly
reduced the number of proliferating cells in lung sections (number/20x field: 54.6±5.5
vs. 77.2±5.9, p<0.05) (Figure 5.5B). The Ozone/-24hr group did not show a
significant difference in proliferating cell number compared to Air/-24hr group
(number/20x field: 66.4±7.9, vs.71.5±6.4), indicating that negative effect of ozone on
proliferation might be prevented by treatment with iPSC-MSCs (Figure 5.4B).
However, the Ozone/-24hr group only showed a trend of increased proliferating cell
number compared to Ozone/saline group which did not reach statistical significance.
The Ozone/+6hr group also did not show a significant difference in the number of
proliferating cells compared with the Ozone/saline group.
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air air ozone ozone ozone0
10
20
30
*
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
† †† †
Ap
op
toti
c C
ells
(n
um
ber/
20x f
ield
)
Figure 5. 4 Effects of iPSC-MSCs on ozone-induced apoptosis in mouse lungs. (A)
Apoptotic cells were identified by TUNEL staining (green) indicated by the arrows.
The nuclei were visualized by DAPI staining (blue). (B) Number of apoptotic cells in
lung sections. Apoptotic cells were counted in five randomly selected 20x fields for
each mouse using a fluorescent microscope. *p<0.05 compared to air/saline; ††
p<0.01
compares to ozone/saline.
A
B
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air air ozone ozone ozone0
20
40
60
80
100
salinesaline SC
-24hr
SC
+6hr
*
SC
-24hr
Pro
life
rati
ve C
ells
(n
um
ber/
20x f
ield
)
Figure 5. 5 Effects of ozone and iPSC-MSCs on cellular proliferation in mouse
lungs. (A) Detection of proliferating cells by Ki-67 staining (red). The nuclei were
visualized by DAPI staining (blue). (B) Number of proliferative cells in lung sections.
Proliferative cells were counted in five randomly selected 20x fields for each mouse
using a fluorescent microscope. *p<0.05 compared to air/saline.
A
B
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5.2.5. Effect of iPSC-MSCs on ozone-induced cellular ROS and mitochondrial
dysfunction in mouse lungs
Intact mitochondria and cytoplasmic fractions were extracted from lung tissue.
The ROS levels in the cytoplasm fractions were measured by DCF assay (Figure
5.6A). Ozone exposure significantly elevated the cytoplasmic ROS levels (RFU/mg
protein: 3131±269.8 vs. 2061±123.9, p<0.01). When treated with iPSC-MSCs 24
hours prior to ozone exposure, the cytoplasmic ROS levels were still significantly
higher compared with the Air/-24hr group (RFU/mg protein: 2787±171.9 vs.
2030±178.9, p<0.05). The Ozone/+6hr group also showed significantly higher level of
cytoplasmic ROS compared with Air/saline group (RFU/mg protein: 2909±194.4 vs.
2061±123.9, p<0.05). No significant reduction was observed in both Ozone/-24hr and
Ozone/+6 groups in comparison with the Ozone/saline group.
The mitochondrial ROS levels in the intact mitochondria were measured by
MitoSOX staining (Figure 5.6B). Mitochondrial ROS level was significantly elevated
in the Ozone/saline group compared with the Air/Saline group (RFU/mg protein:
166.4±9.5 vs. 119.9±8.8, p<0.05). Both Ozone/-24hr and Ozone/+6hr groups
demonstrated significantly reduced mitochondrial ROS levels compared to
Ozone/saline group (RFU/mg protein: 122.8±10.0, p<0.05 and 62.9±10.3, p<0.01
respectively). In addition, the mitochondrial ROS level in Ozone/+6hr group is
significantly lower than the Ozone/-24hr group (p<0.01).
Mitochondrial membrane potential (ΔΨm) of the intact mitochondria was
accessed by JC-1 staining (Figure 5.6C). Exposure to ozone without treatment of
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iPSC-MSCs reduced the ΔΨm in comparison to the Air/saline group (fold to
Air/saline: 0.692±.0.057 vs. 1.00±0.041, p<0.01). The treatment of iPSC-MSCs 24
hours prior to the ozone-exposure significantly improved the ΔΨm compared with the
Ozone/saline group (fold to Air/saline: 1.004±.0.076 vs. 0.692±.0.057, p<0.01).
However, the Ozone/+6hr group didn‟t show significant difference as compared with
Ozone/saline group, and the ΔΨm was still significantly lower than the Air/saline
group (fold to Air/saline: 0.778±.0.060 vs. 1.00±0.041, p<0.01).
171
air air ozone ozone ozone0
1000
2000
3000
4000
5000
**# *
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
Cyto
pla
sm
ic
RO
S
(RF
U/m
g p
rote
in)
air air ozone ozone ozone0
100
200
300
*
**
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
†
††
§§
Mit
och
on
dri
al R
OS
(RF
U/m
g p
rote
in)
air air ozone ozone ozone0.0
0.5
1.0
1.5
** *
salinesaline MSCs
-24hr
MSCs
+6hr
MSCs
-24hr
††
m (
Fo
ld t
o A
ir/s
ali
ne)
A
B
C
Figure 5. 6 Effects of iPSC-MSCs on ozone-induced ROS and mitochondrial
dysfunction in mouse lungs. Intact mitochondria and cytoplasmic fractions were
isolated from mouse lungs. (A) Cytoplasmic ROS was measured by DCF staining. (B)
Mitochondrial ROS was measured in intact mitochondria by MitoSOX staining. (C)
ΔΨm was determined in intact mitochondria by JC-1 staining. *p<0.05, **p<0.01
compared to air/saline; #p<0.05 compared to air/-24hr, †p<0.05,
††p<0.01 compared to
ozone/saline; §§
p<0.01 compared to ozone/-24hr.
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5.3. Discussion
This study demonstrated that intravenously administrated iPSC-MSCs were able
to prevent ozone-induced AHR, airway inflammation, apoptosis, mitochondrial ROS
and reduction in ΔΨm. The therapeutic administration of iPSC-MSCs failed to reverse
ozone-induced AHR, airway inflammation and reduction in ΔΨm, but the
ozone-induced apoptosis and mitochondrial ROS were reversed. Although the data
also showed lack of effects on ozone-induced cytoplasmic ROS and reduction in
proliferation, the findings suggested a protective role of iPSC-MSCs on
ozone-induced lung damage.
Ozone is a potent oxidant. High concentration of environmental ozone was
reported to be associated with worsening of symptoms in patients with COPD and
asthma (18, 74, 84, 173, 197, 239, 257). In mouse models, exposure to ozone is
reported to induce airway oxidative stress, AHR and inflammation (54, 253, 272, 274,
275). Oxidative stress-associated airway inflammation is believed to be a key
component of the pathogenesis of COPD (65). Indeed, repeated exposure to ozone can
lead to airway inflammation accompanied by destruction of alveolar structure, leading
to an emphysema-like phenotype (203, 253). In the present study mice were exposed
to ozone (3 ppm) for a single period of 3 hours. The recruitment of inflammatory cells
to the lungs was robust after 24 hours, as indicated by increased cell counts in the
BAL. Increased levels of inflammatory mediators including MCP-1, eotaxin, IL-6,
IL-5 and MIP-1α were also observed in the BALF. These findings confirmed the
induction of inflammation by ozone in the model. The prophylactic injection of
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iPSC-MSCs prevented ozone-induced inflammation, as indicated by the reduction of
both total cell counts in the BAL and levels of inflammatory mediators including
eotaxin, IL-6, IL-5 and MIP-1α, in the BALF. On the other hand, administration of
iPSC-MSCs after ozone exposure did not reverse the recruitment of inflammatory
cells. However, it did reverse the induction of several inflammatory mediators in the
lungs, including eotaxin, IL-6, IL-5 and MIP-1α, which suggested that iPSC-MSCs
still had an anti-inflammatory effect. Failure to reverse inflammatory cell infiltration 6
hours after exposure may be due to a stronger inflammatory response or due to the
shorter length of treatment time in the therapeutic protocol (18 hours) compared to the
prophylactic protocol (48 hours). These findings are in agreement with my results
showing lack of therapeutic effects of iPSC-MSCs on AHR. Although, the
anti-inflammatory role of iPSC-MSCs has been demonstrated by previous studies in
allergic (244) and CS-induced airway inflammation models (as shown in previous
chapters), the ability of iPSC-MSCs to ameliorate AHR has never been reported. AHR
is a major clinical feature of asthma, however it is also observed in some patients with
COPD (291). A previous study in an acute ozone-exposed mouse model demonstrated
that ozone-induced AHR results from the activation of p38 mitogen-activated protein
kinase (147). The exact mechanism of the action of iPSC-MSCs in this model requires
further investigation.
Excessive protease activity associated with oxidative stress and chronic
inflammation is believed to lead to alveolar destruction in patients with COPD (68, 76,
77, 189). However, accumulating evidence suggest that the imbalance between
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apoptosis and proliferation may also contribute to the gradual alveolar destruction
(67). In this study increased lung apoptosis and reduced proliferation was observed
after ozone exposure, which is consistent with the imbalance between apoptosis and
proliferation in the chronic CS-exposed model described in Chapter 3. Treatment with
iPSC-MSCs, either before or after ozone exposure, reduced ozone-induced apoptosis,
but the ozone-induced reduction in proliferation was not alleviated. These findings
indicate a possibly higher anti-apoptotic ability of iPSC-MSCs than pro-proliferative
ability in a context of oxidative stress.
Mitochondrial dysfunction has been characterized in the ozone-exposed mouse
model in a previous report (273). Ozone exposure can lead to reduced ATP levels,
mitochondrial complex proteins expression and ΔΨm as well as increased
mitochondrial ROS. The mitochondria-targeted antioxidant, MitoQ, was able to
attenuate mitochondrial dysfunction in the model, as well as the ozone-induced AHR
and inflammation, indicating the AHR and inflammation may be partly driven by
mitochondrial dysfunction (273). Moreover, mitochondrial dysfunction can trigger the
cellular apoptosis (90). Therefore, the effects of iPSC-MSCs on AHR, inflammation
and apoptosis may all relate to its effects on ozone-induced mitochondrial dysfunction
in lung. Indeed, the prophylactic administration of iPSC-MSCs reduced
ozone-induced mitochondrial ROS and reduction in ΔΨm, in line with the effects on
ozone-induced inflammation, AHR and apoptosis. The administration after ozone
exposure only reversed the ozone-induced mitochondrial-ROS level, but not the
reduction of ΔΨm. The findings are in agreement with the results of our co-culture
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experiments described in the Chapter 4. There I show that iPSC-MSCs can prevent
and reverse CSM-induced mitochondrial ROS, whereas they can prevent but not
reverse CSM-induced ΔΨm loss.
Despite the reduction in ozone-induced mitochondrial ROS by iPSC-MSCs,
ozone-induced cytoplasmic ROS levels were not modulated by iPSC-MSCs. Such
result may indicate a lack of anti-oxidant effects by iPSC-MSCs in the model. Further
investigation on oxidative stress and anti-oxidant mechanisms in the lung should
better address this issue. On the other hand, the data also suggested that mitochondrial
and cytoplasmic ROS are differentially regulated in cells. Although the mechanisms
by which iPSC-MSCs modulate mitochondrial ROS are unclear, the prevention of
ΔΨm loss and the lack of effect on cytoplasmic ROS suggest that this possibly occurs
through mechanisms that specifically target the mitochondria, such as mitochondrial
transfer.
This study has several limitations and further investigations are required. Firstly,
although the acute exposure of ozone can induce AHR and inflammation, a model
involving multiple ozone exposures would be more relevant to COPD (253).
Investigation of effects of iPSC-MSCs on ozone-induced emphysema, AHR,
inflammation and mitochondrial dysfunction in such model would be an important
experiment to perform. Secondly, the mechanisms by which iPSC-MSCs prevent
ozone-induced mitochondrial dysfunction and the associated inflammation, apoptosis
and AHR remain unclear. Lastly, the effects of iPSC-MSCs on the oxidant and
anti-oxidant balance in the lungs can be further investigated.
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In summary, iPSC-MSCs prevented ozone-induced mitochondrial dysfunction as
well as lung inflammation and apoptosis, and AHR. In the therapeutic protocol,
iPSC-MSCs showed no effect on AHR and airway inflammation but reversed
apoptosis and mitochondrial dysfunction in response to ozone. I demonstrate for the
first time the capacity of iPSC-MSCs to prevent mitochondrial dysfunction and AHR
in the lungs, highlighting iPSC-MSCs as promising candidates for the development of
cell-based therapies for COPD.
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Chapter 6. General Discussion
178
6.1. General Discussion
In this study, I demonstrated a superior efficacy of iPSC-MSCs to attenuate
chronic CS-exposure-induced lung emphysema, inflammation and apoptosis in rats
compared to BM-MSCs, which was associated with a higher capacity for
mitochondrial transfer from iPSC-MSCs to lung epithelium. In vitro studies also
demonstrated a higher ability of iPSC-MSCs to transfer mitochondria to and attenuate
CSM-induced ATP depletion in CSM-treated bronchial epithelial cells. In addition,
iPSC-MSCs-CdM demonstrated better efficacy in attenuating CSM-induced apoptosis
and reducing proliferation in bronchial epithelial cells compared to BM-MSCs, which
was partly mediated by SCF. Therefore, iPSC-MSCs demonstrated higher capacity of
both mitochondrial transfer and paracrine release of anti-apoptotic cytokines to
bronchial epithelial cells. I then investigated whether such activities were able to
restore mitochondrial health under oxidative stress. This study was carried out in vitro
first in a CSM-treated model of ASMCs. Direct co-culture with iPSC-MSCs protected
ASMCs from CSM-induced reduction in ΔΨm and elevation of mitochondrial ROS
and apoptosis. However, when direct cell-cell contact was prevented, iPSC-MSCs
only reduced CSM-induced mitochondrial ROS but not reduction of ΔΨm and
increase of apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs
was observed in direct co-culture which was enhanced by CSM treatment. In line with
the in vitro findings, in a mouse model of acute ozone exposure, intravenous
administration of iPSC-MSCs protected lung tissue from ozone-induced
mitochondrial dysfunction and apoptosis. Ozone-induced AHR and inflammation was
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also reduced by iPSC-MSCs. In summary, I demonstrated that mitochondrial transfer
activity from iPSC-MSCs was able to alleviate oxidative-stress-induced ATP
depletion, mitochondrial dysfunction and apoptosis in target cells. The full capacity of
iPSC-MSCs to achieve these effects may rely on combined mechanisms of both direct
cell-cell contact and release of paracrine factors (Figure 6.1). The capacity of
iPSC-MSCs to transfer their mitochondria to and protect airway cells from oxidative
stress-induced damage is superior to that of BM-MSCs.
The study was initially motivated by three observations regarding current status
of respiratory medicine and stem cells. First of all, current medications for COPD can
only reduce symptoms, exacerbations, and improve health status and exercise
tolerance, and no current medication is able to attenuate the progressive decline of
lung function in COPD (263). As a disease predicted to be the 4th leading cause of
death by 2030 (172) associated with enormous economic and social burden, seeking
new treatments for COPD is an urgent and important task in respiratory medicine.
Secondly, there have been numerous reports on the efficacy of MSCs in animal
models of lung disease (269, 270). Despite a lack of knowledge of the underlining
mechanisms, MSCs from BM, adipose or cord blood have been demonstrated to
attenuate emphysema induced by CS, elastase or VEGF deficiency (94, 112, 151, 222,
269, 270). However, in a clinical trial, intravenously infused BM-MSCs failed to
modulate quality of life and lung function in patients with COPD (271). Although
there are multiple variables that may contribute to the lack of efficacy such as dose
and timing, the results still point out the potential gap between the pre-clinical models
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and patients, and the possibility that current findings of therapeutic effects of MSCs in
COPD-related models may not be necessarily translated into treatments for patients
with COPD. Therefore, despite current studies already having demonstrated adequate
efficacy of MSCs in pre-clinical models, the search for improved MSC therapies with
more profound effects in pre-clinical models should not stop. After all, the clinical
trial demonstrated extraordinary tolerance and safety of intravenously infused
BM-MSCs in patients with severe COPD, leaving the door open for future trials.
Thirdly, the in vitro derivation of iPSC-MSCs from iPSCs has provided a new
opportunity to develop improved MSC therapy against COPD. Compared to
BM-MSCs, iPSC-MSCs are more readily available in abundance and possess a higher
ability of proliferation without loss of differentiation potential and telomerase activity
(153). They may hold the hope of overcoming ageing-associated dysfunction of
BM-MSCs by the induction of pluripotency during their generation. As age is an
important risk factor for COPD, iPSC-MSCs may be a good candidate for getting one
own source of MSCs. Moreover, the process of inducing somatic cells into iPSCs and
differentiating iPSCs into iPSC-MSCs provided an opportunity to genetically
manipulate the cells at different stages. Using MSCs as a vehicle for gene therapy
may also be a future option to develop improved therapy against COPD (122). In
summary, iPSC-MSCs may represent a novel and improved candidate for cell therapy
of COPD. From the data presented in Chapter 3, my study demonstrated better
efficacy of iPSC-MSCs than BM-MSCs in attenuating CS-induced lung emphysema,
inflammation and apoptosis, possibly via higher activity of mitochondrial transfer and
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paracrine secretion of cytokines such as SCF. My study has demonstrated the potential
of iPSC-MSCs as an improved MSC therapy, hence providing a new option that may
lead to a better treatment for COPD.
One striking observation from the study of CS-exposed rats is the mitochondrial
transfer from iPSC-MSCs to epithelial cells, which led to focusing my attention to the
other theme of the thesis, namely the oxidative stress-induced mitochondrial
dysfunction in pulmonary cells. Developing new treatments for COPD relies on the
understanding of the pathogenesis of COPD. One of the recent advances is the
demonstration of mitochondrial dysfunction associated with COPD. Primary
bronchial epithelial cells from ex-smokers with severe COPD have been shown to
exhibit swelling and fragmented mitochondrial morphology which is similar to
CSM-treated BEAS-2B cells (105). Primary ASMCs from patients with COPD were
shown to have elevated mitochondrial ROS levels, and reduced ΔΨm, complex
protein levels and respiratory function (273). Moreover, the mitochondria-targeted
anti-oxidant MitoQ was able to reduce chronic ozone exposure-induced mitochondrial
dysfunction, inflammation and AHR in mice, highlighting mitochondria as a potential
target for COPD treatment (273). Meanwhile, despite a large number of reports on the
efficacy of MSCs in lung disease models, their mechanisms of action remain largely
unknown. One of the latest advances is the in vivo observation of mitochondrial
transfer from BM-MSCs to pulmonary epithelial cells in an acute lung injury model
(118). In other words, mitochondria are a novel target for COPD treatment as well as
a novel player in the action of MSCs. Therefore, the second half of my study aimed to
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investigate whether mitochondria for lung cells are damaged by oxidative stress,
whether this damage can be prevented or reversed by iPSC-MSCs, and whether
iPSC-MSCs exert such protective actions through mitochondrial transfer. The results
demonstrated that CSM led to reduced ATP contents in BEAS-2B cells, and increased
mitochondrial ROS, apoptosis and reduced ΔΨm in primary ASMCs in vitro. Acute
ozone exposure also led to increased mitochondrial ROS and reduced ΔΨm in mouse
lungs in vivo. This oxidative stress-induced mitochondrial dysfunction was alleviated
by iPSC-MSCs either through direct co-culture in vitro, or through intravenous
administration in vivo. Mitochondrial transfer was demonstrated from iPSC-MSCs to
airway epithelium in the CS-exposed model in vivo, and from iPSC-MSCs to
BEAS-2B cells and ASMCs in vitro. CSM enhanced mitochondrial transfer from
iPSC-MSCs to both BEAS-2B cells and ASMCs. The enhanced mitochondria transfer
may be driven by the need for repair, and may be responsible for the alleviation of
CSM-induced mitochondrial dysfunction. Therefore this study demonstrated that
oxidative stress can induce mitochondrial dysfunction of pulmonary cells in vitro and
in vivo, and iPSC-MSCs are able to transfer mitochondria to these damaged cells in
response to the oxidative stress-induced damage and alleviate the mitochondrial
dysfunction.
Improved mitochondrial function may bring other potential benefits to the cell.
Mitochondria serve as the powerhouse of the cells. It has been reported that CS
exposure led to altered lung energy metabolism, with a switch of glucose metabolism
toward pentose phosphate pathway and a reduced supply of substrate to mitochondria.
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The transfer of mitochondria into the lung cells may help maintain the energy supply
(4). In addition, as described in Chapter 1.1.4.7, mitochondria play an important role
in regulating ROS generation and cell fate. Oxidant/anti-oxidant imbalance plays an
important role in the pathogenesis of COPD, with the process of oxidative
phosphorylation in mitochondria being a major source of intracellular ROS (149, 165).
Severe mitochondrial damage may lead to depolarization of mitochondria and release
of cytochrome c, and to mitochondrial pathways of cell apoptosis (90). Mitochondrial
damage can also mediate autophagy and senescence, processes that could contribute
to the development of COPD (11).
In general, mitochondrial dysfunction can be targeted using three different
strategies: repair via scavenging excess ROS, reprogramming via regulatory pathways,
and replacement by exogenous functional mitochondria (5). Mitochondria-targeted
anti-oxidant such as MitoQ is a direct way to reduce mitochondrial ROS, which may
subsequently improve other mitochondrial functions such as ΔΨm and respiration.
This has been demonstrated in the chronic ozone-exposed mouse model (273).
Nutrient overload and metabolic changes associated with obesity are common causes
of mitochondrial stress (5) and improvement of mitochondrial function by exercise,
diet and possibly pharmacotherapy such as metformin has been reported (5). Clinical
trials on COPD have demonstrated the potential of metformin to reduce dyspnea and
increase exercise capacity, but more comprehensive studies are awaited to confirm its
efficacy (225). Both repair and reprogramming relies on the recovery of the current
defective mitochondria, which may not be easy for cells of severely-damaged
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mitochondria. Also, if mitochondrial DNA has been damaged, such strategies may not
work. In such a situation, replacement of damaged mitochondria with fresh
mitochondria may be a better strategy. Mitochondrial replacement can be achieved by
microinjection, incubation with mitocytoplasts or intact purified mitochondria, and
the cell-cell contact-dependent mitochondrial transfer from MSCs (225). The latter
method used in my thesis, namely mitochondrial transfer, is the only way that does
not involve any disturbance of the cells or involve isolating mitochondria and
injecting the cells but involves a natural method of transferring organelles such as
mitochondria between cells. It provides a perfect approach to deliver exogenous
mitochondria into damaged cells. My study confirmed that mitochondrial transfer can
occur in vivo following its first report in 2012 (118). Moreover, as iPSC-MSCs have a
higher expanding ability than BM-MSCs, the approach we used therefore provided a
more abundant source of mitochondrial donor.
Although mitochondrial transfer was demonstrated both in vivo and in vitro in
this study, it should be noted that the recipient cells were human bronchial epithelial
or ASMCs in the in vitro experiments, while in the in vivo study mitochondria were
transferred from human MSCs into rodent lung cells. The functional compatibility
between the human mitochondria and rodent lung cells was not tested in the current
study. Previous reports have demonstrated the transfer of human mitochondria from
human BM-MSCs to rat cardiomyocytes in vitro (204) and to AEC2s in mouse lungs
(118), but the functional compatibility was not discussed. In a further study, despite
reporting efficient mitochondrial fusion between mouse and human mitochondria
185
within 4 hours (288), the study demonstrated low compatibility between mouse
nuclear and human mitochondrial genomes. Therefore the functional significance of
the mitochondrial transfer observed in vivo in this thesis is largely unknown.
Two animal models have been used in this thesis to study the rescuing effect of
MSCs. The first one is a rat model exposed to CS for 1 hour/day for 56 days, while
the second one is a mouse model with a single ozone exposure for 3 hours. The two
models are related in some aspects but also different. Both CS and ozone are potent
sources of oxidants. Therefore, both models can be referred to as oxidative
stress-induced lung damage, which makes them distinct from some other
COPD-related models, such as elastase-induced emphysema in which oxidative stress
is not the direct driving force. Cigarette smoking is the major risk factor of COPD,
and chronic CS-exposure is a classic way to induce COPD-like phenotype such as
airspace enlargement and lung inflammation. Although ozone is not a major cause of
COPD, elevated ozone levels have been reported to increase hospital admission due to
COPD (173). Exposure to ozone twice per week for 6 weeks can induce COPD-like
phenotype in mice including emphysema, chronic inflammation, cell infiltration and
steroid resistance (253, 272). Moreover, the ozone-exposed mouse model has been
reported to exhibit a gene expression signature similar to that seen in human COPD
(273). The major difference between the two models in the present study is that the
CS-exposed model is a chronic model with multiple exposures, while the
ozone-exposed model is an acute model with a single exposure period. The acute
exposure of ozone cannot induce airspace enlargement, but infiltration of neutrophils
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and other inflammatory cells was demonstrated and, as such, could be taken as a
model mimicking an exacerbation of COPD which is a frequent manifestation of
advanced COPD. In general, acute exposure indicates that the animals were
responding to the stimulation based on a normal physiological condition, whereas in
chronic models, along the progression of the damage, the animals respond to the
stimulation based on a damaged, abnormal condition. While the efficacy of
iPSC-MSCs in the chronic CS-exposed model specifically defines iPSC-MSCs as a
promising candidate for MSC therapy of COPD, the efficacy of iPSC-MSCs in the
acute ozone-induced model may provide a more general implication on an acute
oxidative stress insult such as during an exacerbation. Although the acute
ozone-exposed model did not exhibit airspace enlargement, this model has been used
to investigate some parameters that have not been measured in the CS-exposed model,
including AHR, cytosolic ROS, mitochondrial ROS and ΔΨm. Nevertheless, both
models in the study have demonstrated inflammation and apoptosis in the lung. Using
the two different models, I demonstrated the capacity of iPSC-MSCs to alleviate
oxidative stress-induced lung damage in both acute and chronic setups.
The in vitro study involved two different airway cell types. Although both cell
types were treated with CSM, it should be noted that the treatments were optimized
based on different parameters. In the study on bronchial epithelial BEAS-2B cells, the
concentration (2%) and length of treatment (24 hours) was adapted from a previous
study, with an induction of IL-8 release without reduction of viability. As
demonstrated in Chapter 3, such condition was sufficient to induce apoptosis and
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reduce ATP content. In the study of primary ASMCs, the concentration of CS (10% or
25%) and length of treatment (4 hours) was determined by the induction of
mitochondrial dysfunction as measured by mitochondrial ROS and ΔΨm. The CSM
stimulation was more acute and strong in the ASMCs study, leading to a more acute
induction of apoptosis. Such difference may explain why the released cytokines from
iPSC-MSCs (iPSC-MSCs-CdM) were able to attenuate CSM-induced apoptosis in
BEAS-2B cells but not in ASMCs in the study. On the other hand, enhanced
mitochondrial transfer has been detected from iPSC-MSCs to both BEAS-2B cells
and ASMCs under the corresponding CSM treatments. Mitochondrial transfer and
paracrine effects may serve as two different mechanisms of iPSC-MSCs‟ action. By
using apoptosis as a functional output, it is clear that the relative contribution of
mitochondrial transfer and paracrine effects varies among different target cell types
and treatments. In the 24 hour-treated BEAS-2B cells, paracrine effects from
iPSC-MSCs were sufficient to reduce apoptosis. By contrast, in the 4 hour-treated
ASMCs, the anti-apoptotic efficacy fully relied on cell-cell contact that facilitated the
mitochondrial transfer which is a more direct mechanism to rescue mitochondrial
dysfunction. Such differences demonstrated the complexity and the dynamics of
mechanisms of action of iPSC-MSCs.
The in vivo and in vitro data from different chapters complement each other very
well in this study. The mitochondrial transfer from iPSC-MSCs to damaged cells in
vivo in the CS-exposed model was also observed in vitro for both BEAS-2B cells and
ASMCs. The anti-apoptotic effect of iPSC-MSCs in both CS- and ozone-exposed
188
animal models was also demonstrated in both BEAS-2B cells and ASMCs. The
attenuation of mitochondrial dysfunction by iPSC-MSCs was demonstrated in vivo in
ozone-exposed mice as well as in vitro in ASMCs. Moreover, both studies
demonstrated time-dependent effects. In the CSM-treated ASMCs, iPSC-MSCs
prevented but not reversed the CSM-induced mitochondrial dysfunction. Similarly, in
the ozone-exposed mice, iPSC-MSCs prevented but not reversed the ozone-induced
inflammation, AHR and reduction of ΔΨm in the lung. This may be explained by the
fact that ASMCs or mice were treated with iPSC-MSCs for more time in the
preventive protocols, or that the oxidative stress-induced damage has progressed to a
stage that cannot be reversed by iPSC-MSCs. Interestingly, in the ozone-exposed
mice, although the preventive treatment did not attenuate ozone-induced reduction of
ΔΨm, it attenuated ozone-induced mitochondrial ROS in the lung. This is very similar
to the effects of iPSC-MSCs-CdM or iPSC-MSCs in trans-well inserts in vitro, which
also demonstrated reduction of CSM-induced mitochondrial ROS but no change in
ΔΨm in ASMCs. In theory, excess mitochondrial ROS can lead to defective election
transport chain which is responsible for proton transfer across the inner mitochondrial
membrane and defective permeability transition pore which controls the permeability
of inner membrane (170). Both effects lead to ΔΨm reduction. The reduction of
mitochondrial ROS without effects on ΔΨm in the study may be because the reduced
mitochondrial ROS levels were still high enough to induce a reduction in ΔΨm, or
because there are other mechanisms regulating ΔΨm which were less targeted by
iPSC-MSCs, such as cytosolic ROS levels.
189
The most direct clinical implication of the study is that intravenously-infused
iPSC-MSCs may exert a stronger effect in patients with COPD compared to
BM-MSCs which have not been shown to be effective in the clinical trials. However,
it is of concern that the genetic reprogramming may also change the safety profile of
iPSC-MSCs compared to BM-MSCs. In a more general perspective, iPSC-MSCs may
be effective in other oxidative stress-mediated airway diseases such as asthma. In
addition, the higher mitochondria transfer capacity and abundant resource also
suggests that iPSC-MSCs may serve as a useful mitochondrial donor to target
mitochondrial dysfunction in various diseases.
There are several limitations of the work in my thesis. Firstly, due to time
limitations, not all parameters were investigated in all models. As mitochondrial
dysfunction was not the initial focus of the study, mitochondrial ROS and ΔΨm were
not measured in the CS-exposed rat model and CSM-treated BEAS-2B cells.
Secondly, although inflammation was investigated in the in vivo models, there was no
direct measurement of inflammation in the in vitro models. A common inflammatory
measurement used in cell culture studies is measurement of released inflammatory
mediators. However, as both cell types in the co-culture are from human, the secretory
function of one particular cell type is difficult to ascertain. Thirdly, there are other
potential mechanisms involved in iPSC-MSCs action which may also contribute to
the efficacy observed in the study. For example, formation of TNTs may also facilitate
transfer of other organelles such as lysosomes. iPSC-MSCs may also secrete
protective microvesicles to lung cells. Lastly, mitochondrial ROS and ΔΨm may not
190
represent all the functions of mitochondria. A measurement of mitochondrial
respiration profile would have been ideal to address the issue.
6.2. Conclusion
I demonstrated the superior effects of iPSC-MSCs in attenuating CS-induced
emphysema, inflammation and apoptosis in rat lung compared to BM-MSCs, which
was attributed to higher mitochondrial transfer and paracrine release of cytokines
including SCF. iPSC-MSCs can also alleviate oxidative stress-induced AHR,
inflammation, mitochondrial dysfunction and apoptosis in mouse lungs.
Mitochondrial transfer from iPSC-MSCs was able to alleviate
oxidative-stress-induced ATP depletion, mitochondrial dysfunction and apoptosis in
target cells. The full capacity of iPSC-MSCs to achieve this efficacy may rely on
combined mechanisms of both direct cell-cell contact and release of paracrine factors.
These findings support iPSC-MSCs as a promising candidate for the development
of MSCs-based therapy against COPD, which may be driven by multiple
mechanisms.
191
Figure 6. 1 Summary of the effects of iPSC-MSCs on oxidative stress-induced
lung cell damage. The full capacity of iPSC-MSCs to alleviate oxidative
stress-induced mitochondrial dysfunction and apoptosis may rely on combined
mechanisms of both direct cell-cell contact and release of paracrine factors. SCF: stem
cell factor; Mito-ROS: mitochondrial ROS; iPSC-MSCs: induced pluripotent stem
cell-derived mesenchymal stem cells; ASMCs: airway smooth muscle cells; CS:
cigarette smoke; AHR: airway hyper-responsiveness.
192
6.3. Future Studies
Several future investigations can be extended from this study.
In this study, the effects of iPSC-MSCs on mitochondrial dysfunction in animal
models with COPD-like phenotypes have not been demonstrated. It would be
important to further investigate effects of iPSC-MSCs on mitochondria of the lung in
a chronic CS-/ozone-exposed models focusing particularly on the development of
emphysema.
In the study I used cytochalasin B or trans-well membrane to block the formation
of TNTs between iPSC-MSCs and target cells. However, such methods are not
selective blockers of mitochondrial transfer. A recent study has demonstrated that
mitochondrial transfer is mediated by Miro-1 (6). Knock-down or over-expression of
Miro-1 in BM-MSCs leads to reduced or enhanced mitochondrial transfer activity
respectively. This would provide a more selective approach to block mitochondrial
transfer without interruption of TNT formation. Regarding my study, the following
investigations could be proposed. First, whether Miro-1 is also responsible for the
mitochondrial transfer in iPSC-MSCs can be examined. Determination of efficacy of
iPSC-MSCs and mitochondrial transfer after Miro-1 knock-down and over-expression
would address the question. Second, how does CSM enhance mitochondrial transfer?
One hypothesis is that CSM treatment can directly regulate capacity of mitochondrial
transfer of iPSC-MSCs. This can be investigated by measurement of Miro-1
expression in response to CSM and identification of the possible pathways involved,
such as mitogen-activated protein kinase pathways, using specific inhibitors of these
193
pathways. On the other hand, the enhanced mitochondrial transfer may be a response
to signals from injured target cells. How the recipient cells regulate the mitochondrial
transfer will also require further investigation.
Another important follow-up study is to determine whether iPSC-MSCs can
improve mitochondrial function of primary ASMCs from patients with COPD.
ASMCs from patients with COPD already demonstrated mitochondrial dysfunction
compared to normal controls. How this would change the mitochondrial transfer
between the ASMCs and iPSC-MSCs should also be investigated.
194
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