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UNIVERSITY OF OULU P .O. B 00 F I -90014 UNIVERSITY OF OULU FINLAND
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SCIENTIAE RERUM NATURALIUM
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EDITOR IN CHIEF
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University Lecturer Santeri Palviainen
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ISBN 978-952-62-0653-0 (Paperback)ISBN 978-952-62-0654-7 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)
U N I V E R S I TAT I S O U L U E N S I S
MEDICA
ACTAD
D 1271
ACTA
Tuomas M
äkelä
OULU 2014
D 1271
Tuomas Mäkelä
SYSTEMIC TRANSPLANTATION OF BONE MARROW STROMAL CELLSAN EXPERIMENTAL ANIMAL STUDY OF BIODISTRIBUTION AND TISSUE TARGETING
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE,INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF SURGERY,DEPARTMENT OF ANAESTHESIOLOGY;INSTITUTE OF BIOMEDICINE,DEPARTMENT OF ANATOMY AND CELL BIOLOGY;INSTITUTE OF DIAGNOSTICS,DEPARTMENT OF DIAGNOSTIC RADIOLOGY;CLINICAL RESEARCH CENTER
A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 2 7 1
TUOMAS MÄKELÄ
SYSTEMIC TRANSPLANTATION OF BONE MARROW STROMAL CELLSAn experimental animal study of biodistribution and tissue targeting
Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defencein Auditorium 1 of Oulu University Hospital, on 19December 2014, at 12 noon
UNIVERSITY OF OULU, OULU 2014
Copyright © 2014Acta Univ. Oul. D 1271, 2014
Supervised byProfessor Petri LehenkariProfessor Tatu Juvonen
Reviewed byProfessor Yrjö KonttinenDocent Susanna Miettinen
ISBN 978-952-62-0653-0 (Paperback)ISBN 978-952-62-0654-7 (PDF)
ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)
Cover DesignRaimo Ahonen
JUVENES PRINTTAMPERE 2014
OpponentProfessor Ari Harjula
Mäkelä, Tuomas, Systemic transplantation of bone marrow stromal cells. Anexperimental animal study of biodistribution and tissue targetingUniversity of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute ofClinical Medicine, Department of Surgery, Department of Anaesthesiology; Institute ofBiomedicine, Department of Anatomy and Cell Biology; Institute of Diagnostics, Department ofDiagnostic Radiology; Clinical Research CenterActa Univ. Oul. D 1271, 2014University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Bone marrow mesenchymal stromal cells (MSCs) and mononuclear cells (BM-MNCs) haveshown great therapeutic potential in various clinical settings. Although intravasculartransplantation of the cells constitutes the optimal delivery route, massive pulmonary entrapment,with the threat of embolization, remains a major obstacle for using this type of therapy. Becausepulmonary entrapment is at least partially mediated by adhesion molecules, cell surfacemodification could enhance pulmonary passage.
We used a porcine model of allogeneic MSC and autologous BM-MNC transplantation andradionuclide labelling to track the cells. The role of the transplantation route on lung entrapment,biodistribution, safety and BM-MNC targeting to the injured brain was studied. Effects of pronasedetachment on the lung passage of MSCs were studied in porcine and murine models; a rat modelof acute limb injury was used to further evaluate tissue targeting. Treatment with pronase to detachcell surface molecules and the effect on stem cell potential was assessed in vitro.
Intra-arterial administration of MSCs diminishes their lung deposition; intravasculartransplantation did not cause pulmonary embolisms. Intra-arterially transplanted BM-MNCs didnot reach the brain in significant numbers. Transient proteolytic modification of MSCs withpronase decreased lung accumulation and tissue targeting without affecting their therapeuticcharacteristics.
Intra-arterial transplantation increases lung passage of MSCs. Although thromboembolicevents were not observed, further studies are warranted to ensure the safety of this route of MSCdelivery. Pronase detachment is a promising method to enhance the potential of systemic MSCtherapies.
Keywords: biodistribution, imaging, lung entrapment, stem cells
Mäkelä, Tuomas, Luuytimen mesenkymaalisten kantasolujen systeeminenannostelu. Koe-eläintyö kudosjakautumisesta ja -kohdentumisestaOulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Kliinisenlääketieteen laitos, Kirurgia, Anestesiologia; Biolääketieteen laitos, Anatomia ja solubiologia;Diagnostiikan laitos, Radiologia; Kliinisen tutkimuksen keskusActa Univ. Oul. D 1271, 2014Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Luutytimen mesenkymaaliset kantasolut (MSC) ja mononukleaariset solut (BM-MNC) ovatosoittautuneet tehokkaiksi useissa kliinisissä käyttöaiheissa. Solujen systeeminen annosteluverenkiertoon olisi käytännön kannalta paras soluterapian toteutukseen, mutta solujen merkittä-vä taipumus jäädä keuhkoihin loukkuun ja veritulppariski muodostavat haasteen. Keuhkohakeu-tumisen tiedetään ainakin osin johtuvan solujen pintamolekyyleistä ja näiden muokkaaminenvoisi parantaa solujen keuhkoläpäisevyyttä.
Tutkimuksessa käytettiin koe-eläimenä sikaa, jolle istutettiin systeemisesti allogeenisiamesenkymaalisia kantasoluja tai autologisia luuytimen mononukleaarisia soluja; solujen kudos-hakeutumisen seuranta toteutettiin isotooppileimauksella- ja kuvannuksella. Tutkimuksessa arvi-oitiin annostelureitin vaikutusta keuhkoläpäisevyyteen, solujen kudojakautumista, toimenpiteenturvallisuutta sekä mononukleaarisolujen hakeutumista vaurioituneeseen aivokudokseen. Pro-naasikäsittelyn vaikutusta mesenkymaalisten kantasolujen keuhkoläpäisevyyteen arvioitiin sika-ja hiirimallissa; rotan raajavauriomallia käytettiin lisäksi pronaasin kudoshakeutumisvaikutus-ten arvioimiseen. Pronaasikäsittelyn vaikutuksia solujen pintarakenteisiin ja toiminnallisuuteenarvioitiin in vitro- kokeissa.
Mesenkyymalisten kantasolujen annostelu valtimonsisäisesti paransi solujen keuhkoläpäise-vyyttä; tutkimuksissa käytetyt solut eivät aiheuttaneet keuhkoveritulppia. Valtimonsisäisestiannostellut mononukleaarisolut eivät hakeutuneet vaurioituneeseen aivokudokseen sikamallissa.Pronaasikäsittely muovasi solujen pintaproteiineja palautuvasti ja tämä lisäsi huomattavastimesenkymaalisten kantasolujen keuhkoläpäisevyyttä ja kudoshakeutumista vaikuttamatta solu-jen toiminnallisuuteen.
Mesenkymaalisten kantasolujen annostelu valtimonsisäisesti voi parantaa solujen keuhkolä-päisevyyttä. Tutkimuksessa ei todettu keuhkoveritulppaa tai muita tromboembolisia tapahtumia,mutta lisätutkimuksia tarvitaan MSC- terapian turvallisuuden takaamiseksi. Pronaasikäsittelyntulokset mesenkymaalisten kantasolujen systeemisen annostelun parantamisessa olivat lupaavia.
Asiasanat: kantasolut, keuhkoläpäisevyys, kudosjakautuminen, kuvantaminen
to Kristiina
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Acknowledgements
The present study has been conducted at the Cardiothoracic Research Laboratory
of the Department of Surgery, Oulu University Hospital, during the years 2007-
2014.
I am most grateful to my professors, Petri Lehenkari and Tatu Juvonen, for
their continued support during this project. Closely collaborating with Professor
Lehenkari has been a true pleasure. I consider Professor Lehenkari to be an
excellent and innovative scientist; however, the most striking feature about him is
his ability to create hope where none exists. I am most thankful to Professor
Juvonen for recruiting me to his renowned group of experimental surgeons. I have
had the privilege to benefit from his support and his long-time experience with
experimental models, as well as the technical knowledge of his well-established
research group.
I am also most grateful to Professor Yrjö Konttinen and Docent Susanna
Miettinen for acting as the pre-examiners of this manuscript. Finding experts to
review such a specific research subject is not easy and I was fortunate enough that
they agreed to take on this project. Their comments greatly improved this work. I
would also like to thank the members of my follow-up group for their valuable
contribution, Docent Jari Satta and Docent Pekka Rainio.
This project required the effort, sacrificing of free time, patience, brainpower and
skill of many colleagues. I consider that working long hours using such a
demanding experimental model is the ultimate form of teamwork. I would like to
thank Fredrik Yannopoulos, Kirsi Heikkinen, Oiva Arvola, Henri Haapanen and
Johanna Herajärvi for their contributions to this project and the sacrifices that
they made. With the right people, even the most challenging tasks can be
accomplished with ease (we even managed to have a laugh every now and then).
I am also grateful to the older colleagues of our experimental group, Jussi
Mäkelä, Hanna Jensen, Eija Rimpiläinen and Sanna Meriläinen, with whom I had
the privilege to work on their projects. They taught me the principles behind our
experimental models and adopted me as one of their own when I first arrived to
the lab. I also wish to thank our group’s senior reseachers for their valuable
assistance on the project. Docent Vesa Anttila, docent Kai Kiviluoma and
Professor Roberto Blanco all offered expertise in their own clinical fields, which
greatly benefited this project.
I would especially like to thank Reijo Takalo for his collaborative efforts and
expertise in radionuclide medicine. Creating our experimental model and
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interpreting the data was demanding, but noteworthy. Aira Karjalainen and Leila
Mäkelä, both staff members of the Radionuclide Department, offered their
precious assistance. The rest of the radionuclide department also deserves
recognition for tolerating the demanding arrangements for the experiments that
Aira and Leila had to conduct in addition to their daily routines. I am grateful to
the Department of Radiology and Lauri Ahvenjärvi in particular, for arranging the
safety control imaging studies.
Our collaborators in the Finnish Red Cross Blood Service, Docent Erja
Kerkelä and Johanna Nystedt, along with their local team, deserve my deepest
gratitude. Their in-depth knowledge of the bone marrow therapeutic field elevated
our study design and the quality of our written products to a whole new level. It
was an honour for me to collaborate with their team on the publication of my
pronase modification studies.
The entire team of our stem cell laboratory deserves my deepest thanks for
the work involving cell cultivation and processing. Docent Siri Lehtonen, Minna
Savilampi, Henna Ek and Elisa Lehtilahti, your adaptability to our demanding
schedules was amazing and you were always able to provide high-quality cell
products for use in our experiments. Pasi Lepola deserves thanks for carrying out
the electrophysiologic analysis with such a professional touch, despite the long
working hours. Pasi Ohtonen and Risto Bloigu are thanked for their expert
statistical consultation.
A very special thanks goes to Seija Seljänperä for acting as the mother of our
small research family. Docent Hanna-Marja Voipio, Sakari Laaksonen, Veikko
Lähteenmäki and the other members of the Animal Research Centre staff are
warmly acknowledged for providing use of their fine facility, along with their
skilled animal care.
I am grateful to all my friends for their precious company during all these
years and for offering a chance to relax and participate in athletic activities
together. My thanks also go to my current working colleagues in the Lappish
Central Hospital’s Department of Surgery for providing a supportive atmosphere
while I was completing this project and my surgical residency simultaneously.
Pirkko, Mikko, Matti, Kirsti, Sonja, Konsta and Milja, my beloved parents
and family, and not to forget my dear in-laws Marjatta, Heikki and Tommi, thank
you for your endless support. To have all of you in my life is bliss.
Finally, my deepest and sincerest thanks goes to the one person that has
sacrificed even more for this project than I, Kristiina, my love. Countless times
we have had to plan our life according to the demands of this project, with
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countless hours having to be spent apart. Still, you never lost hope and always
provided constant support. Even more amazingly, you still agreed to be my wife.
To have you by my side makes me the happiest man alive. You are the one who
has made all this possible.
This work was financially supported by the Finnish Red Cross Blood Service
and the Finnish Foundation for Cardiovascular Research.
Oulu, October 2014 Tuomas Mäkelä
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Abbreviations
APC Antigen presenting cells
BBB Blood brain barrier
BCAS Bilateral common carotid artery stenosis
BDNF Brain-derived neurotrophic factor
BNGF β-nerve growth factor
BM-MNC Bone marrow mononuclear cell
CBF Cerebral blood flow
CLSM Confocal laser scanning microscopy
CPB Cardio-pulmonary bypass
CSPG-4 Chondroitin sulfate proteoglycan-4
CT Computed tomography
DC Dendritic cells
ECG Electrocardiography
EEG Electroencephalography
ELISA Enzyme-linked immunosorbent assay
EPC Endothelial progenitor cells
ESC Embryonic stem cells
FACS Fluorescent-activated cell sorting
FN Fibronectin
GM-CSF Granulocyte-macrophage colony-stimulating factor
Gmean Geometric mean
GvHD Graft versus host disease
HCA Hypothermic circulatory arrest
HGF Hepatocyte growth factor
hMSC Human marrow stromal cell (mesenchymal stem cell)
mMSC Mouse marrow stromal cell
HSC Hematopoietic stem cell
hUC-MSC Human umbilical cord-derived marrow stromal cell
IA Intra-arterial
IBMIR Instant blood-mediated inflammatory reaction
IDO Indoleamine 2,3-dioxygenase
IV Intravenous
MCA Middle cerebral artery
MCAO Middle cerebral artery occlusion
mDC Myeloid dendritic cells
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MI Myocardial infarction
MRI Magnetic resonance imaging
NGF Nerve growth factor
NK Natural killer cells
NPC Neural progenitor cells
NSC Neural stem cells
MSC(s) Marrow stromal cell (mesenchymal stem cell)
OI Osteogenesis imperfecta
OPC Oligodendrocyte progenitor cells
pDC Plasmacytoid dendritic cells
PELOD Pediatric logistic organ dysfunction score
PET Positron emission tomography
PID Post-ischemic day
ROI Region of interest
SDF-1 Stromal cell-derived factor-1α
SPECT Single photon emission tomography
SPECT/CT Single photon emission tomography- computed tomography
combination
Tc99m-HMPAO Technetium99m-hydroxymethylpropylene amine oxime
TBI Traumatic brain injury
TF Tissue factor
TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP-biotin
nick-end labelling
VOI Volume of interest
15
List of original publications
This thesis is based on the following articles, which are referred to in the text by
their Roman numerals:
I Mäkelä T, Takalo R, Arvola O, Haapanen H, Yannopoulos F, Blanco R, Ahvenjärvi L, Lehtonen S, Kiviluoma K, Kerkelä E, Nystedt J, Juvonen T & Lehenkari P. (2014) Safety and Biodistribution Study of Bone Marrow Derived Mesenchymal Stromal Cells and Mononuclear Cells and the Impact of the Administration Route in an Intact Porcine Model. Manuscript
II Mäkelä T, Yannopoulos F, Alestalo K, Mäkelä J, Lepola P, Anttila V, Lehtonen S, Kiviluoma K, Takalo R, Juvonen T & Lehenkari P. (2013) Intra-Arterial Bone Marrow Mononuclear Cell Distribution after Experimental Global Brain Ischemia Scand Cardiovasc J. 47(2):114-20.
III Kerkelä E, Hakkarainen T, Mäkelä T, Kilpinen L, Lehtonen S, Ritamo I, Pernu R, Pietilä M, Takalo R, Nikkilä J, Tikkanen J, Juvonen T, Laitinen S, Valmu L, Lehenkari P & Nystedt J. (2013) Transient Proteolytic Modification of Mesenchymal Stromal Cells Decreases Lung Entrapment in vivo. Stem Cells Translational Medicine. 2(7):510-20.
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Table of Contents
Abstract
Tiivistelmä
Acknowledgements 9 Abbreviations 13 List of original publications 15 Table of Contents 17 1 Introduction 21 2 Review of the literature 23
2.1 Stem cells ................................................................................................ 23 2.1.1 Bone marrow stromal cells ........................................................... 23 2.1.2 Bone marrow mononuclear cells .................................................. 24
2.2 Mechanisms behind MSC therapeutic properties .................................... 25 2.2.1 Paracrine effects of MSCs ............................................................ 26 2.2.2 Immunomodulation ...................................................................... 28 2.2.3 Effects on T-cells .......................................................................... 30
2.3 Clinical applications of MSCs ................................................................ 32 2.3.1 Ischemic brain injury and stroke .................................................. 33 2.3.2 Other indications .......................................................................... 40
2.4 Intravascular transplantation of bone marrow-derived cells ................... 42 2.4.1 The role of direct transplantation .................................................. 43 2.4.2 Passive biodistribution after intravascular transplantation ........... 44 2.4.3 Active biodistribution by chemotaxis and migration .................... 44 2.4.4 Biodistribution tracking methods ................................................. 45 2.4.5 Acute phase biodistribution .......................................................... 49 2.4.6 Long-term biodistribution ............................................................ 50
2.5 Challenges of intravascular MSC transplantation ................................... 50 2.5.1 Periprocedural damage to MSCs .................................................. 51 2.5.2 Pulmonary adhesion and the lung barrier ..................................... 54 2.5.3 Thromboembolic events ............................................................... 55 2.5.4 Immunologic reactions ................................................................. 58 2.5.5 Development of malignancies ...................................................... 59 2.5.6 Ectopic tissue formation ............................................................... 59
2.6 Optimisation of intravascular MSC transplantation ................................ 60 2.6.1 Type of injury ............................................................................... 60 2.6.2 Cell type ....................................................................................... 61
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2.6.3 Systemic transplantation route ...................................................... 62 2.6.4 Timing of transplantation ............................................................. 62 2.6.5 Cell dose ....................................................................................... 63 2.6.6 Pronase detachment ...................................................................... 65 2.6.7 Vasodilatators ............................................................................... 66 2.6.8 Anticoagulative agents ................................................................. 66 2.6.9 Cellular engineering and culture conditions ................................. 67
3 Aims of the study 69 4 Materials and methods 71
4.1 Experimental animals (Studies I, II, III).................................................. 71 4.2 Perioperative protocol of porcine model (Studies I, II and III) ............... 71 4.3 Porcine MSCs and BM-MNCs (Studies I, II and III) .............................. 72 4.4 Human MSCs (Study III) ........................................................................ 72 4.5 Rat MSCs (Study III) .............................................................................. 73 4.6 Pronase detachment and cell viability (Study III) ................................... 73 4.7 Cell surface analysis by mass spectrometry (Study III) .......................... 73 4.8 Cell surface analysis by flow cytometry (Study III) ............................... 74 4.9 T-cell proliferation assay (Study III) ....................................................... 74 4.10 Angiogenesis assay (Study III) ............................................................... 75 4.11 In vitro rolling assay (Study III) .............................................................. 75 4.12 Labelling of the porcine cells (Studies I, II and III) ................................ 75 4.13 Intravenous transplantation (Studies I, II and III) ................................... 76 4.14 Intraventricular transplantation (Study I) ................................................ 76 4.15 Ischemic brain insult (Study II) ............................................................... 77 4.16 Radionuclide imaging (Studies I, II and III) ........................................... 77 4.17 Image and data analysis (Studies I, II and III) ........................................ 77 4.18 Pulmonary embolism CT- scan (Study I) ................................................ 78 4.19 Tissue biopsies (Studies I, II and III) ...................................................... 78 4.20 Electroencephalography (Study II) ......................................................... 79 4.21 Biodistribution of human MSCs in a mouse model (Study III) .............. 79 4.22 Biodistribution of allogeneic BM-MSCs in a rat model of
inflammation (Study III) ......................................................................... 80 4.23 Statistical analysis (Study I, II, and III)................................................... 80
5 Results 83 5.1 IA versus IV transplantation of BM-MNCs and MSCs (Study I) ........... 83
5.1.1 Computed tomography ................................................................. 83 5.1.2 Haemodynamic data ..................................................................... 87
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5.2 BM-MNC transplantation for global ischemic brain injury during
first 24 hours (Study II) ........................................................................... 88 5.2.1 Proportional counts from biopsies ................................................ 89 5.2.2 Haemodynamic and biochemical data .......................................... 93
5.3 Effects of MSC pronase detachment on lung clearance and organ
targeting (Study III)................................................................................. 94 5.3.1 The effect of pronase detachment on cell-surface protein
epitopes......................................................................................... 94 5.3.2 MSC Functionality ....................................................................... 95 5.3.3 MSCs and lung clearance ............................................................. 96 5.3.4 MSC targeting to an area of injury ............................................... 97
6 Discussion 99 6.1 Biodistribution after intravascular transplantation .................................. 99 6.2 Lung entrapment ................................................................................... 100 6.3 The effect of transplantation route on biodistribution ........................... 101 6.4 Safety assessment .................................................................................. 101 6.5 IA-transplanted BM-MNCs’ lack of homing to ischemic brain ............ 103 6.6 The effect of transplantation timing on homing .................................... 104 6.7 The effect of pronase detachment on MSC lung clearance ................... 105 6.8 Clinical applications .............................................................................. 107 6.9 Limitations of the study ........................................................................ 109 6.10 Summary ................................................................................................110
7 Conclusion 111 References 113 Original publications 129
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1 Introduction
Treatment with stem cells is one of the most promising novel forms of therapies
and is slowly gaining acceptance into clinical practice. An increasing number of
studies have documented successful therapeutic applications of mesenchymal
stromal cells (MSCs) and bone marrow mononuclear cells (BM-MNCs) in both
preclinical and clinical settings. Restorative and immunomodulatory properties of
these cells have debuted in the treatment of various socio-economically high-
impact clinical conditions, such as ischemic brain injury.
Previous research has been understandably directed towards proving the
therapeutic effectiveness of these cells in various clinical conditions and
preclinical success stories have been gradually translated into human use.
However, MSCs remain indisputable as an experimental form of treatment,
because their basic characteristics and behaviour after transplantation are still
incompletely understood.
Intravascular transplantation of MSCs has served as the major delivery route
in this field due to its non-invasiveness and applicability in a variety of clinical
conditions. Researchers have faced unexpected problems with seemingly simple
intravenous and intra-arterial administration of cells, such as undesirable patterns
of organ distribution and failure to reach the therapeutic target. Furthermore,
alarming reports of possible adverse effects resulting from embolus formation
have also been reported. Before MSCs are ultimately able to enter routine clinical
practice as a therapeutic tool, these issues have to be thoroughly researched and
resolved.
The theoretical potential of MSCs, as well as other stem cells, is undeniable
and we are approaching a time when these cells will become available as a
standard therapeutic choice. However, we already have knowledge of what MSCs
can do; now it is of the utmost importance to explore how to actually use them.
This study evaluates the issues of tissue targeting and safety associated with
systemic transplantation of MSCs and offers novel solutions to enhance this form
of cellular therapy. MSC therapy has been investigated in multiple therapeutic
indications, all of which have shared, but also indication-specific, challenges.
Ischemic brain injury is a particularly interesting application of MSCs, as major
issues have been observed when applying MSCs for this indication. Ischemic
brain injury was therefore chosen as an example of an experimental target of the
therapeutic cells.
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2 Review of the literature
2.1 Stem cells
Stem cells are capable of self-renewal and multi-lineage differentiation (Till &
McCulloch 1961). As a result of these properties, stem cells are able to
differentiate into multiple cellular lines, while renewing themselves. Two main
types of stem cells exist: (1) embryonic stem cells, which are found during the
early stages of development, appearing in the inner mass of blastocysts, and (2)
adult stem cells, which can be isolated from various tissues and have a more
limited differentiation capacity. Also, embryonic fetal cells from the genital ridge
and tissue-specific sites can be considered as separate stem cell categories
(Arvidson et al. 2011).
Plastic potency of stem cells determines the capacity of the stem cell
populations to differentiate into other cell types. Totipotent cells are capable of
differentiating into all embryonic and non-embryonic cell types of the human
body and are formed after fusion of spermatocytes and oocytes (Tarkowski 1959).
Totipotent stem cells are able to create an entire organism. Pluripotent stem cells,
which also appear during early stages of embryonic development, are capable of
differentiating into any cell type like totipotent stem cells, but lack the capacity of
forming an entire organism by themselves. Multipotent stem cells are a more
differentiated type of stem cell having less plastic capacity, giving rise to a
limited number of cells of a certain tissue type, such as blood cells or neural cells.
Totipotent, pluripotent and multipotent stem cells arise at different stages of
embryonic development and generally specialise further as a function of time,
with their plastic capacity gradually diminishing.
Adult stem cells are non-specialised multipotent cells present in specialised
tissue that are capable of renewing themselves and forming types of cells of the
tissue from which they originate (Till & McCulloch 1961). Examples of adult
stem cell types include hematopoietic (HSC) (Till & McCulloch 1961, Becker et al. 1963) and neural stem cells (Stemple & Anderson 1992)
2.1.1 Bone marrow stromal cells
It has been discovered that bone marrow serves as a source of non-haematopoietic
multipotent stem cells, called bone mesenchymal stem cells, or now more
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commonly known as marrow stromal cells (MSCs) (Caplan 1991). This
population of cells was originally described by Friedenstein et al. and were first
identified as fibroblast colonies (Friedenstein et al. 1970). These cells are
multipotent stem cells capable of differentiating into osteoblasts, chondrocytes,
adipocytes and myoblasts (Pittenger et al. 1999, Prockop 1997). There is also
evidence that MSCs can even differentiate into astrocytes (Kopen et al. 1999),
glial cells (Sanchez-Ramos et al. 2000) and neurons (Deng et al. 2001, Mezey et al. 2000, Sanchez-Ramos et al. 2000, Woodbury et al. 2000).
There still does not exist a single MSC marker or identifiable characteristic,
which could be used for their unequivocal identification and isolation. Therefore,
there is some controversy as to how to define MSCs, but the commonly accepted
minimum criteria for MSCs include the ability to adhere to plastic and the
potential for in vitro trilineage differentiation into adipogenic, chondrogenic, and
osteogenic cells. In addition, MSCs must also display CD105, CD73 and CD90,
and lack CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface
molecules (Dominici et al. 2006).
Although bone marrow is the first described and mostly commonly used
source of MSCs in clinical and preclinical applications, several other tissues serve
as a source of MSCs, including adipose tissue (Zuk et al. 2002), human placenta
(In't Anker et al. 2004), skin (Shih et al. 2005), human umbilical cord blood
(Erices et al. 2000), human breast milk (Patki et al. 2010), dental pulp (Gronthos et al. 2000), amniotic fluid (Nadri & Soleimani 2007), umbilical cord
perivascular cells (Sarugaser et al. 2005), human umbilical cord Wharton’s jelly
(Wang et al. 2004) and the synovial membrane of the knee joint (De Bari et al. 2001). The MSCs acquired from different sources are likely to have somewhat
varying differentiation potential and cell characteristics.
2.1.2 Bone marrow mononuclear cells
The mononuclear cell fraction isolated from bone marrow aspirates is commonly
referred to as bone marrow mononuclear cells (BM-MNCs). The mononuclear
fraction is a heterogenous mixture containing several populations of stem cells,
such as hematopoietic stem cells, endothelial progenitor cells, MSCs and others
with a greater differentiation capacity (Cuende et al. 2012).
Although BM-MNCs are not technically stem cells, as the population contains
mostly cell types other than MSCs, they are utilized in the same manner as
cultivated MSCs. The main reason use of BM-MNCs have become so popular in
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this field of study is their proven effectiveness in treating various conditions
(including ischemic brain injury) (Iihoshi et al. 2004, Kamiya et al. 2008, Fujita et al. 2010, de Vasconcelos Dos Santos et al. 2010), their easy availability
through a relatively non-invasive puncture procedure and the possibility of
isolating this fraction in just a few hours, which allows cellular interventions in
acute conditions without the need of expansion in cell cultures. The possibility of
rapid isolation and re-transplantation makes BM-MNCs a competitive choice in
various clinical conditions, where better therapeutic results could be expected by
avoiding delays (Chen et al. 2001b, Barbash et al. 2003, Iihoshi et al. 2004, de
Vasconcelos Dos Santos et al. 2010, Yang et al. 2011). In addition, isolation and
re-transplantation is inherently autologous, thus evading a number of possible
immunologic, infection, prion and xenogenic culture medium issues compared to
transplantation of cultured allogeneic or xenogeneic donor cells.
This review concentrates on research conducted using MSCs. Although
studies were also conducted using BM-MNCs, the results obtained are not
directly translatable to MSCs due to the different composition of these two
cellular populations. However, if positive results are shown with MSCs, it can be
hypothesised that BM-MNCs could also show similar therapeutic potential in that
particular clinical disorder. Application of BM-MNCs instead of MSCs should be
considered if rapid deployment of the cellular product is required due to the
nature of the event being treated (e.g. acute ischemic brain injury).
2.2 Mechanisms behind MSC therapeutic properties
A common belief with MSC therapy is that they, as cells capable of division and
transdifferentiation, could serve as a replacement for damaged cells, thereby
forming new tissue (Salem & Thiemermann 2012). If MSCs could be shown to
have the ability to differentiate into functional cells of the damaged, or partially
lost and thus cell-depleted target organs (e.g. cardiomyocytes in myocardial
infarction as reported by Orlic et al. 2001), they could theoretically regenerate
new tissue, thereby restoring normal organ function. However, in case of
myocardial infarction, present studies have challenged this theory because
although the transplanted MSCs start to express certain characteristics of native
cardiomyocytes, they do not differentiate into fully mature, functional
cardiomyocytes (Rose et al. 2008).
Using ischemic brain injury as an example of a therapeutic target of MSCs,
recent studies have reported the localization of only very low numbers of cells in
26
ischemic brain areas during the subacute phase of the developing brain injury
(Harting et al. 2009, Keimpema E. et al. 2009). Poor survival of transplanted
MSCs has been reported (Coyne et al. 2006). Although bone marrow MSCs are
capable of differentiating into astrocytes (Kopen et al. 1999), glia (Bossolasco et al. 2005) and neurons (Mezey et al. 2000, Sanchez-Ramos et al. 2000, Woodbury et al. 2000, Deng et al. 2001, Bossolasco et al. 2005), it appears that the
transplanted MSCs found in the lesional area express neurotrophic factors, but do
not become fully active neurons; however, they may still have therapeutic
potential (Koh et al. 2008). Interestingly, even the transient presence of a small
number of MSCs resulted in a significant reduction in the size of a focal ischemic
brain lesion (Keimpema E. et al. 2009). It thus seems unlikely that the restorative
effect of stem cells is largely based on direct reformation of tissue by the
transdifferentiation of the stem cells at the site of injury.
This theory is supported by a common observation in experimental studies
that the beneficial effects observed after stem cell transplantation are seen far
earlier in ischemic lesions than would be possible by tissue regeneration mediated
by any cell (reviewed by Chopp & Li 2002). Stem cell differentiation and
regeneration may be partially responsible for the therapeutic benefit of MSCs, but
mechanisms other than differentiation and cellular replacement are currently
considered to be of fundamental importance.
2.2.1 Paracrine effects of MSCs
The function of a living organism is based on cellular signalling. Cells constantly
adapt and react to fluctuations in their microenvironments, such as differences in
oxygen content and pH. Cells also react to or receive signals that are generated by
themselves in an intra- or autocrine mode, or by other cells of the organism. Cells
can interact with adjacent cells via direct cell-to-cell contact, which is called
juxtacrine signalling. Cells can also interact with other cells over long distances
by secreting soluble factors. The term paracrine signalling involves local
interactions, while the term endocrine signalling involves systemic interactions.
Secretion of salivary peptide hormones, which affects the gastrointestinal tract, is
an example of an exocrine mode of action. The transmitters of cellular signalling
are mediated by a variety of types of molecules, such as hormones,
neurotransmitters and cytokines. In addition to soluble molecules, cells can also
use direct cell-to-cell and cell-to-extracellular matrix contacts for signalling with
27
their environment. Furthermore, various physiochemical factors play important
roles in regulating cellular behaviour (Kaivosoja et al. 2012).
Cumulative evidence suggests that the effects of MSC therapy, at least in
part, arise from indirect paracrine mechanisms (Salem & Thiemermann 2012). It
has even been shown that administration of medium conditioned with human
MSCs is able to reduce infarct size in a porcine model (Timmers et al. 2007). It is
likely that the paracrine effects of MSCs arise from multiple functioning secreted
substances rather than a single one. This collection of MSC-derived mediators
has recently been referred to as the MSC secretome (Ranganath et al. 2012).
Angiogenesis
In preclinical studies, BM-MNCs have been shown to increase vascular
endothelial growth factor (VEGF) concentration in global (Fujita et al. 2010) and
focal (Chen et al. 2003) ischemic brain injury. VEGF secretion, which promotes
angiogenesis, has been suggested to be a potential mechanism in bone marrow-
derived cell-induced brain protection. VEGF singularly has been shown to affect
the functional outcome in an animal model of cerebral ischemia and was found to
increase blood-brain-barrier (BBB) permeability (Zhang et al. 2000). MSCs have
also been shown to induce angiogenesis in ischemic myocardium (Martens et al. 2006). VEGF-induced angiogenesis could thus reasonably account for some of
the MSC therapeutic properties. Additionally, the number of Ki-67 positive
proliferating cells increased in a model of ischemic brain injury, implying the
induction of angiogenesis (Shen et al. 2006).
Neuroregenerative effects
MSC administration has been demonstrated to increase synaptophysin expression
and the number of oligodendrocyte progenitor cells in the ischemic border or
twilight zone, resulting in improved recovery, which suggests facilitation of
neuronal remodelling (Shen et al. 2006). MSCs may exhibit anti-apoptotic
properties and can protect neurons against apoptotic stress by stimulation of
endogenous survival signalling in neurons (Isele et al. 2007). Human MSCs have
been demonstrated to secrete brain-derived neurotrophic factor (BDNF) (Chen et al. 2002, Crigler et al. 2006), nerve growth factor (NGF) (Chen et al. 2002,
Crigler et al. 2006) and hepatocyte growth factor (HGF) (Chen et al. 2002). It has
been suggested that the neurorestorative effects of MSCs are also based on other
28
factors, in addition to BDNF and NGF, as the effects observed in vitro are more
prominent than those which could be explained by the concentrations of these
secreted neurotrophic factors. Indeed, MSCs have been shown to express a large
number of other neuroregulatory factors (Crigler et al. 2006). MSCs may also be
able to stimulate endogenous neural stem cells (Galindo et al. 2011).
Fig. 1. Marrow stromal cell immunomodulatory effects on different immune cell types.
(original figure by Petri Lehenkari).
2.2.2 Immunomodulation
MSCs affect the immunologic system in a number of different ways, most of
which suppress the system. This phenomenon can be exploited in numerous
therapeutic applications in which the MSC therapy targets, at least partially,
pathological conditions mediated by the innate and adaptive immune system.
These conditions occur in various forms of ischemic organ injury, where a major
proportion of the injury is due to reperfusion of the organ after cessation of blood
29
flow, resulting in a hyper-inflammatory response caused by an influx of white
blood cells (Rao et al. 2014). Graft-versus-host-disease (GvHD) and organ
transplantation rejection are somewhat more obvious targets of MSC therapy,
because the immune system is responsible for the pathogenesis of these
conditions.
Mediators of MSC immunologic effects
It is commonly accepted in the literature that many of the immunomodulatory
effects of MSCs are based on direct cell-to-cell contact or the secretion of soluble
factors. Candidates for MSC mediators are many and it is likely that their
influence is based on a collection of different soluble and cell membrane-bound
substances, rather than a single entity, or in other words, on the MSC secretome
(Ranganath et al. 2012).
Many of the immunomodulatory effects of human MSCs have been
suggested to be mediated by secretion of prostaglandin E2 (PGE2) (Aggarwal &
Pittenger 2005). Interleukin-10 (IL-10) has been suggested as another potential
MSC-derived immunomodulator (Beyth et al. 2005). Interferon-γ (IFN-γ)
production by Th1 type T-cells and NK-cells has been reported to be necessary for
upregulation of indoleamine 2,3-dioxygenase (IDO) in MSCs, leading to
antiproliferative and immunosuppressive effects (Krampera et al. 2006).
Inducible cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS)
are typical enzymes upregulated in inflammatory conditions. Exposure of
astrocytes to MSCs has been shown to substantially downregulate their
expression (Schafer et al. 2012). Tumor necrosis factor-α (TNFα), interleukin-1β
(IL-1β) and interleukin-6 (IL-6) are key proinflammatory cytokines and
conditioning astrocytes with MSCs significantly attenuates their expression,
together with downregulation of anti-inflammatory IL-10, but not anti-
inflammatory transforming growth factor-β (TGFβ) (Schafer et al. 2012). The
role of TGFβ remains controversial, and some studies have reported that it does
not affect immunomodulation (Beyth et al. 2005).
Systemic inflammation
MSCs have been shown to attenuate systemic inflammatory responses in rodent
models of burn wound and lipopolysaccharide- induced endotoxemia (Yagi et al. 2010a). Systemic immunosuppressive effects of MSCs can be due to decreases in
30
serum IFN-γ and increases in IL-10 levels (Parekkadan et al. 2008). Lee et al. demonstrated that hMSCs applied in a mouse model of myocardial infarction
were trapped in the lungs and were activated to secrete anti-inflammatory tumor
necrosis factor-stimulated gene 6 protein (TSG-6). These investigators
demonstrated that secretion of this factor suppressed the inflammatory response
to myocardial infarction without a significant effect on cell migration to the
infarcted heart (Lee et al. 2009c).
However, controversial data about the immunomodulatory effects of MSCs
exist. In a study conducted by Hoogduijn et al. in a mouse model, adipose tissue-
derived MSCs targeted the lungs and induced an inflammatory response, with an
increased expression of monocyte chemoattractant protein-1 (MCP-1), IL1-β, and
TNF-α, as well as an increased macrophage accumulation in lung tissue 2 h after
MSC infusion. They also reported increased serum levels of proinflammatory IL-
6 and chemokines CXCL1 (with a CXC motif) and MCP1, demonstrating
systemic immune activation after MSC infusion. MCP1 and TNF-α levels
increased in liver tissue 4 h after MSC infusion without their migration to the
liver. Only a mild effect on expression of the anti-inflammatory cytokines, TGF-
β, IL-4, and IL-10, was observed. However, three days after MSC infusion the
mice developed a milder inflammatory response to lipopolysaccharide, suggesting
that the in vivo immunomodulatory effects of MSCs originated from an
inflammatory response that were induced by the infusion of MSCs, which were
then followed by a phase of reduced immune reactivity (Hoogduijn et al. 2013).
2.2.3 Effects on T-cells
T lymphocytes or T cells constitute the central white blood cell type associated
with cell-mediated immunity. This class of cells is comprised of multiple
subtypes, each of which has their particular role in the immune system. CD4+ T-
helper-cells, the main mediators of adaptive immunity, assist other cells of the
immune system in their processes and are responsible for general activation of the
immune system following presentation with suitable antigens. Subtypes of CD4+
T-helper-cells include Th1-cells, which support cell-mediated immunity, and Th2-
cells, which support antibody responses while inhibiting several functions of
phagocytic cells. CD4+ regulatory T-cells (Treg) maintain self-tolerance and
suppress reactions to weak immunogenic stimuli by inhibiting T-helper-cell
functions. CD8+ T-cells are cytotoxic cells responsible for destroying other cells,
such as virally infected cells or cells that have undergone malignant
31
transformation, and have been implicated in the rejection of allogeneic transplants
due to the histo-incompatibility between the host and donor organ.
MSCs decrease T-cell proliferation and the percentage of IFN-γ-producing
Th1 T-cells, but do not affect their activation or cytotoxic effector function
(Ramasamy et al. 2008). Some studies have demonstrated that MSCs decrease
IFN-γ secretion by T-cells (Aggarwal & Pittenger 2005, Beyth et al. 2005).
Human MSCs can significantly increase IL-4 production of Th2 cells; MSCs also
increase Th2 differentiation of naïve Th0 T-cells (Aggarwal & Pittenger 2005).
MSCs suppress T-cell proliferation (Bartholomew et al. 2002, Di Nicola et al. 2002, Augello et al. 2005, Krampera et al. 2006), as well as proliferation of both
CD4+ and CD8+ T cells (Di Nicola et al. 2002, Krampera et al. 2006) (Glennie et al. 2005), but some evidence implies stronger suppression of CD8+ than CD4+ T
cells (Krampera et al. 2006). IL-10 has been proposed as a mediator of this effect
and an indirect mechanism has been suggested, whereby T-cell suppression is
established by induction of tolerogenic antigen-presenting cells (APCs) that limit
T-cell activation (Beyth et al. 2005). There is also evidence that T-cell activation
is not suppressed, only proliferation (Glennie et al. 2005). Human MSCs have
been shown to increase the proportion of CD4+CD25+Tregs (Aggarwal & Pittenger
2005).
Direct cell contact is not seen as a requirement for the suppression of T-cell
proliferation (Di Nicola et al. 2002, Krampera et al. 2006). IFN-γ production by
T-cells is reported to be required for MSC IDO upregulation, leading to
antiproliferative and immunosuppressive effects (Krampera et al. 2006). The
exact mechanisms by which MSCs regulate T-cell function remain partially
unsolved, but strong evidence implies suppression of T cell proliferation, thus
explaining the therapeutic results shown in T cell-mediated clinical conditions,
such as reperfusion injury or GvHD.
Effects on natural killer (NK) cells
Human MSCs have been shown to inhibit IFN-γ secretion by NK cells (Aggarwal
& Pittenger 2005) and also their proliferative response to allogeneic stimuli is
diminished (Krampera et al. 2006). IFN-γ production by NK cells has been
reported to be required for MSC IDO-upregulation, leading to antiproliferative
effects (Krampera et al. 2006).
32
Effects on B cells
MSCs suppress B cell proliferation (Augello et al. 2005), but some studies have
also reported no effect on the B cell proliferative response to allogeneic stimuli
(Krampera et al. 2006). MSC-mediated immunosuppression also affects B cell
proliferation in response to CD40L and IL-4 (Glennie et al. 2005).
Effects on antigen-presenting cells
MSCs reduce TNF-α secretion by activating mature myeloid dendritic cells
(mDCs) and increasing IL-10 secretion of mature plasmacytoid dendritic cells
(pDCs) (Aggarwal & Pittenger 2005). MSCs have been shown to condition DCs
and other APCs to suppress T cell activation (Beyth et al. 2005).
Human MSCs have been shown to suppress initial differentiation of monocyte-
derived DCs produced from CD14+ monocytes reversibly, to suppress moderately
mature monocyte-derived DCs and to inhibit immunostimulation of allogeneic T-
cell proliferation by mature DCs. IL-12 production of DCs was also found to be
suppressed (Jiang et al. 2005).
Variation of immunomodulatory effects by species
The mechanisms of immunomodulation observed are not necessarily directly
translatable to clinical situations because there are differences in the
immunosuppressive mediators that are generated in murine- and human-derived
MSCs (Ren et al. 2009). These findings emphasize the need to carefully consider
preclinical results gained from animal studies on the immunomodulatory
properties of MSCs.
2.3 Clinical applications of MSCs
The main therapeutic potential of MSCs lies in their regenerative and
immunomodulatory capacities. Although the range of theoretical clinical
applications of MSCs is broad, past research has focused on different forms of
ischemic injuries of particular organs, with a heavy emphasis on myocardial
infarction and more recently on ischemic brain injury. The role of MSCs in
ischemic organ injury is not solely due to their regenerative capabilities, as was
initially believed; rather MSC-induced immunomodulation is also recognized as a
33
primary mode of action in protecting organs against ischemic-induced injuries
(Salem & Thiemermann 2012).
The immunomodulatory applications of MSCs represent captivating
possibilities. These cells can be seen as multifunctional bioreactors with paracrine
and systemic mechanisms of action actions (Ranganath et al. 2012). The clinical
applications of this attribute, in addition to ischemic organ injuries, cover a range
of diseases, which are at least to some extent characterised by a lack of effective
treatment, such as osteogenesis imperfecta or steroid-resistant GvHD (Horwitz et al. 1999, Horwitz et al. 2002, Le Blanc et al. 2008). MSCs and other types of
stem cells therefore offer an experimental and auspicious approach to the
treatment of such conditions of unmet medical need.
2.3.1 Ischemic brain injury and stroke
Ischemic brain injury is most commonly caused by acute thromboembolic events
involving the cerebral circulation (i.e., stroke). A number of factors predispose to
this major cause of morbidity and mortality among Western society, most
importantly atrial fibrillation, hypertension and carotid arterial stenosis stenosis
(Rerkasem & Rothwell 2011, Lager et al. 2014). Another important cause of
ischemic brain injury results from head trauma.
A number of patients also consistently suffer from ischemic brain injury
having an iatrogenic origin. Modern cardiac surgery requires cessation of the
heart’s systolic function and systemic blood flow to perform essential surgical
manoeuvres, such as replacement of heart valves or coronary artery bypass
surgery. This systemic circulatory arrest inadvertently results in ischemic injury to
sensitive organs ― most importantly the brain (Likosky et al. 2003), the kidneys
(Zanardo et al. 1994) and the heart itself ― despite protective actions taken. As
this form of ischemic brain injury is caused by routine clinical procedures, all
precautions taken to prevent this complication are of the utmost importance, with
MSCs representing a promising approach for treating this condition.
Most experimental studies considering MSC transplantation for ischemic
brain injury have utilised a rat MCAO model (Table 1). Better outcome in
behavioural tests compared to controls has been proven in a number of studies
using different transplantation methods (Table 1). A long-term follow-up study of
intravenous autologous mesenchymal stem cell transplantation in patients with
ischemic stroke showed a trend to improvement, with no obvious adverse effects
related to the MSC transplantation (Moniche et al. 2012). Clinical improvement
34
was correlated with the plasma SDF-1a level at the time of MSC treatment (Lee et al. 2010b). A rodent study with IV MSC transplantation for TBI reported no
improvement in recovery, as the quantity of cell migration was minimal (Harting et al. 2009).
Ischemic brain injury is a particularly interesting application of MSCs due to
the limited effect that the current forms of treatment can offer. The treatment of
stroke mainly consists of thromobolytic agents, which have the disadvantage of
requiring a narrow therapeutic window for treatment. Antihypertensive treatment,
controlling blood glucose levels and medications designed to lower intracranial
pressure comprise the remaining therapeutic options. Ischemic damage to the
brain can be fatal or leave the patient with severe disabilities, with a dependence
on others; hence, developing approaches that lead to even slightest improvements
in treating these conditions can be seen as worthwhile pursuits.
Increasing evidence of the restorative properties of bone-derived stem cells in
treating various disease models is accumulating and ischemic brain injury is no
exception (Table 1-3), although a large gap to pioneer targets of cellular
therapy ― mainly the heart ― exists. Research utilising stem cells in the treatment
of ischemic heart disease and myocardial infarction is advanced, as randomized
controlled trials have already been attempted (Mathiasen et al. 2012, Karantalis et al. 2014).
The brain as a target of cellular therapy differs from many other organs due to
its complex and delicate cellular structure. To date, the possible effects at the
cellular level are seen as a combination of the regenerative, neuroprotective and
immunomodulatory properties of the MSCs
35
Ta
ble
2. E
xp
eri
me
nta
l in
viv
o s
tud
ies in
ve
sti
ga
tin
g M
SC
th
era
py
fo
r is
ch
em
ic b
rain
in
jury
.
Stu
dy
Mod
el
Rou
te
Gro
ups
Spe
cies
C
ell
num
ber
Tim
ing
Follo
w-
up
Ana
lysi
s O
utco
me
Mig
ratio
n
Bao
et al.
2010
MC
AO
D
irect
inje
ctio
n
hMS
Cs
Con
trol
Rat
5
x 10
5 3
days
28
days
Beh
avio
ural
test
s,
His
topa
thol
ogy,
ELI
SA
, TU
NE
L,
Imm
unoh
isto
chem
istry
Red
uced
infa
rct v
olum
e
Neu
rolo
gic
scor
e im
prov
emen
t
Bor
long
an e
t
al.
2004
MC
AO
D
irect
m
MS
Cs
-5 d
iffer
ent
dose
s
-CB
F or
BB
B
-Pla
cebo
Rat
1,
4, 1
0
or 2
0
x105
Imm
edia
te14
days
Lase
r Dop
pler
, Eva
ns
Blu
e B
BB
ass
ay,
GFP
epi
fluor
esce
nce
mic
rosc
opy,
ELI
SA
Dos
e-de
pend
ent C
BF
and
BB
B
rest
orat
ion,
dos
e-de
pend
ent i
ncre
ase
of g
row
th fa
ctor
s
Che
n et al.
2001
a
MC
AO
D
irect
C
ontro
l
MS
Cs
MS
Cs
+ N
GF
Rat
4
x105
PID
1
14
days
Beh
avio
ural
test
s,
His
topa
thol
ogy,
Imm
unoh
isto
chem
istry
Neu
ral d
iffer
entia
tion
of M
SC
s,
impr
oved
reco
very
Che
n et al.
2001
b
MC
AO
IV
E
arly
/Del
ayed
MS
C/P
lace
bo
Low
/Hig
h
dose
Rat
1
x106
3 x1
06
24h
or
PID
7
7 da
ys
or
35
days
Beh
avio
ural
test
s,
His
topa
thol
ogy,
Imm
unoh
isto
chem
istry
,
CLS
M
Impr
oved
reco
very
with
hig
h do
se
MS
Cs
at P
ID 1
and
PID
7
trans
plan
tatio
n
+
Che
n et al.
2003
MC
AO
IV
hM
SC
s
plac
ebo
Rat
1
x106
1 da
y 14
days
ELI
SA
, His
topa
thol
ogy,
Imm
unoh
isto
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istry
Incr
ease
d V
EG
F, in
crea
sed
angi
ogen
esis
36
Stu
dy
Mod
el
Rou
te
Gro
ups
Spe
cies
C
ell
num
ber
Tim
ing
Follo
w-
up
Ana
lysi
s O
utco
me
Mig
ratio
n
Det
ante
et
al.
2009
MC
AO
IV
M
CA
O
Con
trol
Rat
3
x106
PID
7
PID
7
MR
i, G
amm
a ca
mer
a,
Tiss
ue c
ount
s,
His
topa
thol
ogy
Incr
ease
d br
ain
upta
ke in
MC
AO
grou
p, lu
ng a
ccum
ulat
ion
+
Har
ting
et al.
2009
TBI
IV
TBI
Sha
m
Rat
4
x106
or 1
04 -
106
24h
2 or
14
days
Imm
unoh
isto
chem
istry
,
Infra
red
cell
track
ing,
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avio
ural
test
s
Alm
ost n
o ce
lls in
the
brai
n at
PID
14,
no im
prov
ed re
cove
ry, p
rom
inen
t lun
g
accu
mul
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mal
l num
ber o
f cel
ls
reac
hing
arte
rial c
ircul
atio
n
Kei
mpe
ma
E. e
t al.
2009
MC
AO
IA
M
SC
s
Pla
cebo
Rat
1
x 10
6 2h
P
ID 1
4 H
isto
path
olog
y,
Imm
unoh
isto
chem
istry
CLS
M
Red
uced
lesi
ons,
onl
y a
few
cel
ls
dete
cted
afte
r 2 w
eeks
+
Koh
et al.
2008
MC
AO
D
irect
hU
C-M
SC
s
Con
trol
Rat
6
x 10
5 P
ID 1
4 P
ID 3
5 B
ehav
iour
al te
sts,
Imm
unoh
isto
chem
istry
,
MR
i
Impr
oved
reco
very
, red
uced
lesi
on
size
, neu
rotro
phic
fact
or e
xpre
ssio
n
Lee
et al.
2010
a
Uni
late
ral
caro
tid
occl
usio
n
IA
(intra
-
card
iac)
hMS
Cs
or
plac
ebo
Rat
,
neon
atal
1 x
106
72h
PID
40
Beh
avio
ural
test
s,
His
topa
thol
ogy
Impr
oved
reco
very
, red
uced
lesi
on
size
Par
k et al.
2011
TBI
IV
Trau
ma
Con
trol
Rat
1
x106
24h
24h
+
4h
Gam
ma
cam
era,
Tiss
ue c
ount
s
Incr
ease
d br
ain
upta
ke in
trau
ma
grou
p by
gam
ma
coun
ter,
gam
ma
cam
era
upta
ke in
diffe
rent
She
n et al.
2006
MC
AO
IA
M
SC
s
Pla
cebo
Rat
2
x 10
6 P
ID 1
P
ID 2
8 B
ehav
iour
al te
sts,
His
topa
thol
ogy,
Imm
unoh
isto
chem
istry
Impr
oved
reco
very
, inc
reas
ed O
PC
coun
t and
mar
kers
rela
ted
to a
xon
and
mye
lin re
mod
ellin
g
37
Stu
dy
Mod
el
Rou
te
Gro
ups
Spe
cies
C
ell
num
ber
Tim
ing
Follo
w-
up
Ana
lysi
s O
utco
me
Mig
ratio
n
de
Vas
conc
elos
Dos
San
tos
et al.
2010
Foca
l
corti
cal
isch
emia
IV
BM
-MN
Cs
MS
Cs
Pla
cebo
Rat
B
M-
MN
Cs:
3 x1
07
MS
Cs:
3 x1
06
1, 7
, 14
or
30 d
ays
77
days
Beh
avio
ural
test
s,
Imm
unob
lotti
ng
Impr
oved
reco
very
whe
n B
M-M
NC
s
trans
plan
ted
at 1
or 7
day
pos
t-op,
MS
Cs
at 1
day
, BM
-MN
Cs
and
MS
Cs
equa
l
38
Ta
ble
3. E
xp
eri
me
nta
l in
viv
o s
tud
ies in
ve
sti
ga
tin
g B
M-M
NC
th
era
py
fo
r is
ch
em
ic b
rain
in
jury
.
Stu
dy
Mod
el
Rou
teG
roup
s S
peci
esC
ell n
umbe
r Ti
min
g Fo
llow
-
up
Ana
lysi
s O
utco
me
Mig
ratio
n
Fujit
a et al.
2010
BC
AS
BC
AS
-BM
-MN
Cs
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m
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se
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day
s La
ser s
peck
le
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imag
ing
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men
ted
cere
bral
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od
flow
, les
s w
hite
mat
ter
dam
age,
incr
ease
d V
EG
F
Iihos
hi e
t a
l.
2004
MC
AO
IV
B
M-M
NC
s: 3
-
72h
Pla
cebo
: 3-
72h
Sha
m
Rat
1
x107
3, 6
, 12,
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2h
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ehav
iora
l tes
ts,
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topa
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on in
all
grou
ps,
earli
er ti
min
g m
ost e
ffect
ive,
impr
oved
reco
very
+
Kam
iya
et
al.
2008
MC
AO
IV
IA
IV, I
A
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cebo
Rat
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x107
Imm
edia
te
7 da
ys
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topa
thol
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avio
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ests
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rct v
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IV
and
IA g
roup
s, im
prov
ed
reco
very
in IA
gro
up
de
Vas
conc
elos
Dos
San
tos
et al.
2010
Foca
l
corti
cal
isch
emia
IV
BM
-MN
Cs
MS
C
Pla
cebo
Rat
B
M-M
NC
s: 3
x 10
7
MS
Cs:
3 x
106
1, 7
, 14
or 3
0
days
77 d
ays
Beh
avio
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ests
,
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oved
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very
whe
n B
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trans
plan
ted
at P
ID 1
or 7
, MS
Cs
at 1
day
BM
-
MN
Cs
and
MS
Cs
equa
l.
39
Ta
ble
4. C
lin
ica
l stu
die
s i
nv
esti
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ng
BM
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C o
r M
SC
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dy
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num
ber
Tim
ing
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w-
up
Ana
lysi
s O
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me
Mig
ratio
n
Bar
bosa
da
Fons
eca
et
al.
2009
MC
A
Stro
ke
BM
-
MN
Cs
IA
No
cont
rol
n =
1 A
utol
ogou
s5
x108
PID
67
2, 2
4,
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ma
cam
era
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T
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l acc
umul
atio
n in
lesi
on, n
o
adve
rse
effe
cts
+
Bar
bosa
da
Fons
eca
et
al.
2010
MC
A
Stro
ke
BM
-
MN
Cs
IA
No
cont
rol
n =
6 A
utol
ogou
s1.
25 x
108
to 5
x 1
08
< P
ID
90
120
days
Gam
ma
cam
era,
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EC
T
Cel
l acc
umul
atio
n in
lesi
on, n
o
adve
rse
effe
cts
+
Cor
rea
et
al.
2007
Stro
ke
BM
-
MN
Cs
IA
No
cont
rol
n =
1 A
utol
ogou
s3.
0 x
107
PID
9
4 mon
ths
SP
EC
T C
ell a
ccum
ulat
ion
in le
sion
at 8
h,
no a
dver
se e
ffect
s
+
Cox
et al.
2011
TBI i
n
child
ren
BM
-
MN
Cs
IV
No
cont
rol
A
utol
ogou
s6
x106 /k
g B
efor
e
PID
2
6 mon
ths
PE
LOD
scor
ing,
Neu
rolo
gic
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very
, MR
i
No
adve
rse
effe
cts,
neu
rolo
gic
scor
e im
prov
ed
n/a
Mon
iche
et
al.
2012
MC
A
Stro
ke
BM
-
MN
Cs
IA
BM
-
MN
Cs
Con
trol
n =
10
n =
10
Aut
olog
ous
1.59
x 1
08
(mea
n)
PID
6.4
(±1.
3)
6 mon
ths
GM
CS
F,
BN
GF,
MR
i
Neu
rolo
gy
Impr
oved
reco
very
40
2.3.2 Other indications
Although ischemic brain injury is an interesting application for MSCs, several
other indications have shown promising results with cellular therapy. Some of the
indications offer a possibility for either direct or systemic transplantation of the
cells, but only systemic transplantation is an option with certain diseases.
Myocardial infarction
Myocardial infarction resulting from coronary arterial disease is the single most
important cause of mortality and morbidity in western countries. The therapeutic
potential of MSCs is well documented in the treatment of myocardial infarction in
experimental models, with the number of clinical trials growing steadily.
Intravascular delivery, either intravenously or via the intracoronary route, is
shown to reduce the infarct volume and preserve the ejection fraction of the heart,
thus protecting the vital function of the heart while maintaining the integrity of
circulatory homeostasis (Chou et al. 2014). Due to the vast number of cases and
the potentially lethal nature of ischemic cardiac events, MSCs represent a
promising approach if they can be applied to the prevention of ischemic damage
or the regeneration of previously injured myocardium.
Osteogenesis imperfecta
Osteogenesis imperfecta (OI) is a rare hereditary disorder of bone development
characterized by fragile bony structures that are prone to fractures. The
underlying defect is a mutation in one of the two genes encoding type I collagen,
the primary structural protein of bone. The severity of the disease varies. Some
variants of OI cause inevitable perinatal morbidity, whereas patients carrying
milder forms of the disorder survive into adulthood, but suffer from pathologic
fractures and skeletal deformities. No curative treatment currently exists for the
disorder. Clinical case reports with MSCs have offered encouraging results in the
treatment of OI, showing trends toward increased bone mineral content and
decreased occurrence of fractures (Horwitz et al. 1999), and even evidence of
accelerated growth (Horwitz et al. 2002). Graft-versus-host-disease
Organ transplantation is an advanced procedure for treating various severe
clinical conditions and is usually performed as a last resort due to the complexity
41
of the required surgical operation, the shortage of suitable organ donors and the
inherent problems associated with allogeneic organ transplantation. The
postoperative complications consist of organ rejection by the recipient’s immune
system and GvHD, in which the allograft starts to attack the host. Both conditions
have severe consequences, including loss of the allograft or even host mortality in
severe GvHD cases. MSCs have emerged in the treatment of steroid-resistant
GvHD following HSC transplantation. A number of patients were reported to
completely respond in a clinical Phase II trial, as reported by LeBlanc et al. (Le
Blanc et al. 2008).
Renal transplant rejection
Renal failure and ultimately end-stage renal disease is caused by a variety of
aetiologic clinical conditions, conditions that are both acquired, such as diabetes,
or inherited (e.g. polycystic kidney disease). The loss of renal function requires
patients to undergo dialysis multiple times per week to filter excess urea and
creatinine from the blood. Renal allograft transplantation replaces the need for
dialysis and significantly decreases the treatment dependency of the patient, but
allograft rejection is a common complication, with a risk of graft loss and patient
mortality.
A Phase I clinical trial reported the usage of autologous MSCs for renal
transplant rejection as being safe; potential therapeutic effects were observed, but
possible mechanisms behind this effect remain unknown (Reinders et al. 2013). A
randomised controlled trial reported that autologous MSCs, compared with anti-
IL-2 receptor antibody induction therapy, resulted in a lower incidence of acute
rejection and a better outcome (Tan et al. 2012).
Other indications
A constantly growing number of MSC clinical trials are registered to treat various
other disorders in addition to those previously mentioned. Mesenchymal stem
cells have been shown to migrate to damaged lung tissue (Lee et al. 2009a, Lee et al. 2009b). A Phase I/II double-blinded, randomized, placebo-controlled clinical
trial utilizing locally injected MSCs showed positive trends among ankle-brachial
pressure index and ankle pressure in patients suffering from critical lower limb
ischemia (Gupta et al. 2013). Universal arterial stenosis is a common
manifestation of atherosclerotic arterial disease, often leading ultimately to
42
amputations of lower limbs, resulting in severe disability. Use of MSCs have also
emerged in the treatment of liver cirrhosis patients, who showed positive effects
in a Phase I/II clinical trial (Kharaziha et al. 2009). Huntington’s disease
(Bantubungi et al. 2008) and tissue engineering of artificial bones (Petite et al. 2000) are yet additional examples of the range of possible MSC applications.
2.4 Intravascular transplantation of bone marrow-derived cells
The optimal method to deliver stem cells in vivo is a matter of debate. In
preclinical animal studies, numerous delivery routes have been attempted,
including direct injection into the target area and intravascular delivery (either via
intravenous or intra-arterial routes).
For direct injection, it can be theoretically assumed that this route would
result in the delivery of a large effective cell dose to the target area. However,
direct injection (to the heart or the brain for example) is a considerably invasive
procedure with inherent risks and no direct evidence supports such an approach
for stem cell transplantation for these indications. Intravascular transplantation
could be considered a more clinically relevant approach. Intravenous
transplantation is a particularly appealing method due to its simplicity, non-
invasiveness and assumed safety. In addition, this route would naturally be the
method of choice in clinical conditions, where a systemic approach would be
reasonable, such as in GvHD.
Intra-arterial transplantation should theoretically overcome the issues
associated with using an IV route to a certain extent. Delivering the cells through
an arterial route would allow the cells to avoid the first passage through the
pulmonary circulation. The cells could either be administered systemically to the
arterial circulation via large central arteries to allow natural distribution in the
vascular compartment or they could be targeted very specifically to the lesion
using modern catheterisation techniques. Catheterisation would allow the cells to
be administered directly to coronary arteries or cerebral arteries, thereby allowing
a precise targeting of MSC therapy to the subject.
Preclinical trials with animal models offer the opportunity to explore more
invasive transplantation methods other than intravascular routes. However, issues
of safety are critical when dealing with actual patients and it is of the utmost
importance that the risks of the transplantation procedure do not exceed the
potential therapeutic benefits gained with the transplantation.
43
2.4.1 The role of direct transplantation
In addition to intravascular routes, direct transplantation could also offer some
advantages in certain clinical settings, as their paracrine effects can take place at
the site of injury; an example for such indication would be intramyocardial
injection during cardiac surgery (Lehtinen et al. 2014). Direct intracranial
transplantation has shown positive effects in animal studies (Chen et al. 2001a).
When comparing direct BM-MNC transplantation and IV transplantation
effectiveness in a rat spinal cord lesion model, direct injection resulted in more
potent remyelination with smaller cell doses, but the same effect could also be
achieved with larger IV cell doses (Inoue et al. 2003). Direct intramyocardial
transplantation has been shown to improve lesional homing in a porcine MI
model compared with intracoronary transplantation (Makela et al. 2009). Some
other possible indications for local transplantation could be in the treatment of
Huntington’s disease (Bantubungi et al. 2008) and in the tissue engineering of
artificial bones (Petite et al. 2000). The potential benefits naturally come at the
expense of considerably more invasive approaches.
Novel approaches of direct transplantation include different cell
encapsulation techniques, whereby therapeutic cells are transplanted within
biocompatible-carrying materials such as gels. Encapsulation aims to enhance the
survival and retention of cells in the target area. Ideally, the encapsulation
materials protect the cells from mechanical stimuli, but the porosity of the
material allows the secretome to exert its paracrine function (Goren et al. 2010,
Levit et al. 2013).
Another factor to be considered with direct transplantation relates to
observations of possible therapeutic benefits due to the accumulation of cells in
the lung (Lee et al. 2009c), raising the question as to whether some of the
systemic therapeutic characteristics are lost due to local transplantation.
Presumably, MSCs that are directly transplanted to the target of therapy do not
reach the systemic circulation in relatively large numbers (Makela et al. 2009),
thus resulting in lung deposition. In addition, some of the systemic secretome-
based effects could theoretically even arise from migration to certain organs other
than the lung, although there is currently no evidence supporting this hypothesis.
Inoue et al. (2003) compared BM-MNC dose on transplantation-dependent
remyelination in a rat spinal cord injury model. Although direct transplantation
had a better remyelination rate, comparable results could also be achieved with IV
transplantation; the equal dose reported in the study was 1 × 107 autologous bone
44
marrow cells IV and direct intraspinal injection of 1 × 105 cells (Inoue et al. 2003). This finding would favour the use of an intravascular transplantation route,
as no neurosurgical intervention for cell transplantation would be needed and
comparable results could still be achieved.
2.4.2 Passive biodistribution after intravascular transplantation
Intravenous transplantation can be executed by cannulating peripheral or central
vessels. Intravenously transplanted substances, such as bone marrow-derived cells
diluted with a solution, circulate through the venous system, passing through the
right atrium and ventricle of the heart to the pulmonary circulation, inevitably
encountering the lung capillaries. After passing through the lung vasculature, the
cells reach the systemic circulation after they are ejected from the left ventricle.
The arterial circulation begins from the left ventricle, which ejects the blood
volume through the whole systemic vasculature, reaching arteries of diminishing
calibre all the way down to the peripheral capillaries. After passing through the
capillary level, the blood passes again through peripheral veins, only to reach the
heart once again.
If mechanisms of active migration are not taken under consideration, it could
be assumed that the cells are passively targeted to various organs with such
distribution as the blood stream follows. Certain organs (i.e, the brain and the
kidneys) use the majority of the heart’s minute volume and this proportion is not
necessarily commensurate to the organs weight. Circulating blood volume
distribution is also greatly affected by the systemic state of the body, being
regulated by constriction or relaxation of the vessel walls.
2.4.3 Active biodistribution by chemotaxis and migration
A common observation noted in various studies is that systemically administered
MSCs are targeted to injured tissues. Acute inflammation due to organ damage
increases local circulation, but the results found suggest an active migration of the
transplanted cells.
The production of SDF–1 (Stromal cell derived factor–1α), is a chemokine,
which production increases as a result of damage to cellular DNA (Ponomaryov et al. 2000). The CXCR4 receptor binds to SDF-1 and increases stem cell migration
to the SDF-1-secreting tissue (Bhakta et al. 2006). A lung contusion model
performed with rats showed an increased bone marrow-derived cell migration to
45
the damaged area after SDF-1 injection and resulted in improved healing
(Hannoush et al. 2011). It has been demonstrated with a myocardial infarction
model that SDF-1 enhances stem cell homing to the damaged area, where SDF-1
is forcibly overexpressed in myocardium, but not in the absence of myocardial
infarction (Abbott et al. 2004).
Controversial mechanisms of tissue migration have also been suggested. The
role of CXCR4, as well as integrin α4 in MSC engraftment in a MI model has
been suggested to be of less importance, whereas integrin β1 has been shown to
facilitate migration and engraftment in MI (Ip et al. 2007). A broader spectrum of
chemokines and their receptors has been hypothesised to mediate MSC tissue
migration, with CXCL8 (interleukin-8) having also been verified to increase
migration (Ringe et al. 2007). The impact of SDF-1 on MSC migration has been
questioned in the study of Ries et al. 2007. Instead, the inflammatory cytokines
TGF-β1, IL-1β, and TNF-α function by acting as chemoattractants for MSCs,
augmenting their invasive capacity by up-regulation of MMP-2, MT1-MMP,
and/or MMP-9 activity in these cells (Ries et al. 2007). CD44 interaction with
extracellular hyaluronic acid has been reported to facilitate MSC migration (Zhu et al. 2006).
Bone marrow stem cell-specific mechanisms of migration are not yet
completely resolved, but similar mechanisms are suggested to be involved, as
seen with white blood cells. It has been shown that MSCs are capable of
coordinated rolling behaviour and adhesion to endothelial cells, as they bind to P-
selectin and VCAM-1, thus fulfilling the basic requirements of extravasation to
various organs (Ruster et al. 2006). Cell adhesion of MSCs mediated by the
adhesion molecules ICAM-1 and VCAM-1 is considered critical for mediating
the immunosuppressive effects of MSCs (Ren et al. 2010).
2.4.4 Biodistribution tracking methods
In order to assess how the transplanted MSCs distribute among the vasculature
and engraft into organs, studies must be conducted using methods that allow the
monitoring of the fate of the cells. An ideal tracking method would accurately
reveal global cell deposition throughout the whole body, the method would not
interfere with the cells naïve biological characteristics and therapeutic potential
and the method should be safe to the subject and be as minimally invasive as
possible to avoid introducing effects of entry trauma, possibly biasing cell
distribution. Preclinical animal studies can compromise certain safety measures if
46
more precise information of the cellular fate is gained in return; however,
methods applied with human subjects must be safe for the patient.
Radionuclide imaging
Radionuclide imaging is a widely used method in which a radioactive substance is
injected into the subject and tracked with equipment able to detect the emitted
radiation, followed by reconstruction of images from the data. In a variety of
clinical applications a common method is to intravascularly inject the substance,
such as glucose or leukocytes that have been labelled with a radioactive isotope.
The clinical applications cover a large range of different studies, including
leukocyte scintigraphy or metabolic mapping used in cancer diagnostics.
Different imaging methods exist; generally, the more advanced methods have
higher spatial resolution and accuracy, but study costs are significantly higher and
study execution more elaborate. Scintigraphy is a basic form of radionuclide
imaging in which the gamma radiation emitted from the radiotracer is detected by
a gamma camera, forming two-dimensional planar images from the subject. In
single photon emission tomography (SPECT), the gamma-radiation is measured
from multiple directions, thereby allowing reconstruction of three-dimensional
images. SPECT possesses the ability to combine the acquired imaging data with
computed tomography (CT) images taken from the same subject. The
combination of these two imaging methods is called SPECT/CT. Positron
emitting tomography (PET) allows more precise spatial resolution; PET-scanners
detect pairs of gamma rays emitted indirectly by a positron-emitting radionuclide
and it can also be combined with other forms of tomographic imaging, such as
CT.
Radionuclide imaging provides the possibility to dynamically assess the fate
of the transplanted therapeutic cells (Kraitchman et al. 2005). Experimental
radionuclide imaging methods are adopted from clinical practice, where the
application most transferable to stem cell imaging is the labelling of leukocytes.
This procedure, combined with imaging methods, is primarily used in whole body
mapping of infection foci. The most feasible radiotracers for MSC labelling are
technetium99m-hydroxymethylpropylene amine oxime (Tc99m-HMPAO) or indium-
111 oxine. The isotope half-life of these tracers are 6.01 hours and 2.80 days,
respectively.
Radionuclide imaging is considered to be the only method for MSC
biodistribution mapping available and provides the opportunity to acquire global
47
body biodistribution imaging data. The disadvantage of radionuclide methods is
the limited half-life of the radiotracer, resulting in diminishing amounts of emitted
gamma-radiation, which restricts the monitoring of the transplanted MSCs.
Additionally, it is possible that radiolabelling of the transplanted cells could affect
their biological characteristics or compromise their viability. In addition, this
method naturally exposes the target to a certain dose of radiation, but the dose can
generally be considered tolerable as the exposure resulting from the radiotracers
would presumably be the same as that applied in daily clinical practice.
Tissue counts
In addition to actual radionuclide imaging modalities, labelling the transplanted
MSCs with radiotracer isotopes enables the quantitation of tissue counts. Organic
material, such as biopsies from target organs or even whole organs in the case of
small animal models, can be placed in a gamma detector. The counts measured
can be normalised to the weight of the sample, thus making samples obtained
from various organs comparable.
The method can be considered very specific, as the samples are visually taken
from the target organ, thus avoiding bias of scattering radiation from surrounding
organs or limitations set by imaging resolution. An obvious weakness of the
method is that biopsies have to be relatively large, hence limiting the use for post-
mortem analysis and naturally preclinical animal experiments. This means that
using tissue counts, MSC distribution can only be assessed at one time point in a
single subject. Another possibly biasing factor is that if biopsies are obtained from
a large organ, they may not represent the whole organ’s MSC density if the cells
are unevenly distributed throughout the organ. Assessment of overall distribution
of the transplanted cells is challenging with tissue counts, as representative tissue
samples would have to be obtained from all organs. However, tissue samples are a
feasible tracking method if MSC quantification is limited to selected targets and
overall distribution is not of a concern.
Magnetic resonance imaging
Magnetic resonance imaging (MRi) is an imaging technique that uses the
application of a strong magnetic field. Imaging equipment detects radiofrequency
signals emitted by excited hydrogen atoms in the subject’s body. The resulting
48
images are accurate and the MRi does not expose the subject to any ionizing
radiation.
MRi can be applied in the assessment of the biodistribution of MSCs after
labelling of the cells with radio-contrasting agents. Super paramagnetic iron oxide
labelling has been used in MSC studies, which makes the transplanted cells
detectable from MRi images. MRi is not as sensitive as radionuclide imaging
methods in detecting the transplanted cells, as reported in a comparative analysis
in a canine MI model. In this study, MRi failed to detect ferumoxine- and
indium-labelled IV transplanted MSCs in the subject’s myocardium, which were
detectable by SPECT/CT (Kraitchman et al. 2005). This questions the feasibility
of using MRi as an overall MSC biodistribution tracking method, as even a small
number of transplanted cells migrating to the target organ could possess
therapeutic significance, as described in a model of ischemic brain injury
(Keimpema E. et al. 2009). However, even with limited sensitivity to detect the
transplanted cells, MRi offers a range of features not available with other tracking
methods, such as assessment of myocardial ischemia and different forms of
functional brain imaging.
Optical tracking methods
Labelling the transplanted cells with self-illuminating substances, such as
luciferase, offers a method of non-invasive imaging. Self-illuminating proteins
can be introduced into target cells by viral vectors. The transplanted cells can then
be detected in a bioluminescence chamber with an ultrasensitive camera. The
method is non-invasive and allows longitudinal study setups (Wang et al. 2009).
The transplanted MSCs can be labelled with histologic stains, which allow
them to be detected in histopathologic samples (e.g. organ biopsies). This makes
the transplanted cells detectable in microscopic samples. However, this method is
not feasible in the quantification of the cells and is limited only to confirming the
presence of the cells. Assessment of overall biodistribution encounters the same
challenges as tissue count biopsies, as representative samples are hard to obtain
for comparison between organs. The value of histopathology and
immunohistochemistry methods lies in their ability to evaluate the cellular fate,
differentiation, survival and even functionality of the transplanted cells. Possible
labels that allow MSC tracking include β-galactosidase (β-gal), Green
Fluorescence Protein (GFP), chloromethyl-dialkylcarbocyanine (DiI), Hoechst
dyes, 4',6-diamidino-2-phenylindole (DAPI), 5-bromo-2'-deoxyuridine (BrdU)
49
and 5-ethynyl-2’-deoxyuridine (EdU) (Lin et al. 2013). Another novel approach
for MSC labelling is the use of quantum dots, which can be applied in the
imaging of intra- and inter-cellular communication (Pietila et al. 2013).
Polymerase chain reaction
DNA of the transplanted cells can be detected from tissue samples by application
of the polymerase chain reaction (PCR). This method can be applied in
xenogeneic transplantation. Usually the transplanted cells are of human origin,
thus rendering the possibility to isolate human DNA from the experimental
animal’s tissues. PCR allows the potential to confirm the presence of the
transplanted cells after prolonged follow-up (Meyerrose et al. 2007).
2.4.5 Acute phase biodistribution
A common pattern of bone marrow-derived cell biodistribution after intravascular
transplantation consists of strong early lung deposition (Gao et al. 2001, Barbash et al. 2003, Detante et al. 2009, Barbosa da Fonseca et al. 2010, Kraitchman et al. 2005, Allers 2011, Vasconcelos-dos-Santos et al. 2012). The rest of the cells
localise mostly in the liver and spleen (Gao et al. 2001, Barbash et al. 2003,
Kraitchman et al. 2005, Detante et al. 2009, Barbosa da Fonseca et al. 2010,
Vasconcelos-dos-Santos et al. 2012), followed by kidneys (Table 4).
Biodistribution is considered as a dynamic effect and lung clearance occurs
during the first few days. This redistribution results in a proportional increase of
cells deposited in the liver, spleen, kidneys (Chin et al. 2003, Kraitchman et al. 2005) and spine (Kraitchman et al. 2005). Redistribution appears to occur
relatively quickly following transplantation, with an initial 80 % of cell
entrapment for the BM-MSCs decreasing to 40 % after 12h (Nystedt et al. 2013).
Thus, it could be hypothetically estimated that the “half-life” of MSC lung
entrapment is roughly 12 hours.
Unfortunately, little is known about the naïve biodistribution of the
transplanted MSCs, as nearly all the studies employed some form of target organ
(Table 4). Usually ischemic organ damage is induced by experimental methods in
preclinical studies (e.g. ligation of coronary vessels or MCAO occlusion) or, in
the case of human studies, organ damage has already occurred as a result of a
natural disease process, such as atherosclerosis or embolisation. Ischemic injury
of a major organ alters the inflammatory state of the entire system, which
50
presumably affects MSC immunologic characteristics, thereby causing MSC
migration to the injured organ as observed previously (Abbott et al. 2004, Bhakta et al. 2006, Hannoush et al. 2011). It is currently unknown how the organ injury
and resulting alterations in the immune system affect the MSC deposition in
organs other than the injured one. Devine et al. reported a study in which two
unconditioned baboons received autologous and allogeneic MSCs. These
transplanted cells could be detected in significant amounts in gastrointestinal
tissues, as well as in kidneys, lungs, liver, thymus, and skin, as assessed by real-
time PCR (Devine et al. 2003).
2.4.6 Long-term biodistribution
Although, the studies offer somewhat comprehensive insights into the acute
biodistribution of the transplanted cells, the long-term fate of the transplanted
cells remains partially unknown. Radionuclide methods, which potentially offer
the most accurate way of assessing overall distribution, cannot be used to observe
long-term engraftment due to the limited half-life of the radioactive isotopes.
Some studies have attempted to elucidate long-term engraftment. It appears that
adipose tissue-derived hMSCs persist in the tissues of a murine model for up to
75 days and can be found mainly in the lungs and the spleen, regardless of the
route of administration, while retaining their multilineage potential and
clonogenic capacity (Meyerrose et al. 2007). IV or intramuscularly transplanted
human adipose tissue-derived mesenchymal stem cells showed long-term
engraftment to the liver in a murine model after an 8-month follow-up, as
assessed by bioluminescence imaging, and no tumour formation was observed
(Vilalta et al. 2008). Interestingly, human MSCs have shown long-term deposit
into lungs when intravenously transplanted into unconditioned mice, as human
DNA could be detected 4 months after transplantation (Allers et al. 2004). It can
thus be concluded that the transplanted cells may survive for prolonged periods
after transplantation and engrafting into multiple organs.
2.5 Challenges of intravascular MSC transplantation
Although intravascular transplantation is a promising method of delivering the
therapeutic cells, certain complications related to the procedure have to be taken
under consideration. The transplantation can either be unsuccessful or even
posess potential hazard to the patient.
51
2.5.1 Periprocedural damage to MSCs
The bone marrow-derived cell’s natural environment (namely the bone marrow) is
characterized by relatively low oxygen content, high cell density and consistency
varying from solid trabecular bone to highly viscose and fatty bone marrow. The
MSCs are located in perivascular niches, where they constantly interact and co-
exist with other types of cells, such as HSCs and macrophages.
Compared to the natural environment of MSCs, the intravascularly
transplanted cells are subjected to various circumstances that can be considered
unnatural and injurious. These conditions may lead to cellular death or damage of
the cells, which could affect their therapeutic capacity due to mechanical and
biochemical damage.
Mechanical damage to MSCs can be caused by various reasons. Cells are
usually aspirated into syringes and injected from them when they are harvested
from the bone marrow, handled and relocated in cultivation procedures or
transplanted into target organisms. MSCs have a strong tendency to attach onto
materials, such as cultivation plates, which requires the use of detaching
substances and mechanical detachment. Mechanical forces caused by the
circulatory system can also expose the cells to physical damage, especially in the
arterial circulation as the cells are forcefully pushed by the blood stream through
the small-diameter capillary structures of the lungs, liver and various other
organs.
The MSCs are suspended into various solutions during the isolation and
cultivation process, which also can be considered as non-physiological conditions
compared to their natural environment. Additionally, the intravascular
transplantation of the cells cultivated on culture dishes leads to a drastic increase
of surrounding oxygen content that could cause chemical damage. The lack of
maintenance cells, namely macrophages, in culture dishes prevents the clearance
of unfit or damaged cells from the culture population. Compared to signal-
induced activation and migration of MSCs from the bone marrow to the systemic
circulation, mechanical harvesting, culturing and direct injection into target
tissues or the circulation truly challenges the survival potential of the MSC to
elude cellular death due to damage, as well as the retaining of their therapeutic
characteristics.
52
Ta
ble
5. S
tud
ies
in
ve
sti
ga
tin
g b
on
e m
arr
ow
de
riv
ed
ce
ll a
cu
te b
iod
istr
ibu
tio
n.
Stu
dy
Mod
el
Spe
cies
Trac
king
C
ells
N
umbe
r R
oute
Tim
ing
Qua
ntita
tion
Follo
w-
up
Gro
ups
Bra
inLu
ng
Live
rS
plee
n K
idne
yH
eart
Not
e
Alle
rs e
t a
l.
2004
Hea
lthy
Mou
se
Tc99
m-
HM
PA
O
PC
R
hMS
Cs
0.2–
2x10
5 IV
n/
a W
hole
bod
y
scan
, PC
R
15m
in,
3h, 2
4h
+++
+++
+
Bar
bash
et
al.
2003
MI
Rat
Tc
99m
-
HM
PA
O
MS
Cs
4x10
6 IV
, IA
PID
2,
10 o
r 14
Imag
ing
afte
r 4h
Who
le b
ody
scan
Tis
sue
coun
ts
IV
LV
- -
+++ +
+ ++
+ ++
+ +
+ +
Bar
bosa
da
Fons
eca
et
al.
2009
Stro
ke
Hum
an
Tc99
m-
HM
PA
O
BM
-MN
Cs
5 x1
08 IA
SP
EC
T 2h
, 24h
,
48h
++
+
++
++
-
Chi
n e
t a
l. 20
03
MI
Pig
11
1In
oxin
e
MS
Cs
n/a
IV
n/a
SP
EC
T
Gam
ma
cam
era
Tiss
ue
coun
ts
++
+/++
+/++
+/++
+
-
Det
ante
et
al.
2009
MC
AO
R
at
Tc99
m-
HM
PA
O
hMS
Cs
3 x1
06 IV
2h
or
20h
MC
AO
Con
trol
+ ++
+ +/
- +
+
Kra
itchm
an e
t
al.
2005
MI
Dog
MS
Cs,
allo
gene
ic
1.13
x108
IV
72h
SP
EC
T P
ID
1,2,
5
and
8
MI
Con
trol
n/a
+++/
+++/
+++/
++
+/++
+
Mitk
ari e
t a
l.
2013
MC
AO
R
at
Indi
um-
111-
oxin
e
MS
Cs,
allo
gene
ic
1.1
x 10
6 or
0.5
x 10
6
IA
24h
SP
EC
T 30
min
to 2
4h
MC
AO
/
Sha
m +
mod
ified
/
nonm
odifi
es
cells
+ +
+++
+++
++
+ Lu
ng a
ctiv
ity
decr
ease
d
over
tim
e
53
Stu
dy
Mod
el
Spe
cies
Trac
king
C
ells
N
umbe
r R
oute
Tim
ing
Qua
ntita
tion
Follo
w-
up
Gro
ups
Bra
inLu
ng
Live
rS
plee
n K
idne
yH
eart
Not
e
Vas
conc
elos
-
dos-
San
tos
et
al.
2012
Stro
ke
Rat
Tc
99m
- B
MM
Cs
3 x
107
IV, I
A
24h
Gam
ma
coun
ter,
Tiss
ue c
ount
s
S
troke
/
cont
rol +
IV/
IA
+/-
++/-
+++
+ ++
-
Lung
act
ivity
decr
ease
d
over
tim
e
54
2.5.2 Pulmonary adhesion and the lung barrier
Multiple studies have reported robust lung accumulation of the transplanted cells.
(Gao et al. 2001, Barbash et al. 2003, Detante et al. 2009, Barbosa da Fonseca et al. 2010, Kraitchman et al. 2005, Allers 2011, Vasconcelos-dos-Santos et al. 2012) This phenomenon is considered to be potentially harmful, as pulmonary
adhesion of the cells could lead to a lesser proportion of the cells reaching the
arterial circulation and their intended therapeutic target, hence hypothetically
attenuating their effectiveness and migration to other organs (Barbash et al. 2003,
Fischer et al. 2009, Freyman et al. 2006, Lee et al. 2009c). Lung entrapment is
especially observed after IV transplantation (Allers et al. 2004, Barbash et al. 2003, Barbosa da Fonseca et al. 2010, Detante et al. 2009, Gao et al. 2001,
Kraitchman et al. 2005, Makela et al. 2013, Vasconcelos-dos-Santos et al. 2012)
and it appears that the majority of the transplanted cells first target the lungs in
large numbers. Intravenous transplantation of MSCs was even shown to cause
lethal pulmonary emboli in preclinical studies (Lee et al. 2009d). Schrepfer et al. named this prominent lung deposition as lung entrapment (Schrepfer et al. 2007).
Physical lung entrapment purely due to the size of the transplanted cells has
been suggested by some recent studies. Fischer et al. observed a 30-fold greater
lung accumulation in MSC transplantation compared to BM-MNCs and based on
their larger diameter (5 to 8 µm cell size vs 13 to 19 µm, respectively), MSCs do
not fit through the lung capillaries (Fischer et al. 2009). The diameter of MSCs
has also been suggested to function as a physical obstacle for their lung passage
by Schrepfer et al. (Schrepfer et al. 2007). Interestingly, Aicher et al. (2003) reported no lung accumulation of In111-labelled endothelial progenitor cells
(EPCs) when transplanted intravenously or intraventricularly in a rat open-chest
MI model (the physical size of EPCs is considerably smaller in comparison with
the latter two cell types).
In addition to lung entrapment due to the physical size of MSCs and their
relation to the diameter of the pulmonary vasculature, several studies have
suggested involvement of adhesion molecules resulting in attachment of the
transplanted cells to the pulmonary endothelial cells. MSCs are capable of
coordinated rolling behaviour and adhesion onto endothelial cells by binding to P-
selectin and VCAM-1, (Ruster et al. 2006). It has also been suggested that
VCAM-1 is more involved in MSC pulmonary adhesion than P-selectin (Fischer et al. 2009). When comparing the lung clearance of human BM-MSC’s and UCB-
55
MSC’s in a murine model, the UCB-MSCs had higher expression levels of α4
integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6) and the hepatocyte
growth factor receptor (c-Met), as well as a higher general fucosylation level. The
levels of CD49d and CD49f expression could be functionally linked with an
increased lung clearance rate (Nystedt et al. 2013).
Additional evidence supporting the involvement of adhesion molecules in
mediating lung entrapment is that administration of MSCs via two boluses
significantly increased pulmonary passage in a rat model of allogeneous IV
transplantation. The number of MSCs given during the second bolus was
significantly higher as compared to the first bolus, suggesting saturation of
receptors during the first bolus, which allowed more cells to pass through the
vasculature with the second bolus (Fischer et al. 2009). Another factor to be
considered when assessing the relation of physical and adhesion molecule-
mediated lung entrapment of the transplanted cells is that larger cells have larger
cell surface areas with possibly more adhesion molecules expressed per cell,
thereby creating a higher probability for cell-cell contact through adhesion
molecules and their cognate ligands on the alveolar endothelium (Nystedt et al. 2013).
Presumably the lung accumulation of the intravascularly transplanted cells is
a combination of the actual physical entrapment and cell-cell interactions. Passive
circulation of the cells to the lungs can be assumed to lead to the deposition of the
cells within the lungs, but it has to be taken under consideration that MSCs are
shown to actively migrate to injured lung tissue (Lee et al. 2009a, Lee et al. 2009b). As the lungs are a natural barrier with constant exposure to pathogens and
particles of the breathed air, it can be hypothesized that the lungs are under a
constant state of inflammation, thereby facilitating active adhesion of the
transplanted MSCs. What makes understanding the mechanisms and
consequences of the lung entrapment even more complicated are the observed
positive effects after lung entrapment. For example, Lee et al. showed that
hMSCs trapped in mouse lungs attenuated myocardial infarction and the effect
was mediated by TSG-6 (Lee et al. 2009c).
2.5.3 Thromboembolic events
The well documented tendency of the IV transplanted MSCs to accumulate to
lungs in large numbers raises obvious concerns of safety. A number of studies
56
have confirmed these suspicions, indicating the occurrence of thromboembolic
events after intravascular MSC transplantation.
Thromboembolus formation and MSCs
Blood coagulation results from a cascade that normally begins after the
occurrence of endothelial damage to the vessel wall. Subendothelial layers
contain tissue factor (TF) and certain collagens that trigger the coagulation
cascade. The coagulation cascade consists of primary hemostasis, in which
activated platelets form a clot. Primary hemostasis is followed by secondary
hemostasis, whereupon multiple coagulation factors interact with each other,
forming a cascade that reinforces itself, resulting in the formation of an insoluble
fibrin clot. The secondary hemostasis is initiated by subendothelial collagenases
in the intrinsic pathway or by TF via the extrinsic pathway (van der Windt et al. 2007).
Moll et al. conducted in vitro and in vivo studies investigating interactions
with the coagulation system. MSCs express TF and collagen I, which are both
prothrombotic factors normally found in the subendothelial compartment that
could induce an instant blood-mediated inflammatory reaction (IBMIR) in which
transplanted cells trigger the coagulation cascade. The prothrombotic effects of IV
MSCs in vivo were weak when infused in clinically relevant doses and resulted in
a decrease in thrombocyte counts. The prothrombotic effects increased when late
passage (P5-P8) cells were used (Moll et al. 2012). Intravascularly transplanted
adipose tissue-derived MSCs have been shown to lead to possible formation of
pulmonary emboli triggered by tissue TF (Tatsumi et al. 2013).
Several animal studies have demonstrated adverse thromboembolic events
associated with MSC transplantation. IV transplantation of murine MSCs in a
murine model displayed aggregates of MSCs in the lung as early as one day post-
transplantation, which in many cases persisted thereafter to result in tissue
damage with development of fibrotic tissue, as well as impaired organ function
(Anjos-Afonso et al. 2004). IV transplantation of hMSCs in a murine model led
to lethal pulmonary emboli (Lee et al. 2009d). MSC entrapment at the pre-
capillary level and even microembolus formation has been reported with intra-
arterial transplantation (Toma et al. 2009). Intracoronary injection has also led to
decreased coronary blood flow in porcine models and even myocardial infarction
in canine subjects (Freyman et al. 2006, Vulliet et al. 2004). Clotting and
57
decreased capillary blood flow is also reported in an intravital microscopy study
(Furlani et al. 2009).
The consequences of MSC-related thromboembolism
Past studies led to an assumption that IV transplanted cells can cause a pulmonary
embolism in a human subject. The consequences of pulmonary embolization vary.
Embolization of pulmonary capillaries decreases the effective ventilation area of
the lungs, leading to hypoxia. Embolization of main pulmonary arteries or a
sufficiently large amount of pulmonary capillaries can lead to failure of the right
ventricle due to increased working load, ultimately resulting in circulatory
collapse collapse (McIntyre & Sasahara 1971).
The effects caused by IV transplanted cells depend on whether they are able
to cause the formation of a large clot, causing embolization of the main
pulmonary artery truncus, or the formation of microemboli in the pulmonary
capillaries. Microembolisation of the capillaries could be tolerated to a certain
extent, depending on the condition of the subject’s heart and ventilation function
collapse (McIntyre & Sasahara 1971). Massive embolization of the main
pulmonary arteries, on the other hand, could cause lethal consequences on an
otherwise healthy subject. Pulmonary embolization that leads to haemodynamic
instability could be treated with anticoagulative agents or thromobolytic therapy
in a normal clinical setting, but these treatment options could be unavailable
under certain clinical conditions with inherent risks of bleeding that MSC therapy
could otherwise cover (e.g. traumatic brain injury).
Thromboembolic events caused by cell transplantation could have other
severe consequences when applied via the IA route. If the cells were targeted to a
certain organ with catheterization techniques and emboli formed emboli (Anjos-
Afonso et al. 2004, Vulliet et al. 2004, Freyman et al. 2006, Furlani et al. 2009
Lee et al. 2009d, Toma et al. 2009), this would naturally result in ischemic
damage and loss of function of the target organ. Again, the type of damage would
depend on whether the cells caused macroclotting or reached the vasculature at
the capillary level. Macroembolus formation after central IA delivery, such as via
the intraventricular route, would resemble the thromboembolic events caused by
atrial fibrillation, in which the embolus is mostly limited to the cerebral
vasculature, ultimately leading to stroke. Other possible consequences of central
macroembolisation would be acute limb ischemia, acute mesentery ischemia or
kidney infarction. If cells were to cause microembolisation after central delivery,
58
this would presumably lead to sporadic ischemic events throughout the body that
would otherwise hypothetically cause reversible damage, but as the brain is
recognized as the primary target of the central emboli, this could have severe
consequences.
Contrary to the preclinical studies reporting possible associated threats, the
clinical trials with MSCs or BM-MNCs have not revealed the occurrence of
thromboembolic events, but Moniche et al. have reported the occurrence of
seizures during follow-up on two patients who received IA BM-MNC
transplantation for acute stroke, although clear causality could not be indicated
(Moniche et al 2012). Central venous route for BM-MNC administration was
used in a human trial with pediatric patients suffering from traumatic brain injury
(Cox et al. 2011), with no adverse effects. Intravenous transplantation of
autologous MSCs for patients after myocardial infarction also did not reveal
safety threats (Hare et al. 2009). Current clinical studies with intravascular MSC
or BM-MNC transplantation have neglected the lack of sufficient information
regarding the risks of thromboembolic events and have, in fact, endangered the
trial participants, as no salvage plan, in case of occurrence of adverse effects, is
presented. Human MSC transplantation showed no manifestations of acute or
chronic toxicity in adult unconditioned mice in a 13-month follow-up study
(Allers et al. 2004).
2.5.4 Immunologic reactions
Transplantation of allogeneic MSCs could potentially result in adverse
immunologic reactions. The transplantation subject’s immune system could
interpret the transplanted cells as hostile and attack them. As the transplanted cells
are shown to persist in the subject’s tissues for extended periods of time (Allers et al. 2004, Meyerrose et al. 2007, Vilalta et al. 2008), this hypothetically could lead
to the development of a GvHD-type of condition if the transplanted cells targeted
the subject’s tissues. Donor MSC transplantation has been reported to lead to an
inflammatory response in healthy rat graft rejection within 7 days after direct
stereotactic transplantation (Coyne et al. 2006) and dose- and antigenic mismatch
degree-dependent immunogenic reactions were also seen when MHC class I
mismatched MSCs were intracranially transplanted in primates (Isakova et al. 2010). However, when autologous MSCs were transplanted in human subjects
that had developed GvHD after HSC transplantation, only weak immunogenic
potential was observed (Sundin et al. 2009). These immunologic issues can be
59
avoided by choosing autologous transplantation, but in the case of MSCs, this
would practically rule out the possibility of transplantation in the hyperacute
phase after ischemic injury due to time needed for cell cultivation.
2.5.5 Development of malignancies
Concerns of potential malignant transformation of MSCs have arisen regarding
their transplantation in clinical trials. The MSCs are multipotent progenitors with
clonogenic capabilities and thus possess a theoretical possibility to form
malignant clones. Since the cells are shown to persist in host tissues, an
unavoidable question is whether they can generate malignancies at some point
during their life span. Possible malignant transformation of the engrafted cells
could occur with a delay of decades, which makes it hard to study this potential
threat. Human adipose tissue-derived MSCs in a murine model with an 8 month
follow-up period showed no tumour formation (Vilalta et al. 2008). Long-term
engraftment of MSCs in human subjects appears to be low, hence suggesting the
risk of malignancies to be unlikely (von Bahr et al. 2012). It has been speculated
that autologous cells could have a lower risk of malignant transformation, as they
may be eventually rejected despite their immunoprivileged nature. Currently no
reported malignant transformation of MSCs is known (Barkholt et al. 2013).
In addition to malignant transformation of the transplanted cells, the cells
could accelerate the development of host malignancies (Barkholt et al. 2013). A
common clinical example of this potential cancer cell-MSC interaction is adipose
tissue transplantation for reconstructive breast surgery after mastectomy resulting
from breast cancer. There is evidence supporting the theory that adipose tissue-
derived MSCs can promote the growth of breast cancer cells (Zimmerlin et al. 2011) and their immunosuppressive effects are shown to favour tumour formation
in a murine model (Djouad et al. 2003). It is known that the cells induce
angiogenesis (Chen et al. 2003, Fujita et al. 2010), which is a well-recognized
factor involved in growth of malignant tumours. Immunosuppressive properties
of the MSCs could also interfere with the naïve immune system’s anti-oncogenic
mechanisms.
2.5.6 Ectopic tissue formation
The MSCs are stem cells of mesenchymal origin and a natural concern is that they
are capable of differentiating into various types of mesenchymal tissues. As the
60
transplanted cells become affected with an uncontrollably varied number of
different cellular signals released by the targeted tissue or the host’s unaffected
system, there is a possibility that the MSCs could differentiate into impractical
tissues, which are unsuitable for the target organ’s function. Indeed, in a murine
model of MI, direct lesional injection of allogeneic MSCs led to the formation of
calcification and ossification, demonstrating a potential safety threat (Breitbach et al. 2007).
The clinical consequences of ectopic tissue formation would depend on the
type of tissue formed and the host organ’s function. Osteogenic differentiation in
myocardial tissue could potentially affect contractile function of the heart and
ectopic formation of neural tissue could hypothetically cause abnormal
conduction circuits within the myocardium, potentially resulting in arrythmias
although no adverse effects in a placebo controlled clinical trial with an
intramyocardial injection was present (Lehtinen et al. 2014). Ectopic tissue
formation within the cerebral parenchyma could potentially lead to seizure
formation. Low long-term engraftment of MSCs in human participants is reported
(von Bahr et al. 2012) and thus it can be considered relatively unlikely that
formation of ectopic, MSC-derived tissue would be a threat, unless this would
happen in critical sites of physiologic function.
2.6 Optimisation of intravascular MSC transplantation
Past research has revealed certain challenges concerning the intravascular
transplantation of MSCs. A range of studies have focused on ways to overcome
these particular issues and improve the efficacy and safety of the transplantation
procedure.
2.6.1 Type of injury
When applying MSCs for ischemic organ injury, the type of injury can be
assumed to remarkably affect the chance of the transplantation’s success.
Individual organs have different microenvironments and secretomes that ischemic
injury greatly affects. Thus, the behaviour of the MSCs should first be studied
with organ-specific preclinical settings and generalized assumptions of MSC
therapeutic functions should be avoided until their exact biological characteristics
are fully understood.
61
Another possible factor possibly affecting the success of cellular therapy is
the severity of the ischemic injury. Shindo et al. compared transplanted neural
stem cell (NSC) survival between a rat model of mild and severe TBI, concluding
poorer survival with more severe injury (Shindo et al. 2006). This can also be
hypothesized to apply to bone marrow-derived stem cells. It is crucial to explore
the limits of MSCs’ restorative effects, as the therapeutic gain should always
exceed the possible adverse effects. If the severity of the ischemic injury in a
clinical setting prevents the MSCs having any therapeutic benefit, then the use of
cellular therapy should be avoided.
2.6.2 Cell type
The type of the therapeutic cells could have a major impact on the biodistribution
and therapeutic capacity of the cell transplantation. The possibility for rapid re-
transplantation of the BM-MNCs is recognized as a favourable factor for their
use. Again, it is obvious that the cultivated MSC population clearly contains more
actual therapeutic stem cells, leading to a natural presumption that this population
possesses more therapeutic capacity. BM-MNCs also contain HSCs, which have
been suggested to have neuroprotective effects independently due to their
immunomodulatory properties, such as a reduction of immune cell migration to
the ischemic lesion in stroke (Schwarting et al. 2008).
Studies comparing the effectiveness of MSCs and BM-MNCs are rare. The
MSCs and BM-MNCs have been reported to have similar effects when
transplanted early after the onset of focal cortical ischemia (de Vasconcelos Dos
Santos et al. 2010). MSCs have been reported to be more effective in a model of
chronic myocardial infarction (Mazo et al. 2010). The small number of studies
thus favours the use of MSCs for their therapeutic capacity, but again the factors
to be considered are the timing and safety of the transplantation, as the BM-
MNCs could have better pulmonary passage (Schrepfer et al. 2007, Fischer et al. 2009).
The source of MSCs could also affect their biodistribution and therapeutic
capacity. Nystedt et al. compared the characteristics and pulmonary passage of
human umbilical cord MSCs (hUC-MSCs) and BM-MSCs. The hUC-MSCs had
higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f,
VLA-6) and the hepatocyte growth factor receptor (c-Met), as well as a higher
general fucosylation level, and the level of CD49d and CD49f expression could
be functionally linked with increased lung clearance rate (Nystedt et al. 2013).
62
Although bone marrow acts as an easily accessible source for MSCs, this finding
suggests that MSCs of other sources could possess different biodistribution
capacities.
It is currently not certain whether allogeneic cells have an inferior therapeutic
capacity compared to autologous cells. The allogeneic transplantation could
interact with the host immune system by possibly limiting their biological
function. Antiproliferative effects on lymphocytes have been observed to occur
with both autologous and allogeneic MSCs and involvement of MHCs with
MSCs is speculated to not be required (Le Blanc et al. 2003). This finding
encourages the use of cells of autologous origin as precultivated cells could be
available during the acute phase of the ischemic injury.
2.6.3 Systemic transplantation route
Intravascular transplantation can be performed via IV or IA routes, both of which
could have advantages and disadvantages concerning biodistribution, therapeutic
capacity and also safety. The effectiveness of IV transplantations has been
questioned due to the lung entrapment effect (Fischer et al. 2009).
Although the number of studies comparing these two transplantation routes is
limited, the results generally favour the IA route. IA transplantation resulted in a
better outcome than IV in a rodent model of MCAO (Kamiya et al. 2008). In a
murine model, IA administration of allogeneous adult multipotent progenitor cells
is shown to result in decreased lung accumulation and an increased amount of
cells in the liver, kidneys and brain at 30 days compared with IV transplantation
(Tolar et al. 2006). Choosing to transplant the therapeutic cells by an IA route
could thus enhance their therapeutic effects and biodistribution. Again, the
possible disadvantages of the IA route (i.e. more invasive approach and
embolization), would then be encountered.
2.6.4 Timing of transplantation
The therapeutic window of bone marrow cell transplantation can be hypothesised
to affect cell biodistribution. Using ischemia- reperfusion injury of the brain as an
example, certain factors related with transplantation timing can be recognized.
A rodent study compared the effects of early versus delayed transplantation of
MSC migration in damaged myocardium after intra-arterial transplantation,
showing a statistical trend of higher uptake at 48h after the insult compared to 10-
63
14 days following transplantation (Barbash et al. 2003). A study utilising a rat
model of multiple focal cortical ischemic lesions demonstrated improvement of
neurologic outcome when BM-MNCs where administered at post-ischemic day
(PID) 1 or 7, but not at PID 14 or 30; the same study also used MSCs with
positive outcome when transplanted at PID 1 but not at PID 30 (de Vasconcelos
Dos Santos et al. 2010). A rat model of MCAO also reported improved recovery
of MSC transplantation at PID 1 and 7 (Chen et al. 2001b). A rat study with IV
transplantation for MCAO presented strong evidence for earlier transplantation
superiority; 3h post-ischemic transplantation had the most therapeutic effect, with
the effect linearly decreasing to 72h (Iihoshi et al. 2004). A rodent cerebral
ischemia model with BM-MNC transplantation investigated the optimal delivery
time, comparing 72h with 7 days administration, and observed neurologic
improvements with earlier transplantation (Yang et al. 2011).
It appears that greater therapeutic effect is achieved if the cells are
administered in the acute phase after the ischemic insult. Favourable results are
generally seen when transplantation is performed within the first 24 to 72 hours
(Barbash et al. 2003, Iihoshi et al. 2004, de Vasconcelos Dos Santos et al. 2010,
Yang et al. 2011), although some benefit may be seen when the cells are
administered within one week after the insult insult (Chen et al. 2001b, de
Vasconcelos Dos Santos et al. 2010). However, the evidence supporting even
earlier transplantation at 3 hours after cerebral ischemia (Iihoshi et al. 2004)
strongly emphasizes the need to consider the use of BM-MNCs due to their
availability without cultivation.
2.6.5 Cell dose
Optimal therapeutic cell dose is not yet fully understood, but there are certain
factors that must be considered. Firstly, the used cell dose has to have clinical
effect. Secondly, the dose should be safe and the consequences of increasing the
dose beyond the minimal effective amount should be carefully considered.
Increasing the dose could result in more therapeutic potential, but this would also
be likely to increase the probability of possible adverse effects related to the
transplantation.
Several studies support the hypothesis that the therapeutic effects have a dose
response. A rat study with MCAO reported improved recovery with high dose of
MSCs for transplantation, but no statistically significant effect was seen with a
lower dose (Chen et al. 2001b). Mouse MSC transplantation in rat MCAO
64
showed dose-dependent cerebral blood flow (CBF) and blood brain barrier (BBB)
restoration in addition to dose-dependent increases of growth factor secretion;
subjects with cell doses of 2 x105 or 105 cells transplanted directly into the lesion
showed better results than those with cell doses of 104 or 4 x 104 (Borlongan et al. 2004). Wolf et al. conducted a porcine study experimenting with dose ranges of 1
x 103 to 1 x 106 IV transplanted autologous or allogeneic MSCs and demonstrated
a dose-dependent improvement in left ventricular function after MSC delivery,
statistically significant at 1 x 105 and 1 x 106 cells per body weight (kg), as
assessed by echo cardiography (Wolf et al. 2009). A proliferative effect on
lymphocytes is reported in very low MSC doses in vitro, but a dose-dependent
antiproliferative effect was seen with larger MSC quantities (Le Blanc et al. 2003).
On the contrary, some studies have witnessed a lack of dose response or
limitations of dose response by further increasing the dose. Hare et al., conducting a human trial with MI patients receiving IV allogeneic hMSC
transplantation, reported a decrease in ventricular arrhythmias and improved
pulmonary function. Dose response was also evaluated in 3 placebo-controlled
dose escalation cohorts of 0.5, 1.6, and 5.0 × 106 hMSCs/kg body weight, but no
dose-response was observed except for the number of premature ventricular
contractions, which decreased among higher hMSC doses (Hare et al. 2009).
Yang et al. compared 1, 10 or 30 x 106 doses of BM-MNCs transplanted at 24
hours in a rat model of cerebral ischemia and reported no therapeutic benefits
from the 1 x 106 dose. Additionally, increasing the dose up to 30 x 106 from 10 x
106 did not improve the therapeutic benefits (Yang et al. 2011).
Albeit higher cell doses can be assumed to result in better therapeutic
outcome, as the transplanted cells would likely have a higher capacity for
releasing beneficial soluble factors, larger quantities could also have adverse
effects due to the nature of cell biodynamics. Intravascular transplantation of
human MSCs in a mouse model is reported to cause embolus formation in
capillaries and the effect was more prominent with higher cell concentrations
(Furlani et al. 2009). Moll et al. investigated the prothrombotic effects of MSCs
and although the adverse effects were weak when median 1,6 x 106 cells per
kilogram were given at a low cell-passage number (P1-4), they concluded that
higher doses could have more prothrombotic potential (Moll et al. 2012).
Optimal cell dosage is a factor to be determined when considering
intravascular stem cell transplantation. Low dose transplantation has even resulted
in a lack of therapeutic results, although further increasing the number of
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transplanted cells in high dose transplantation appears to not offer greater
therapeutic benefit (Yang et al. 2011). It could be speculated that the dose-
response of MSCs could bring greater therapeutic benefit until a certain plateau is
reached. In addition to the lack of a greater benefit gained by further increasing
the dose, the increased risk of thromboembolic events possibly connected with
higher doses becomes a concern. The balance between embolus risk and
biologically potent cell quantity is an important factor that needs to be solved in
future studies.
Another factor to be considered when defining the optimal cell dose is that
fresh bone marrow aspirates of BM-MNCs collected from each individual
contains a varying amount of therapeutic cells and thus it is necessary to
determine whether the amount of re-transplanted cells needs to be manipulated to
optimise the therapeutic outcome. If MSCs are used for treating an ischemic
organ injury, it would be likely that their isolation and cultivation would begin as
the patient would be admitted to the point of care. Recognizing that earlier
transplantation leads to greater therapeutic effects (Barbash et al. 2003, Iihoshi et al. 2004, de Vasconcelos Dos Santos et al. 2010, Yang et al. 2011), it is essential
to specify the cell dose that leads to optimal therapeutic effects, as extending the
cultivation time to produce more cells potentially sacrifices the therapeutic benefit
of earlier transplantation.
Usually, the MSC transplantation is executed using a slow infusion. However,
it is still unclear whether multiple boluses or infusions could have beneficial
results. The administration of 4 x 106 MSCs via two boluses significantly
increased pulmonary passage in a rat model of allogeneous IV transplantation
(Fischer et al. 2009). The result was explained by saturation of lung adhesion by
the first dose. This finding suggests that performing the MSC transplantation
using more than one dose or infusion could offer certain advantages. Qian et al. reported no decrease in pulmonary entrapment with low MSC concentrations
(Qian et al. 2006).
2.6.6 Pronase detachment
Pronase, used as a dispersing agent of cultured cells in the 1970s, is currently
commonly used in protein analysis and antigen retrieval protocols (Foley &
Aftonomos 1970). Pronase has mostly been replaced by trypsin as a dispersing
agent. Given the fact that cell surface adhesion molecules clearly effect lung
deposition, substances that affect cell attachment to surfaces, such as dispersing
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agents, could have an impact on cell adhesion characteristics, thereby potentially
affecting lung clearance (Nystedt et al 2013).
2.6.7 Vasodilatators
Due to the fact that pulmonary entrapment of the intravascularly transplanted
therapeutic cells has been correlated to their diameters in relation to lung
capillaries capillaries (Schrepfer et al. 2007, Fischer et al. 2009), vasodilating
agents could thus increase the pulmonary clearance of the transplanted cells by
allowing more cells to pass through the narrow capillaries. Application of sodium
nitroprusside has shown to dilate lung capillaries, thus increasing the lung passage
of MSCs (Gao et al. 2001, Schrepfer et al. 2007). The use of sodium nitroprusside
also increased the MSC accumulation in the liver and long bones as a result of
greater lung clearance (Gao et al. 2001). These are encouraging results, as
vasodilators are commonly used in various clinical settings and their possible
adverse effects, mainly hypotension, is familiar to clinicians, clinically tolerated
and reversible.
2.6.8 Anticoagulative agents
The reports suggesting possible thromboembolic manifestations after MSC
transplantation (Anjos-Afonso et al. 2004, Freyman et al. 2006, Furlani et al. 2009, Lee et al. 2009d, Tatsumi et al. 2013, Toma et al. 2009, Vulliet et al. 2004)
are a safety threat to be considered. Possible pharmaceutical interventions to
prevent thromboembolus formation would primarily consist of using
anticoagulative agents. Tatsumi et al. reported successful suppression of MSC’s
procoagulative properties by applying anti-TF antibody or using factor VII
(Tatsumi et al. 2013). Suspension of IV transplanted MSCs in heparin in a mouse
model was highly beneficial, whereas application of MSCs suspended in
PBS/EDTA or control buffer caused severe pulmonary reactions and, in some
cases, death (Deak et al. 2010).
The use of anticoagulative agents could serve as a promising tool for
reducing the risk of MSC-induced thromboembolic complications. The possible
adverse effects of the thromoboembolic agents could be the risk of bleeding. The
clinical impact related to the increased risk of bleeding would depend on the
clinical setting in which the cells were used; the risk would be well tolerated in
physiological circumstances, but conditions, such as trauma, could alter the
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body’s coagulative system’s balance, thus resulting in the restricted use of
anticoagulative substances because of their potential hazards.
2.6.9 Cellular engineering and culture conditions
Altering the characteristic of the transplanted cells prior to the transplantation
could provide a way to improve their biodistribution and therapeutic capacity.
One interesting option to increase the targeting of therapeutic cells would be to
add a specific tag on the cell surface. For example, chemical modification of the
BM-MSC surface with sialyl Lewisx, the moiety for selectin recognition,
improved the homing of MSCs to inflamed tissue (Sarkar et al. 2011). The
homing of MSCs to ischemic myocardium has been successfully increased by
applying a peptide-based tag (Kean et al. 2012), which also improved E-selectin-
mediated adhesion and rolling of MSCs (Cheng et al. 2012). The bone marrow
homing of BM-MSCs was shown to increase with glycan engineering of CD44
(Sackstein et al. 2008).
Altering culture conditions provide another method of manipulating MSC
biodistribution capacity. Low-density culturing increased the expression of
podocalyxin-like protein and CD49f on the cell surface, resulting in better
migration in a MI model (Lee et al. 2009d). Cytokine pretreatment and hypoxic
culture conditions induced migration, engraftment, and therapeutic potential of
MSCs (Hemeda et al. 2010, Hung et al. 2007, Rosova et al. 2008). These
applications could offer potential benefit in the future and enhance the therapeutic
properties and tissue targeting of the MSCs.
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69
3 Aims of the study
I How do BM-MNCs and MSCs distribute to organs when transplanted
intravenously or intra-arterially in an intact porcine model and does the intra-
arterial route decrease lung accumulation? Does intravascular transplantation
of these cells cause pulmonary embolisms?
II Can intra-arterially transplanted BM-MNCs reach the brain after a global
ischemic brain injury and is there a culmination point of migration during the
first 24 hours?
III Does transient proteolytic modification of MSCs with pronase decrease lung
accumulation and improve migration to target organs and how does pronase
affect cell characteristics?
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71
4 Materials and methods
4.1 Experimental animals (Studies I, II, III)
The porcine studies (Studies I-III) were conducted with female domestic pigs
weighing between 18-35 kg. Study III used female Wistar Han rats and 7–8-
week-old female mice. All the studied animals received humane care in
accordance with the “Principles of Laboratory Animal Care” formulated by the
National Society for Medical Research and the “Guide for the Care and Use of
Laboratory Animals,” prepared by the Institute of Laboratory Animal Resources,
National Research Council (published by the National Academy Press, revised in
1996). The porcine studies were approved by the Research Animal Care and Use
Committee of the University of Oulu, Oulu, Finland. The Study III rodent
protocols were approved by the ethical committee of Helsinki University Central
Hospital, the Finnish Red Cross Blood Service and the National Animal
Experiment Board in Finland.
4.2 Perioperative protocol of porcine model (Studies I, II and III)
The animals were sedated with ketamine hydrochloride (15 mg/kg) and
midazolam (2 mg/kg). Anaesthesia was induced with thiopental and fentanyl
followed by endotracheal intubation. The anaesthesia was maintained by a
continuous infusion of fentanyl (25 µg/kg/h), midazolam (0.25 mg/kg/h),
pancuronium (0.2 mg/kg/h), and inhaled isoflurane (1.0%). The animals received
intravenous Ringer’s Acetate (15 mL/kg/h) throughout the experiment.
An arterial catheter was positioned into the right femoral artery for arterial
pressure monitoring and blood sampling. A thermodilution catheter (CritiCath, 7
French [Ohmeda GmbH & Co, Erlangen, Germany]) was installed via the right
femoral vein into the pulmonary artery, allowing sampling of blood, monitoring
of central venous pressure, pulmonary artery pressure and pulmonary capillary
wedge pressure, and the recording of blood temperature and cardiac output. Mean
arterial pressure was maintained at a minimum level of 65 mmHg with fluids and
norepinephrine infusion.
Arterial blood gases, pH, electrolytes, serum ionised calcium, glucose,
haemoglobin levels (i-STAT Analyzer, i-STAT Corporation, East Windsor, NJ),
basic blood count, leukocyte differential count (Cell-Dyn Analyzer [Abbot, Santa
72
Clara, CA]), creatine kinase-MB ([Hydrasys LC-electrophoresis, Hyrys-
densitometry, Sebia, France]) and troponin-I (Immunoassay, Innotrac AIO, Turku,
Finland) were measured at the baseline.
4.3 Porcine MSCs and BM-MNCs (Studies I, II and III)
Animals assigned to receive BM-MNC’s underwent bone marrow harvesting after
the induction of anaesthesia resulting in autologous transplantation. Bone marrow
was harvested from both tibial bones with a sternal puncture needle and the
aspirate was collected into syringes containing 10 000 IU’s of heparin sulphate.
The bone marrow aspirate (1:5 dilution in phosphate-buffered saline [PBS],
Sigma-Aldrich) was subjected to density gradient centrifugation (Ficoll-Paque
Plus, Amersham Biosciences) to exclude erythrocytes and granulocytes. Bone
marrow mononuclear cells were collected from the interphase and washed 3 times
with PBS. The cells were suspended in a saline solution at a density of 25-50 x
106 cells/mL. Trypan blue dye, which was added to a fraction of isolated cells,
confirmed viability of more than 99% of the transplanted cells.
Bone marrow aspirate was collected and MNCs were isolated from a single
healthy donor animal, as described in the previous section. BM-MNCs were
plated in a cell culture flask at a density of 300 000 cells per cm2 in Minimum
Essential Medium Eagle alpha modification (Sigma-Aldrich) supplemented with
10% fetal calf serum (Promo Cell), 100 U/ml penicillin, 0,1 g/l streptomycin, 20
mM HEPES and 2 mM L-glutamine (all reagents from Sigma-Aldrich unless
otherwise indicated). The cells were allowed to attach for two days after which
the medium was replaced. Half of the medium was replaced every 3-4 days and
the cells were passaged at near confluence. The cells were used for
transplantation at passages 3 or 4.
4.4 Human MSCs (Study III)
Umbilical cord blood (UCB) collections were performed at the Helsinki
University Central Hospital (HUCH) and Helsinki Maternity Hospital (Finland).
Bone marrow (BM) was collected from an unaffected bone site from patients
operated on for osteoarthritis (> 50 years of age) or scoliosis (< 20 years of age).
All donors gave their informed consent prior to sample collection, and the study
protocols were approved by the ethical committee of HUCH, the Finnish Red
Cross Blood Service (FRCBS), and Oulu University Hospital (Finland). Human
73
UCB-MSC cultivation was performed, as previously described (Laitinen et al. 2011). BM-MSC cultivation was performed, as described previously (Leskela et al. 2003).
4.5 Rat MSCs (Study III)
Wistar rat MSCs (Cyagen Biosciences Inc., Sunnyvale, CA, http://www.cyagen.
com) were cultured according to the manufacturer’s instructions.
4.6 Pronase detachment and cell viability (Study III)
Pronase is a nonspecific protease mixture isolated from Streptomyces griseus. The
subconfluent cells were detached with 0.5% pronase (Roche, Mannheim,
Germany, http://www.roche.com) in phosphate-buffered saline/0.25 mM EDTA.
Different concentrations of pronase, ranging from 0.05 to 1%, were also tested.
Trypsin (TryPLe Express; Life Technologies, Paisley, U.K., http://www.lifetech.
com) detachment was always used as a control. As with trypsin detachment,
pronase detachment was always stopped with excess culture medium within 4
minutes from the time of the addition of the enzyme to protect the detached cells.
Subsequent centrifugation and cell re-suspension were performed to remove the
enzymes. Cell viability was determined by trypan blue exclusion or analysis using
a Nucleocounter NC-100 (Chemometec, Lillerod, Denmark, http://www.
chemometec.com).
Viability was routinely determined in 30 individual pronase-detached cell
samples. Cell morphology was observed and documented by microscopy. For the
protein recovery studies, cells were incubated in culture medium for 5–7 hours at
37°C after detachment with subsequent cell viability measurements. Gentle
agitation of cells was done to prevent the attachment of cells. In addition, the
depolarization of mitochondrial inner membrane potential (ΔΨm) was analyzed
from differently detached BM-MSCs. Re-adherence to culture vessels and growth
kinetics of pronase-detached cells were also investigated and compared with
trypsinized cells.
4.7 Cell surface analysis by mass spectrometry (Study III)
UCB-MSCs were used for mass spectrometry (MS) analysis of cell surface
proteins after trypsin and pronase detachment and processed directly or after 5
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hours of post-detachment recovery. The cell surface proteins were biotinylated.
The labelled proteins were harvested with magnetic beads prior to liquid
chromatography (LC)-MS analysis.
4.8 Cell surface analysis by flow cytometry (Study III)
UCB-MSCs and BM-MSCs were analysed for cell surface epitope expression
immediately after use of different detachment protocols and after 5–7-hours of
post-detachment recovery. The antibodies against the following proteins were
used: CD44, CD49d, CD49e, CD73, CD90, CD105, HLA-DR, CD14, CD19,
CD34, CD45, CD13, CD29, CD49c, CD54, CD59, CD106, CD146, CD147
(Abcam, Cambridge, U.K., http://www.abcam.com), CD166, CD184, fibronectin
(FN) (Abcam), galectin-1 (Acris Antibodies, Herford, Germany, http://www.acris-
antibodies.com), and chondroitin sulfate proteoglycan (CSPG)-4. All antibodies
were purchased from BD Biosciences (San Diego, CA, http://www.bdbiosciences.
com) unless stated otherwise.
The cells were labelled with 2 µl or 0.5–1 µg (CD147, FN, galectin-1,
CSPG4) of the antibodies per 1 x 105 cells. Secondary antibody staining was
performed for CD147, FN, galectin-1, and CSPG4. The labeled cells were run
with a FACS Aria (BD Biosciences) flow cytometer, and the results were
analysed with FACSDiva Software (BD Biosciences). Appropriate isotype control
antibodies were also used.
4.9 T-cell proliferation assay (Study III)
Peripheral blood mononuclear cells (PBMNCs) were isolated from buffy coats
from anonymous blood donors (FRCBS) by density gradient centrifugation
(Ficoll-Paque Plus, GE Healthcare, Piscataway, USA) and labeled with 5 µM
5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) solution
(Molecular2 Pronase Detachment of MSCs Probes, Eugene, OR,
http://probes.invitrogen.com). CFSE-labeled PBMNCs were cocultured with
trypsin- or pronase-detached UCB-MSCs (n = 2) in RPMI with 10% fetal bovine
serum (FBS), 100 U/ml penicillin + 0.1 mg/ml streptomycin (Life Technologies).
MSC:MNC ratios of 1:10, 1:20, 1:50, and 1:100 were used. To activate T-cell
proliferation, 100 ng/ml of the anti-human CD3 antibody clone Hit3a
(BioLegend, San Diego, CA, http://www.biolegend.com) was used. Non-
stimulated CFSE-labelled cells and stimulated/nonstimulated non-labelled
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PBMNCs were used as controls. T-cell proliferation was analysed after 4 days as
dilution of CFSE intensity by FACSAria flow cytometry (BD Biosciences) and
FlowJo software (version 7.6.1; Tree Star, Ashland, OR, http://www.treestar.com).
4.10 Angiogenesis assay (Study III)
Angiogenesis was studied in a validated angiogenesis co-culture model (Sarkanen et al. 2011) using human fibroblasts (American Type Culture Collection, Boras,
Sweden, http://www.atcc.org) and human umbilical cord vein endothelial cells
(HUVECs). Fibroblasts and HUVECs were co-cultured for 7 days before adding
differently detached UCB-MSCs. UCB-MSCs were also plated in inserts
(Scaffdex, Tampere, Finland, http://www.scaffdex.com). The following media
were used: basic test medium (BTM) (Endothelial Basal Medium-2 with 2.0%
FBS, 0.1% Gentamicin, Amphotericin-B, 1% L-glutamine; Lonza Group Ltd.,
Basel, Switzerland, http://www.lonza.com), BTM with angiogenic growth factors
for positive controls, and UCB-MSC medium. Technical validity was ensured
with positive (n= 4) and negative controls (BTM, n= 3; UCB-MSC medium, n=
2). On day 13, tubules were visualized with anti-von Willebrand factor staining
and analysed as described (Sarkanen et al. 2011).
4.11 In vitro rolling assay (Study III)
A Diaphot TMD inverted-stage microscope (Nikon, Tokyo, Japan,
http://www.nikon.com) configured with phase contrast, objective lens (4x),
MicroPublisher 5 megapixel camera (QImaging, Surrey, BC, Canada, http://www.
qimaging.com) and MCID Core image analysis program (InterFocus Imaging
Ltd., Cambridge, U.K., http://www.mcid.co.uk) was used to count the attached
cells and their average rolling speed on FN-coated (0.5 µg/µl) capillaries
(diameter, 0.93 mm; length, 12.5 cm). An Alaris-Asena syringe pump
(CareFusion Corp., Palm Springs, CA, http://www.carefusion.com) was used for
perfusion with a calibrated flow speed (5 ml/hour). The cells (20,000 cells per
milliliter) were imaged for 2 minutes in each analysis.
4.12 Labelling of the porcine cells (Studies I, II and III)
The cells were labelled with Tc99m-HMPAO (Ceretec®, Amersham Healthcare)
using a standard technique for leukocyte labelling (Vorne et al. 1989). In brief,
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534 – 702 MBq 99mTc-HMPAO was added to the BM-MNC suspension, and left
for 15 – 20 min at room temperature. The cell suspension was centrifuged in
sterile tubes at 300 x G for 10 min. The supernatant was then separated from the
BM-MNCs, and the cells were re-suspended in 2 ml saline.
The whole BM-MNC population was first labelled, then the dose
standardised to the weight of the animal (5 x 106 cells/kg) was isolated and
suspended in 10 cc of saline. In the subjects with MSC transplantation, the
amount of cells that first underwent labelling was 3-6 x106 and the standardised
dose was 5 x 105 cells/kg. Control samples of 50 000 and 100 000 were
administered into vials for later use as gamma counter references. After the
standardised dose was rationed, the activity of the syringe was measured. An
activity level of 90 MBq was set as a requirement, as lower activity could
compromise radionuclide imaging quality. If the threshold activity level was not
reached, the experiment was terminated.
4.13 Intravenous transplantation (Studies I, II and III)
An intravenous route was established by installing a Schwan-Ganz-type
triluminal pulmonary arterial catheter. An incision was made in the left groin in (I,
III) or the right groin (II). Femoral artery and vein were exposed and ligated. The
catheter was carefully inserted from an incision to the vessel wall. Location of the
tip of the catheter was confirmed by typical pressure curve and wedge curve
caused by an inflated balloon (this described method is commonly used in clinical
practice). The cells were administered by applying the lumen with an opening
positioned in the right atrium. After slow injection, the catheter was then rinsed
with 50 cc of saline.
4.14 Intraventricular transplantation (Study I)
An intraventricular route was established by installing a coronary arterial catheter
into the left ventricle. The catheter was inserted through the left femoral artery
after open surgical preparation. Arterial pressure was measured in these animals
using the catheter instead of an arterial needle. Fluoroscopic imaging and typical
pressure curve were used to confirm the intraventricular location. The cells were
transplanted through the arterial catheter followed by rinsing of the catheter with
saline. After the transplantation, the tip of the catheter was then retracted from the
ventricle to avoid prolonged interference of aortic valve function.
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4.15 Ischemic brain insult (Study II)
A left thoracotomy was performed from the second intercostal space. The
brachiocephalic trunk and the left subclavian artery were clamped for a period of
7 minutes after systemic application of heparin. The response to sustained
cerebral blood flow was assessed from the rapid depression of BSR in EEG,
which was considered as validation of significant brain ischemia. After releasing
the clamps, 120 mg papaverinehydrophosphate was applied to the vessels.
4.16 Radionuclide imaging (Studies I, II and III)
The animals were kept under anaesthesia for 8 hours followed by transfer for
radionuclide imaging. Hemodynamic parameters and vital functions were
monitored and maintained during the follow-up period. Radionuclide images were
acquired using a hybrid camera combining a gamma camera with a CT camera
within the same cantry (Symbia T2; Siemens Medical Solutions USA, Inc.). A
planar whole-body scan was performed after the follow-up period, with both
anterior and posterior images being acquired at a speed of 5 cm/min using low-
energy high-resolution collimators, a 256 × 1024 matrix, and a 140 keV ± 20%
photopeak (I, II and III). After a planar scintigraphy, the SPECT study was
obtained over a 360º arc, using 96 frames at 90 s per frame. The images were
acquired into a 128 × 128 matrix. The CT part was acquired at a slice step of 3
mm, a current of 120 mAs, and a voltage of 130 kV. Whole body SPECT (I, III)
or head SPECT only were acquired (II).
4.17 Image and data analysis (Studies I, II and III)
Dual-head whole-body acquisitions were used to calculate geometric mean
(Gmean) datasets to estimate organ uptake of BM-MNCs (I, II) and MSCs (I, III).
Regions of interest (ROI) were drawn around the lungs, liver, spleen, kidneys,
and whole body. Gmean was calculated for the different ROIs using the formula
Gmean = square root (anterior × posterior), in which the anterior is the number of
counts from the anterior projection and the posterior is the number of counts from
the posterior projection. Mean activity of individual organ ROIs were normalised
to whole-body mean activity to achieve comparable data between subjects.
SPECT images were analysed by Hermes Hybrid PDR software (Hermes
Medical Solutions, Stockholm, Sweden). ROIs were drawn around target organs
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according to anatomic location, as assessed by CT. The ROIs were then copied to
the SPECT data, so their positions and areas were equal. Target organ ROIs were
drawn on preset single slices with an aim to minimize variation between subjects;
the slices were chosen based on clear recognition for repeatability and maximal
avoidance of scattering activity of nearby organs. Mean activity values of ROIs,
provided by analysis software, were used in data analysis to compare individual
organs.
When assessing the lung accumulation (I, III), the ROIs were drawn in all
slices presenting lung tissue. The ROIs were then combined to form a volume-of-
interest (VOI), which was copied to SPECT data, and VOI’s mean activity was
calculated using VOI’s total activity and cell volume. Formation of atelectasis
was confirmed by a radiologist’s expert opinion and VOI of atelectasis areas were
individually measured. To assess the atelectasis’ impact on lung accumulation,
lung VOI mean activity was also calculated after reducing the atelectasis’ VOI
total activity and volume from the corresponding whole lung values, resulting in
mean activity of non-atelectatic lung parenchyma.
4.18 Pulmonary embolism CT- scan (Study I)
In addition to the twelve animals forming the primary study groups, a total of four
pigs underwent a lung embolism CT-scan with radiocontrasting agent infusion
after performing the radionuclide imaging and preceding study protocol. Two (2)
of the animals received BM-MSC transplantation intravenously and two (2)
underwent sham operation with cell transplantation replaced with a similar
volume of saline infusion. The animals were euthanized after the CT-scan and no
tissue biopsies were collected because the execution of the CT-scan could
compromise the acquired data due to infusion of radiocontrasting agent with
possible adverse effects on kidneys that could bias the cell distribution.
4.19 Tissue biopsies (Studies I, II and III)
The animals were euthanized by intravenous injection of pento-barbital. An
autopsy was performed and biopsies were acquired from target organs. Biopsies
were collected from both cerebral hemispheres, the cerebellum, the thymoid
gland, the right atrium of the heart, the apex of the heart, the liver (2 biopsies), the
spleen (2 biopsies), the mesenteric lymph nodes and both kidneys. The biopsies
were inserted into vials and placed in an automated gamma counter (Wallac
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Wizard 1480, Perkin Elmer, Gaithersburg, MD, USA). Counts were also
measured from the control samples containing 50 000 and 100 000 cells and used
to calculate biopsy activity index to compare subjects based on biopsy sample
weight and measured activity of control samples and the biopsy sample (results
expressed as counts per minute) (CPM) (I, III).
The following formula was used:
Activity index = (organ CPM / organ weight) / ((50 000 cells CPM / 50 000) + (100 000 cells CPM / 100 000) / 2) = cell count / weight (g) of target organ.
4.20 Electroencephalography (Study II)
Electroencephalography (EEG) electrodes were inserted into the skull. Four
electrodes consisted of two recording electrodes, a reference electrode and a
grounding electrode. Recording began 5 minutes before the ischemic insult and
this period was used as a baseline reference of the burst-suppression ratio (BSR).
Data from EEG were constantly recorded with a NeuroScanSynamps Amplifier,
equipped with NeuroScan 4.0 Software (Neuro Soft Inc., Sterling,VA), using a
sampling rate of 5000 Hz. Isoflurane anaesthesia was maintained constant at 1%
to prevent alteration in BSR. Recovery was assessed by the time it took the
subjects to reach 50 and 100% of the baseline BSR.
4.21 Biodistribution of human MSCs in a mouse model (Study III)
Differently detached UCB-MSCs (p4) and BM-MSCs (p6) werelabelled with
Tc99m-HMPAO (Ceretec; GE Healthcare, http://www.gehealthcare.com) for 15
minutes at room temperature followed by determination of cell viability.
Subsequently, 7–8 week-old female mice received 5 x 105 cells IV in 100 µl of
saline (UCB-MSCs: n= 3; BM-MSCs: n= 5). One hour or 15 hours post-injection
mice were sacrificed, and lungs, heart, liver, spleen, pancreas, kidneys,
gastrointestinal (GI) tract, and femur were collected. Radioactivity of the tissues
was determined with a gamma counter (Wallac Wizard 1480; PerkinElmer,
Gaithersburg, MD, http://www.perkinelmer.com). The relative radioactivity for
each tissue was calculated as a percentage from the total amount of radioactivity
in all the tissues measured. Additionally, the biodistribution of UCB-MSCs
labelled with the fluorescent dye PKH-26 was studied in mice.
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4.22 Biodistribution of allogeneic BM-MSCs in a rat model of inflammation (Study III)
Female Wistar Han rats were used in the study. λ-Carrageenan (Sigma-Aldrich,
St. Louis, MO, http://www.sigmaaldrich.com) was dissolved in saline to give a
4% solution, and 150 µl was administered into the plantar region of the left hind
paw immediately before cell injection. Subsequently, animals received either
trypsin-detached (n= 3) or pronase-detached (n=4), Tc99m-HMPAO-labelled rat
BM-MSCs (1 x 106) into the tail vein. Whole-body SPECT-CT (nanoSPECT/CT;
Bioscan Inc., Washington, DC, http://www.bioscan.com) images were acquired
50 minutes, 3 hours, 5 hours, and 24 hours post-injection.
Only one animal per group was imaged at the 24-hour time point, and it was
excluded from the statistical analysis. The volume of the inflamed paw was
measured with a plethysmometer (Ugo Basile, Comerio, Italy, http://
www.ugobasile.com) immediately before and 1 minute after the injection and
directly after imaging. SPECT images were reconstructed with HiSPECT NG
software (Scivis GmbH, Gottingen, Germany, http://www.scivis.de) and fused
with CT data sets using InVivoScope software (Bioscan). VOIs were drawn
around lungs, kidneys, paw, and liver. The same VOIs were used for all
subsequent analyses of corresponding organs. The radioactivity in each VOI was
calculated and corrected for decay. Results are presented as a percentage of the
injected dose.
4.23 Statistical analysis (Study I, II, and III)
IBM SPSS Statistics version 20.0 was used for statistical analysis of the data. The
study groups were compared on transplantation route and cell lineage as 6 versus
6- setup with nonparametric Mann- Whitney U-test for calculating p-values.
Statistically significant differences were considered at p < 0,05. Medians with a
25-75 percent interquartile range were used when reporting values with 6 versus
6- setup. Further comparison was made between subgroups of the same cell
lineage or transplantation route with 3 versus 3- setup; value ranges were reported
and used to assess differences between the subgroups. Data for Study III were
processed with Mascot Distiller (version 2.3; Matrix Science Ltd., Boston, MA,
http://www.matrixscience.com) and searched with Mascot Server (version 2.2.06;
Matrix Science) against human proteins in the UniProtKB database (version
15.12). LC-MS differential expression analysis was performed with Progenesis
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LC-MS software (version 2.6; Nonlinear Dynamics Ltd., Newcastle upon Tyne,
U.K.).
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83
5 Results
5.1 IA versus IV transplantation of BM-MNCs and MSCs (Study I)
There were only small differences in the biodistribution patterns between the
three nuclear medicine methods (Tables 5-7, Figures 2 and 3). The effect of the
administration route was smaller in the BM-MNC lineage than in the BM-MSC
lineage. The most striking difference between the two lineages was the
accumulation of the cells in the spleen and vertebrae, with the MNCs having a
much higher accumulation in those organs. The BM-MSCs, in turn, had much
higher uptake in the kidneys. As expected, the majority of the IV-transplanted
BM-MNCs localised to the lungs, followed by the liver, spleen and kidneys. The
IV administration of BM-MSCs resulted in the highest deposition in the lungs,
followed by kidneys, liver, with an equal accumulation being found in the spleen
and vertebrae. The IA route decreased BM-MSC uptake in the lungs (Figure 2)
and increased it in other organs, especially the liver. The IA administration route
did not have such a major impact on BM-MNC lung deposition and its effect on
the other organs was variable. Interestingly, BM-MNC and BM-MSC uptakes
were much higher in the right lung than in the left lung. The atelectic areas had an
approximately two-fold deposition of BM-MNCs and BM-MSCs compared to the
non-atelectic areas. There was no evidence of between-group differences in the
number of atelectic areas.
5.1.1 Computed tomography
Those animals, which underwent helical contrast-enhanced CT did not show any
signs of pulmonary embolism, but they had some atelectic lung areas, as
confirmed by two radiologists.
84
Table 6. Biodistribution pattern, as assessed from the planar whole-body images.
Lineage Route Lungs Liver Spleen Kidneys
BM-MNCs IV 24-30 4-9 3-7 1-2
IA 16-21 11-14 5-7 1-1
BM-MSCs IV 27-31 2-3 0-2 3-4
IA 13-17 9-12 1-2 6-7
Geometric means were used to assess percentage organ activity of the whole body geometric mean. BM-
MNCs: bone marrow-derived mononuclear cells; BM-MSCs: bone marrow-derived mesenchymal stem cells; IV:
intravenous; IA: intra-arterial (n=3 in each group).
Table 7. Biodistribution pattern, as assessed from SPECT/CT images (percentage of
input activity).
Lineage Route Lungs Liver Spleen Kidneys Vertebrae
BM-MNCs IV 4.7-11.3 1.6-2.8 2.9-4.8 0.4-1.5 2.2-3.5
IA 3.6-6.4 2.5-4.1 2.8-4.7 0.6-1.0 1.2-2.5
BM-MSCs IV 3.6-7.0 0.3-0.5 0.1-0.1 1.1-3.2 0.1-0.1
IA 2.3-3.2 1.3-2.0 0.3-1.0 1.8-4.2 0.2-0.4
BM-MNCs: bone marrow-derived mononuclear cells; BM-MSCs: bone marrow-derived mesenchymal stem
cells; IV: intravenous; IA: intra-arterial (n=3 in each group).
Table 8. Biodistribution pattern, as assessed from activity in tissue biopsies (activity
index).
Lineage Route Right lung Left lung Liver Spleen Kidneys
BM-MNCs IV 7296-11840 3675-7835 1586-2158 3556-6161 731-1119
IA 3138-6618 2446-6579 2620-3418 3885-6363 1051-1618
BM-MSCs IV 8348-10450 2497-11010 209-348 63-77 2709-3689
IA 3118-4943 1155-2323 1246-1759 515-1807 3209-4622
BM-MNCs: bone marrow-derived mononuclear cells; BM-MSCs: bone marrow-derived mesenchymal stem
cells; IV: intravenous; IA: intra-arterial (n=3 in each group)
85
Fig. 2. Posterior whole body gamma camera images acquired after intravenous (IV) or
stromal cells (BM-MSC) or bone marrow mononuclear cells (BM-MNC).
86
Fig. 3. SPECT- CT analysis of accumulated cells in the lungs and bone at 8 h. Range
bars in A demonstrate the range of lung accumulation. In panel B, a similar analysis
was performed for the L3 and L5 vertebrae.
87
Fig. 4. Summary of biodistribution data from planar scan images, SPECT/CT, and
biopsy gamma counter analysis. The biodistribution of transplanted cell at 8 h was
analyzed in all major organs. Range bars represent individual organ values for each
subgroup.
5.1.2 Haemodynamic data
Comparison of subgroup baseline characteristics is presented in Table 8, with no
major differences being observed. None of the study animals showed evidence of
pulmonary hypertension in combination with other symptoms of embolus
formation; thus it was concluded that none of the animals developed
haemodynamically significant pulmonary emboli. Hypotension was observed in
some of the animals after prolonged anaesthesia and blood pressure was
maintained constant with crystalloid infusion and L-norepinephrine and dopamine
infusion if needed. Inotropic support was given to 2 of the animals in the IV BM-
MNC group, 2 of the animals in the IA BM-MNC group and 1 of the animals in
the IV BM-MSC group. The underlying cause of hypotension was clinically
diagnosed to result from possible infection or reaction to the used anaesthetic
agents, and therefore did not appear to be related to cell administration in any of
the animals.
88
Table 9. Baseline characteristics of study groups (Study I).
Route IV IA
Lineage BM-MNCs BM-MSCs BM-MNCs BM-MSCs
Weight 22.3-25.3 20.6-25.6 20.5-23.8 18.1-23.3
Heart rate 106-117 73-132 96-139 89-150
RRsys 85-101 101-151 69-133 100-140
RRdia 37-54 52-95 57-85 62-77
MAP 54-71 71-118 63-108 78-106
PAsys 14-19 19-29 27-57 20-22
PAdia 7-14 11-22 21-44 10-14
CVP 3-7 4-9 8-9 5-10
PCWP 3-4 1-9 12-14 3-8
C.O. 2.66-3.16 2.47-6.50 2.19-3.97 2.75-3.52
TPA 38.8-39.7 38.4-40.5 36.6-38.5 38.1-38.9
pH 7.52-7.55 7.42-7.53 7.35-7.55 7.52-7.85
pCO2 5.02-5.89 4.90-7.21 5.130-6.43 3.130-5.08
pO2 29.7-34.0 24.6-29.6 32.5-42.0 22.4-38.1
Weight (kg), Heart rate (beats per minute), RRsys = systolic blood pressure (mmHg), RRdia = diastolic blood
pressure (mmHg), MAP = mean arterial pressure (mmHg), PAsys = pulmonary artery systolic blood
pressure (mmHg), PAdia = pulmonary artery diastolic blood pressure (mmHg), CVP = central venous
pressure (mmHg), PCWP = pulmonary capillary wedge pressure (mmHg), C.O. = cardiac output, TPA =
pulmonary arterial temperature (C°), pCO2 = arterial blood carbon dioxide partial pressure (kPa), pO2 =
arterial blood oxygen partial pressure (kPa).
5.2 BM-MNC transplantation for global ischemic brain injury during
first 24 hours (Study II)
Severe but non-lethal ischemia was chosen based on 4 animals that were used as
pilots; clamp intervals tested were between 5 and 15 minutes. Rapid burst
cessation was observed after clamping in all animals. Median time to reach
complete EEG silence was 0:00:20 (0:00:18-0:00:23). The BSR remained at zero
level in all animals after the insult, slowly recovering to 50% at a median time of
0:30:00 (0:25:00-0:45:00) and to a level of 100% at a median time of 1:45:00
(1:25:00-2:00:00). Two animals with a noticeably faster BSR silencing rate also
failed to recover BSR to baseline levels. Recovery data was lost from one subject
due to data corruption.
89
5.2.1 Proportional counts from biopsies
Activity was divided by biopsy weight, and results were used to form an activity
index for the purpose of easing interpretation and comparability between subjects.
Mesenteric lymph node biopsies had minimal variation between subjects and
were thus chosen as a reference. The activity index was calculated by dividing the
target biopsy index by the mesenteric index and then multiplying by one hundred.
Brain activity proportion remained clearly below 0.6 % for all organs in all
animals. There was no trend towards a higher level of migration nor were major
differences in biodistribution observed in any of the follow-up groups at any
given time. At 4 and 6 hours, however, the number of cells was the highest. The
most up-take was observed in the lungs, the liver and the spleen (Table 9, Figure
5). No visual sign of migration to brain tissue could be detected from either planar
scintigraphy or SPECT-CT reconstructions of the head using the previously
described imaging equipment and image reconstruction software (Figure 6).
Activity could be observed in the skull bones and vertebrae, but no sign of the
transplanted cells could be detected within the brain parenchyma in any of the
subjects. Organ Gmean values were compared with the whole body values (Table
10).
90
Ta
ble
10
. N
orm
ali
sed
bio
ps
y c
ou
nts
Follo
w-u
p R
ight
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R
ight
lung
Le
ft lu
ng
Thym
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Live
r S
plee
n
2h
4.1
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3,
6 0.
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3,9
0.1%
12
6036
%
332
10%
62
1.
8%
552
16%
12
70
36%
2h
7.4
0.1%
5,
1 0.
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6,0
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19
9018
%
3940
35
%
188
1.7%
14
2013
%
3730
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%
4h
10.7
0.
2%
13,3
0.2%
33
,80.
6%
1820
30%
14
80
24%
78
1.
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1010
17%
16
50
27%
4h
13.8
0.
1%
15,5
0.1%
18
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1%
5120
31%
74
40
45%
56
0.
3%
1240
8%
2790
17
%
6h
51.6
0.
2%
38,4
0.2%
26
,60.
1%
8590
33%
10
600
41%
46
0.
2%
1530
6%
4980
19
%
6h
37.5
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7,
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3730
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42
30
33%
28
0.
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658
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4318
.133
%
12h
7.4
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6,
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8,4
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13
0023
%
2240
40
%
19
0.3%
39
6 7%
16
80
30%
12h
10.7
0.
1%
10,1
0.1%
13
,10.
1%
1100
10%
55
20
52%
53
0.
5%
1350
13%
25
90
24%
91
Follo
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20.7
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31.7
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30
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7470
34%
79
10
36%
43
0.
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1780
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4500
21
%
24h
11.1
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9.0
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13
.70.
1%
3410
31%
36
80
33%
12
8 1.
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367
3%
3500
32
%
Mea
n 10
.9
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11
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27
0028
%
4090
35
%
54
0.8%
11
2010
%
3150
27
%
Row
s re
pres
ent e
ach
of th
e ex
perim
enta
l sub
ject
s. E
ach
subj
ect's
org
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dexe
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tivity
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the
sum
of e
ach
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ect’s
inde
xes
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disp
laye
d on
the
right
. col
umns
92
Fig. 5. Box-plots demonstrate each organ’s percentage of the whole body value
calculated from the radionuclide planar image in Study II
Fig. 6. Head SPECT-CT acquired from a subject with cell transplantation at 24h time
point shows no sign of cell accumulation within the brain parenchyma in Study II. The
image is scaled to it’s individual maximum, which results cell uptake to the vertebrae
appear prominent.
93
Table 11. Percentages by organs of the whole body activity
Follow-up Brain Lungs Liver Spleen Kidneys
2h 0% 43.5% 14.3% 2.2% 4.2%
2h 0% 31.0% 16.4% 11.3% 1.7%
4h 0% 34.7% 16.9% 1.8% 2.7%
4h 0% 28.2% 12.1% 8.4% 3.2%
6h 0% 53.4% 12.6% 3.6% 1.2%
6h 0% 33.1% 14.2% 9.4% 2.1%
12h 0% 29.5% 11.8% 11.4% 3.6%
12h 0% 32.3% 17.9% 4.9% 2.3%
24h 0% 36.5% 20.9% 6.2% 2.8%
24h 0% 31.9% 9.0% 11.2% 2.0%
Mean 0% 32.7% 14.2% 7.3% 2.5%
5.2.2 Haemodynamic and biochemical data
The mean arterial pressure, central venous pressure, cardiac output and
pulmonary capillary wedge pressure in all follow-up groups remained comparable
throughout the experiment. No significant differences were detected between
study groups in leukocyte counts, haemoglobin, glucose, serum ionised calcium,
sodium, potassium or cardiac enzymes.
94
5.3 Effects of MSC pronase detachment on lung clearance and organ targeting (Study III)
The pronase detachment protocol produced viable, single-cell MSC suspensions
without any cell aggregates with > 90% viability (n > 30), which is equal to the
trypsin control. All tested pronase concentrations (0.05–1%) detached the cells
effectively within 4 minutes. The highest concentration (1 %) affected the cell
morphology slightly, although cell viability remained at > 90%. A concentration
of 0.5% pronase was chosen as a standard concentration, as it detached cells
rapidly with high viability and effectively cleaved certain cell-surface proteins.
Pronase-detached cells also adhered normally to culture vessels, proliferated, and
reached confluence similarly to trypsinized cells.
In addition, one BM-MSC line was selected for the analysis of ΔΨm.
Trypsin- and pronase- detached cells exhibited identical 5,5’,6,6’-tetrachloro-
1,1’,3,3’ tetraethylbenzimidazolcarbocyanine iodide (JC-1) staining patterns, and
most of the cells showed energized or partly depolarized mitochondria compared
with control (protonophore carbonyl cyanide m-chlorophenylhydrazone-treated
cells exhibited fully depolarized mitochondria as expected). Accordingly, this data
indicates that the use of pronase as a substitute for trypsin detachment does not
compromise the viability of MSCs.
5.3.1 The effect of pronase detachment on cell-surface protein epitopes
For 19 cell-surface proteins, pronase treatment decreased the abundance of
corresponding peptide features compared with trypsin treatment (e.g., CD166,
CD49e, CD44, CSPG4, CD29, CD105, CD49c, CD147, CD146, CD99, and
CD90). Also, secreted galectin-1 was digested by pronase. CD29, CD49c, and
CD59 had many novel, pronase-generated peptides, thus revealing a characteristic
cleavage pattern of pronase. Unfortunately, it was not possible to determine in
detail how the cleavage of these peptides affected the actual structure of the
proteins. Also, some peptides from cytosolic proteins had diminished peptide
feature abundance after the pronase detachment, which is probably due to the
cleavage of their interaction partner on the cell surface.
The flow cytometric analysis supported the MS analysis, as there was a clear
decrease in the binding of CD166, CD49e, CD44, CSPG4, and CD146 antibodies
in the pronase-detached cells compared with trypsin-detached cells. The intensity
of anti-CD105 binding was also slightly decreased. Instead, the cell-surface
95
expressions of galectin-1, CD29, CD49c, D147, CD59, and CD90 were similar
between trypsin- and pronase- detached cells, although the MS analysis had
indicated cleavage of those proteins by pronase. This discrepancy indicates that
the antibodies could recognize the protein epitopes that were still left on the cell
surface after the pronase detachment. Furthermore, the peptides with
glycosylation or other surface modifications could not be detected with MS.
The pronase cleavage of additional adhesion or migration-related proteins
was studied. Significant FN expression on MSCs has been previously described
(Nystedt et al. 2013), and this was further verified in immunofluorescence
staining of BM-MSCs. Pronase always consistently cleaved FN from the cell
surface. Also, CD49d and CD54 expression was lower on the cell surface after
pronase detachment compared with control cells. Their expression levels varied
slightly, even within the same cell line, most likely reflecting the effect of the
culture conditions on expression. The expression of CD106 and CD184 was low
on MSCs. The expression of CD73 was similar in both samples.
The recovery of cleaved cell-surface proteins was studied by flow cytometry.
CD44, CD49e, CSPG4, and CD166 showed almost full recovery after a 7-hour
recovery period. Recovery of proteins was also seen in the MS peptide data for
both intracellular and membrane-bound proteins.
5.3.2 MSC Functionality
To study the effect of pronase on the multipotency or functional properties of
MSCs, the differentiation capacity, immunosuppressive properties, and
angiogenic potential of pronase-detached cells were compared with those of
control cells.
Differentiation
Pronase detachment did not affect the multipotency of MSCs, since both
adipogenic and osteogenic differentiation was successful and comparable to
results after trypsin detachment.
Immunosuppression
Based on the T-cell proliferation assay, the pronase-detached cells inhibited the
proliferation of activated T cells in a dose-dependent manner and just as
96
effectively as the trypsin-detached cells. This suggests that pronase detachment
does not weaken the immunosuppressive properties of MSCs. T-cell proliferation
was tested with two UCB-MSC lines against allogeneic PBMNCs from two
donors.
Angiogenesis
Both trypsin- and pronase-detached UCB-MSCs were able to strongly stimulate
angiogenesis in the in vitro assay, both directly after detachment and after a 5-
hour recovery. UCB-MSCs required their own culture medium to stimulate tubule
formation. The medium alone was also favourable for angiogenesis, as it
contained growth factors (e.g., platelet-derived growth factor-BB, which is a mild
inducer in this assay). In the test medium, the angiogenic effect of MSCs
remained rather weak. Furthermore, the angiogenic effect was clearly increased
when MSCs were grown in contact with the angiogenesis assay cells compared
with MSCs cultured in inserts.
In Vitro Rolling Assay
Contrary to other functional assays, the pronase detachment had a substantial
impact on BM-MSC adherence and rolling speed in an in vitro flow perfusion
assay. The number of cells having a rolling speed of greater than 220 μm/second
(fast rollers) was clearly higher (p = .056 with t test) in the pronase group (n =35;
mean rolling speed, 261 μm/second) compared with the trypsin group (n = 19;
mean, 221 μm/second). No difference was observed between the groups for the
slowly moving cells (slow rollers).
5.3.3 MSCs and lung clearance
The biodistribution of radioactively labelled BM-MSCs and UCB-MSCs was
studied in mice. More than 75% of the total radioactivity was detected in the
lungs in both cell detachment groups and for both cell types 1 hour after injection.
The rest of the activity was detected in the liver, kidneys, and GI tract, followed
by low radioactivity in spleen, pancreas, heart, and femur. At 15 hours post-
injection, most of the radioactivity was detected in the liver in both cell
detachment groups and for both cell types (49% in the BM-MSC control group
vs. 70% in the BM-MSC pronase group, p = 0.001). There was, however, a
97
significant difference in total radioactivity measured in the lungs at this time
point, with 32% for control BM-MSCs versus 7% for pronase BM-MSCs (p =
0.001). The amount of radioactivity in the lungs was per se lower for control
UCB-MSCs (3.6%), but in line with the BM-MSCs, as there was clearly less
radioactivity in the lungs for pronase UCB-MSCs (0.6%). Radioactivity was low
in all other organs studied, but generally slightly more radioactivity was observed
when using pronase-detached cells.
The reduced lung retention for pronase-detached cells was confirmed with
fluorescently labeled UCB-MSCs. Twenty hours post-injection, there were one-
fifth as many cells in the lungs in the pronase group than in the control group (p =
0.01), consistent with the results observed using the radioactively labelled cells.
Interestingly, the number of pronase-detached cells was higher in the spleen and
peripheral blood compared with control cells 1 hour post-injection (p = 0.001 and
p = 0.05, respectively). Pronase detachment had no visible effect on cell numbers
in spleen, BM, or blood 20 hours post-injection. Unfortunately, it was not possible
to analyze the liver with this method because of its high enzymatic activity.
Finally, the effect of pronase detachment on lung clearance was verified in a
large-animal model. Consistent with the mouse data, a significant reduction of
lung retention was observed for pronase- detached BM-MSCs in the homologous
porcine model (p= 0.05). In line with the rodent data, prominent radioactivity was
detected in the lungs, kidneys, liver, and spleen in both cell detachment groups
after the 8-hour follow-up. There was, however, more variance in the
biodistribution of the cells in the large animals compared with the mouse model.
No adverse effects due to cell injections were observed, and the porcine groups
had comparable hemodynamic and biochemical data. The lung CT scans revealed
perioperative atelectasis in some individuals in both groups. The effect of the
potentially damaged areas was evaluated, and it was concluded that the atelectasis
did not significantly impact the results.
5.3.4 MSC targeting to an area of injury
The therapeutic advantage of pronase detachment was further studied in a rat
model of carrageenan-induced inflammation. Remarkably, pronase-detached rat
MSCs migrated significantly more effectively to the inflamed area compared with
control cells (p = 0.001). Twice as much radioactivity was detected in the
inflamed paw in the pronase-group compared with the trypsin-group both 50
minutes (0.25 vs. 0.13%, respectively) and 3 hours (0.22 vs. 0.11%) post-
98
injection. In line with the previous observations made in mice, significantly (p =
0.01) less radioactivity was detected in the lungs of the rats receiving pronase-
detached MSCs at all time-points studied.
99
6 Discussion
6.1 Biodistribution after intravascular transplantation
The biodistribution pattern seen in Studies I, II and III is consistent with earlier
studies. A common pattern consists of strong lung deposition followed by lower,
but detectable, liver and spleen accumulation (Allers et al. 2004, Barbash et al. 2003, Barbosa da Fonseca et al. 2010, Chin et al. 2003, Detante et al. 2009,
Kraitchman et al. 2005, Makela et al. 2013, Vasconcelos-dos-Santos et al. 2012).
Commonly, the renal accumulation varies across studies. So far, all previous
studies have mainly focused on a specific organ as a therapeutic target and
investigators, including ourselves, have additionally reported overall
biodistribution findings. This lack of unbiased biodistribution studies without any
major ischemic event or other injury led us to conduct our current study with a
porcine model.
Strong BM-MNC homing to bone was observed in Study I. The BM-MNCs
are a mixture of different bone marrow lineage cells and it is possible that
different cell types are responsible for active accumulation within various organs.
BM-MNCs contain haematopoietic cells (HSCs) that are known to home to bone
marrow stem cell niches after administration (Nilsson et al. 1997). Interactions
between HSCs and BM-MSCs have been shown to occur in stem cell niches
(Mendez-Ferrer et al. 2010) and this phenomenon could affect each cell type’s
biological potency in different forms of restorative therapies in as yet unknown
ways. Other major differences in cell biodistribution, when comparing cell
material, were seen in the pattern of migration to the liver, the spleen and the
kidneys. The liver and spleen act as a blood cell reservoirs and so it could be
hypothesized that labelled white blood cells that form a major portion of BM-
MNC population, naturally migrate to these organs. It must also be considered
that the increased liver and spleen accumulation observed in our study after intra-
arterial administration could decrease cellular therapy effectiveness similarly as
lung accumulation – especially so, because we don’t know the exact cell
population that is responsible for the therapeutic effect. BM-MSC administration
resulted in higher activity in the kidneys; it can hypothesised that activity
measured from kidneys can either be a result of real cell migration or homing or
alternatively, Tc99m-HMPAO metabolism. This radiolabel is known to be secreted
via the kidneys, which is confirmed by visualisation of the urinary bladder.
100
6.2 Lung entrapment
Prominent lung entrapment was present in our porcine specimens in Study I, II
and III, as well as in murine and rat models used in Study III. Study II did not
reveal any obvious differences in lung deposition as a function of follow-up time.
This observation was in contrast to other works reporting diminishing lung
deposition (Chin et al. 2003, Kraitchman et al. 2005, Nystedt et al. 2013).
MSC lung retention may arise from passive entrapment of cells in small-
diameter lung capillaries (Schrepfer et al. 2007, Vilalta et al. 2008) or may be
related to an adhesion molecule-mediated phenomenon (Fischer et al. 2009, Karp
& Leng Teo 2009), as the cell-surface structures appear to at least partially
explain MSC lung retention and clearance (Nystedt et al. 2013).
Whatever the cause for lung adherence, it is obvious that this leads to
diminished biodistribution to other target organs (Fischer et al. 2009, Schrepfer et al. 2007). Intravenously injected cells always encounter the lung capillaries
before reaching the systemic arterial circulation, which leads to the natural
assumption that more cells are found in the lungs after IV administration. An
earlier study has shown that this lung entrapment might even lead to extravasation
and engraftment (Nystedt et al. 2013). We find it interesting that when injecting
the cells intra-arterially, the cells reach the lung after just a single pass through the
circulatory system and still major differences in their organ distribution can be
observed. It could be hypothesised that the cells are sequestered effectively and
specifically by the organ they primarily reach. If this uptake is specific, it might
lead to a change in the freely circulating cell population, especially in the case of
BM-MNCs. Direct cell migration to any target organ can be assumed to be
required for the stem cell paracrine effects (Ranganath et al. 2012). Thus, it could
be crucial that the cells are targeted as accurately as possible to the organ’s
vasculature.
A study by Lee et al. with TSG-6-mediated cardioprotection by hMSCs
entrapped in the lungs is a particularly interesting observation (Lee et al. 2009c).
These results imply that the therapeutic effects of the transplanted MSCs are at
least partially mediated by the portion of the cells trapped in the lungs. Studies
also imply that triggering of IBMIR may be needed for MSC paracrine effects
(Moll et al. 2012). It is still unclear whether decreasing the portion of the cells
deposited in the lungs could have some adverse effects on the outcome of the
cellular therapy. Future studies must compare the actual therapeutic effects with
natural or enhanced lung clearance-pronase detachment methods. Such methods,
101
which were used in Study III, would offer the opportunity for this comparison, as
we showed increased targeting of the cells to the damaged area after increased
lung clearance.
6.3 The effect of transplantation route on biodistribution
Study I demonstrates that the administration route and cell lineage have major
effects on the acute biodistribution of the transplanted bone marrow-derived cells.
Intra-arterial administration effectively decreases lung accumulation of
transplanted BM-MSCs and appeared to increase their proportion in the liver, the
spleen and the kidneys. When using BM-MNCs, similar variations of
biodistribution could also be observed, but the effect was not as distinct as seen
with the BM-MSCs. The BM-MSCs had a tendency to deposit in the lungs in
larger numbers compared to the BM-MNCs.
Vasconcelos-dos-Santos et al. and also Detante et al. compared BM-MSC
biodistribution differences between IV and IA routes in a rat model of cerebral
ischemia and both reported early lung accumulation and subsequent clearance
with very similar biodistribution to other organs (Detante et al. 2009,
Vasconcelos-dos-Santos et al. 2012). MSC biodistribution was also investigated
in humans. Rosado-de-Castro et al. conducted a biodistribution study on stroke
patients using delayed administration of BM-MNCs through IV or IA routes
(Rosado-de-Castro et al. 2013). They reported lower lung uptake and higher liver
and spleen uptake at the 2 hour time point when using the IA route. Similarly to
animal studies, the high lung uptake of the IV group diminished during the 24
hour follow-up and liver and spleen uptake increased. All these results are
confirmed by our data, which also shows that intra-arterial administration results
in lesser cell accumulation to the lung. Intra-arterial transplantation has also been
suggested as having more therapeutic potency than an intravenous transplantation
(Kamiya et al. 2008). Our observed BM-MNC distribution after the injection in
Studies I and II was comparable with other studies using an intra-arterial
transplantation setup (Correa et al. 2007, Makela et al. 2009).
6.4 Safety assessment
The lung manifestations seen in Study I were most likely due to atelectasis caused
by prolonged mechanical ventilation and the use of muscle relaxants, as both are
rather common phenomenon in clinical practice. Increase of labelled cells in the
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lung tissue consolidations could easily explain the activity peaks observed in this
study setup. Both cell lineages have been proven to migrate to injured lung tissue
(Araujo et al. 2010, Rojas et al. 2005). Study I did not reveal any evidence of
pulmonary embolism, as assessed by the contrast enhanced CT. Radiological
diagnostics has its intrinsic limitations and after observance of pulmonary
consolidations, natural concerns of safety arose, as microembolus formation has
also been reported (Furlani et al. 2009, Toma et al. 2009). Possible microemboli
at the subsegmental level cannot be totally excluded with CT-angiography, but the
method’s accuracy should reveal any life threatening emboli.
Recently, it has been suggested that tissue factor could activate thrombotic
events, which has been reported in one case to have led to the death of an
individual who received adipose tissue MSCs (Tatsumi et al. 2013). Pulmonary
hypertension, a classic haemodynamic manifestation of pulmonary embolism,
when physiologic abnormalities of pulmonary embolism patients are assessed
(McIntyre & Sasahara 1971), could not be observed in any animal in Study I. The
porcine, murine and rat models of Studies II and III also did not reveal any
obvious safety issues associated with transplantation of UCBs or bone marrow
MSCs. In addition, other clinical symptoms of pulmonary embolism, such as
decreased arterial oxygen tension, decreased cardiac output or systemic
hypotension, were absent. CT-angiography did not show embolus formation in
the safety controls as well. It remains to be elucidated whether the embolus issue
is only limited to adipose tissue-derived cells, as has already been demonstrated
in mice (Furlani et al. 2009). To the best of our knowledge, there are no reported
adverse effects from the use of BM-MSCs or MNCs. This issue should be
investigated further, but theoretically adipose tissue might be more prone to have
active tissue factor, as it represents cells from the peripheral tissue and bone
marrow cells that also naturally co-exist with blood in the bone marrow.
In Study I, we found notably more lung accumulation of MSCs in the
animals with IV administration. It is likely that the administered cells scatter
throughout the lungs, thereby occluding the microvasculature, as described in
previous studies (Freyman et al. 2006, Furlani et al. 2009, Toma et al. 2009,
Vulliet et al. 2004). Instead of evaluating whether this microembolization occurs,
it is even more important to evaluate the impact on haemodynamics by measuring
increased pulmonary vascular resistance and right ventricular overload, which
could lead to circulatory collapse. Such an effect was not present in our study, and
a similar central venous route for BM-MNC administration was used in a human
trial with pediatric patients suffering from traumatic brain injury (Cox et al.
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2011), with no adverse effects being reported. Intravenous transplantation of
autologous MSCs for patients after myocardial infarction also did not reveal any
safety threats (Hare et al. 2009) and similar observations were seen in a study
with ischemic stroke (Lee et al. 2010b).
Rigorous attempts to reveal possible safety threats concerning the
intravascular MSC and BM-MNC transplantation are required. A realistic
approach is essential and possible safety factors should be assessed without
deprecating the possible hazards to ensure patient safety, but exaggerating the
possible adverse effects should also be avoided, as the therapeutic benefit of
cellular therapy could exceed them. When translating the data gained from
preclinical settings to clinical practice, the clinician should be aware of the
rationale for intravascular MSC transplantation to fully capitalise on the
therapeutic potential of these cells in a controlled way.
6.5 IA-transplanted BM-MNCs’ lack of homing to ischemic brain
In Study II, we were unable to detect any significant cerebral migration, which is
in conflict to earlier rodent studies (Li et al. 2001, Correa et al. 2007, Barbosa da
Fonseca et al. 2009, Barbosa da Fonseca et al. 2010). The number of other studies
reporting a negative result is small. No selective administration or focal ischemia
was used, which could explain the differences in the results. In a porcine model
this, however, would require overcoming problems related to selective
intravascular embolization, as the rete mirabilis arterial maze practically prevents
efforts of cerebrovascular cathetisation (Burbridge et al. 2004).
Before conducting Study II, we experimented on a porcine bilateral common
carotid artery occlusion model, which appeared to cause global ischemia
unreliably, and resulted in no alteration in EEG activity. This finding was
hypothesised to originate from circulatory anastomoses and led us to clamp both
the left subclavian artery and the brachiocephalic trunk. The chosen method
resulted in rapid BSR silencing followed by continuous attenuation and slow
recovery. Decreased BSR was associated with ischemic injury in previous studies
(Pokela et al. 2003). We attempted to produce a simplified and reproducible
model of global ischemia with limited biasing factors for the purpose of primarily
observing the behaviour of the transplanted BM-MNCs.
Systematically low results compared with expected migration levels resulted
in discontinuing Study II, as the lack of migration was evident. Our hypothesis
that no higher migration tendency, or statistically significant differences, would
104
have been detected in this experimental setup if the study was extended to further
time points or conducted with more animals. Lack of migration has also been
reported in a rodent study with a comparable experimental setup (Park et al. 2011). Our study was not conducted further with healthy control animals due to
ethical reasons, as no significant result could be observed in any of the
experimental animals.
An alternative explanation for the lack of homing in this model is the damage
threshold. In global ischemia, the brain tissue oxygen deprivation is life-
threatening after only a short exposure time. This might be insufficient in causing
the required change in the brain vascular endothelium, which ultimately controls
the final homing. With smaller ischemic exposure, the effect is more chronic and
hence more readily sufficient for endothelial activation without a fatal loss of the
brain function.
6.6 The effect of transplantation timing on homing
Intra-arterial transplantation after a stroke has shown to result in BM-MNC
migration to brain tissue at the 2h time point, but not at 24h in a rodent model
(Barbosa da Fonseca et al. 2010). Other studies investigating the impact of timing
on the transplantation’s therapeutic capacity have also generally suggested the
superiority of early transplantation (Barbash et al. 2003, Chen et al. 2001b, de
Vasconcelos Dos Santos et al. 2010, Iihoshi et al. 2004, Yang et al. 2011). This
led us to focus on observing potential cerebral migration during the first 24 hours
of the ischemic insult. Our study did not reveal any obvious culmination point of
migration. The twelve hour time point has been suggested as the peak of
migration in a rodent study with comparable setup (Keimpema E. et al. 2009
Feb).
An optimal window of transplantation is yet to be determined, but we can
postulate that the migration should have occurred during the follow-up in Study
II. The lack of cerebral migration does not rule out the possible therapeutic
benefits of the transplanted cells, as the involvement of indirect paracrine effects
is documented in various clinical settings (Timmers et al. 2007, Keimpema E. et al. 2009, Lee et al. 2009c,).
105
6.7 The effect of pronase detachment on MSC lung clearance
Study III results show that MSCs can be detached with pronase without altering
the MSC therapeutic capacity. It appears that pronase detachment transiently
modifies MSC surface adhesion proteins, which significantly accelerates the
clearance of these therapeutic cells from the lungs, as shown with both hMSCs in
a xenogeneic mouse model and also in homologous rat and porcine models. In
addition, increased number of pronase-detached cells could be observed in the
inflamed tissue compared with trypsin- detached cells, which suggests potential
beneficial effects of pronase detachment on targeting of MSCs to areas of injury.
As seen in earlier studies (Allers et al. 2004, Barbash et al. 2003, Fischer et al. 2009, Gao et al. 2001, Meyerrose et al. 2007, Nystedt et al. 2013, Schrepfer et al. 2007, Tolar et al. 2006, Vilalta et al. 2008), a majority of the MSCs accumulated
in lungs in Studies I, II, and III, which makes it even more important to optimise
the IV delivery of the MSCs. Despite these obvious issues, the IV route can be
considered as an optimal method of cell transplantation due to its rapid and non-
invasive nature. IV delivery would also be the most suitable for off-the-shelf cell
therapy products.
Existing evidence supports the role of adhesion molecule-mediated lung
deposition of MSCs (Fischer et al. 2009, Nystedt et al. 2013, Ruster et al. 2006)
and, as observed in Study III, pronase accelerated the lung clearance of MSCs.
This finding suggests a specific mechanism linked to cell-surface protein
modification. Pronase also had an impact on the MSC rolling behaviour in a flow
perfusion assay, which further supports the important role of MSC adhesion
characteristics. The functional effect of pronase detachment on lung clearance
was evident in three different animal models in Study III. Although reduced lung
retention was observed in all models, the initial cell retention was more prominent
in a xenogeneic experimental setting used in Study III.
FN is shown to have a strong impact on the adhesion and migration
characteristics of MSCs (Nystedt et al. 2013). Several FN binding receptors are
expressed on lung endothelium, including integrins VLA-4 (CD49d/CD29) and
VLA-5 (CD49e/CD29) and as the MSCs also express these integrins, they
therefore could be able to bind to FN present in the lung endothelium. VLA-4/FN
interactions have even been observed to be involved in migration of MSCs (Yagi et al. 2010b). The blocking of CD49d on MSCs is shown to increase pulmonary
clearance (Fischer et al. 2009). A similar effect has been observed with MSCs
dissolved in heparin, which is known to bind FN (Deak et al. 2010). Study III
106
showed that FN and CD49d were cleaved by pronase from the cell surface, and
hence this effect could be responsible for the increased pulmonary clearance of
pronase-detached MSCs.
Additionally, pronase cleaved CD44, as indicated by MS and flow cytometry
data. CD44 is observed to affect leukocyte rolling and adhesion to endothelial
cells (Murai et al. 2004) and to also have an impact on MSC migration and
adhesion to endothelium (Brooke et al. 2008, Zhu et al. 2006). CD44 cleavage
could provide a mechanism of mediating pronase-induced pulmonary clearance.
CD54 and CD166 are also involved in endothelial adhesion of MSCs (Brooke et al. 2008); both proteins are also shown to be cleaved by pronase in Study III. It
can be hypothesized that MSC pulmonary adhesion and increased pulmonary
clearance after pronase detachment is a result of manipulation of a range of
different adhesion molecules.
The number of cells did not continuously increase in the injured area after the
earliest time point post-injection. In fact, there was a decrease in the number of
cells in the paws between 50 minutes and 5 hours post-injection. This was also
evident with trypsinized cells and in the control paws, but it was most notable
with pronase-detached cells in the inflamed paw. Cleavage of adhesion molecules
after enzymatic detachment may play a role in the adherence of MSCs to the
injured area. Nevertheless, the amount of pronase-detached cells in the inflamed
paw was always the highest at all time points studied as compared with the
number of cells detected in the other study groups. The amount of pronase-
detached cells remained stabilized from 5 hours onward, suggesting possible
engraftment to the tissue, which coincided with the recovery of the cell-surface
proteins seen in vitro. The acute nature of carrageenan-induced inflammation can
also affect cell recruitment. There is a strong blood flow to the inflamed paw and
rapid formation of edema in the initiation phase of inflammation with a variety of
cytokines attracting the cells, including MSCs, to the site (Hargreaves et al. 1988,
Morris 2003). It may in fact be advantageous that therapeutic cells are recruited to
the inflammation rapidly along with infiltrating neutrophils. After 5 hours, the
edema started to decrease, and it is possible that the paracrine nature of MSCs, via
secretion of beneficial factors that reduce the inflammation, is partly contributing
to diminishing the edema.
Although we have preliminary data to support a beneficial therapeutic effect
of pronase-detached cells in an inflammatory condition, further studies should
explore this in more detail. The results of this study show that pronase detachment
of MSCs resulted in significantly faster clearance of the cells from the lungs due
107
to a transiently altered cell surface. This was detected with both BM-MSCs and
UCB-MSCs using both small and large animal models in xenogeneic and
homologous experimental setups. In addition, targeting of inflammation was
significantly improved with pronase detachment in a rat model of peripheral
inflammation, most likely because of increased bioavailability. Beneficial
functional consequences of a transiently modified cell surface by pronase were
also supported by the in vitro rolling assay data. Pronase-detached cells retained
therapeutically relevant functional properties, since both the immunosuppressive
and angiogenic potential was equal to that of control cells in vitro. Furthermore,
pronase detachment did not affect the multipotentiality of MSCs. No adverse
effects due to cell injections were observed in the large-animal model, and the
porcine groups had comparable haemodynamic and biochemical data during the
follow-up. Combining pronase detachment with other methods, such as optimal
culture conditions for migratory properties or adding a tag to induce targeting,
could be one way to further develop MSC preparations.
6.8 Clinical applications
Despite the possible systemic effects that the therapeutic cells possess, the
targeting of MSCs to the site of injury is currently considered relevant for some of
their paracrine and restorative functional effects. Commonly, transplanted MSCs
are deposited in the lungs, liver and spleen, as seen in Studies I, II and III; MSCs
targeting to the site of injury generally appears inaccurate (Barbash et al. 2003,
Chapel et al. 2003, Devine et al. 2003, Sackstein et al. 2008, Karp & Leng Teo
2009). We observed in Study I that MSC administration through IA decreased the
pulmonary entrapment of these cells. IA transplantation of MSCs is more invasive
than the IV route, but technical establishment of an IA transplantation route could
be easily performed using pre-existing facilities, such as angiography equipment
or perioperative administration in surgical settings.
Cardiac surgery-related ischemic brain injury would be a promising candidate
for bone marrow-derived stem cell therapy for a number of reasons. The surgical
operations are commonly elective; the occurrence of brain injury can be predicted
in advance. This allows collection and separation of autologous bone marrow
stem cells and the transplantation can be timed in the very early phase, as it is
precisely known when the brain is exposed to ischemia. This also would allow the
use of cultivated autologous MSCs, which have been suggested to have better
clinical effect than BM-MNCs. Cardiac surgery would also allow intra-arterial
108
transplantation during the operation with minimal additional risks; higher cell
concentrations to the brain and less lung entrapment could be achieved. No
cerebral migration of the BM-MNCs was present in Study II, but this does not
rule out the possible systemic benefits of the therapeutic cells. Future studies
should focus on investigating the occurrence of functional recovery after cell
administration.
In Study III, we showed that pronase detachment increased pulmonary
clearance of the IV-transplanted MSCs and even further increased their
recruitment at a peripheral site of injury. Although certain systemic effects of
MSCs are recognized, the natural assumption is that the cells should target the site
of injury in order to fulfil their paracrine regenerative function.
Immunomodulatory functions of MSCs are well recognized and could have a
major effect in prevention of ischemia-reperfusion injury. In addition, the pronase
detachment procedure is performed without any additional effort compared to
traditional MSC cultivation with trypsin detachment. Futhermore, we observed
that the mechanism behind the enhanced pulmonary passage is based on transient
cell surface modification and the cell viability and differentiation potential is not
compromised, suggesting that their therapeutic function is preserved in this
process.
IV-transplanted MSCs travel through the lung vasculature which could
function as a filtering mechanism against possible thromboemboli caused by
blood contact of the MSCs. Although IA transplantation is another potential
method of increasing tissue targeting of MSCs, pronase detachment could have a
superior safety profile, as injecting possible emboligenic material into the arterial
vasculature could be avoided.
Pronase detachment could provide a viable method of transplantation
optimisation in such clinical conditions, where tissue targeting of MSCs would be
beneficial; classically, this would mean ischemia-related tissue injury at a specific
focus. Overall, the optimal therapeutic indications for pronase-detached MSCs
would be those that lack effective forms of treatment by other means and which
might otherwise be severe for the patient. The possible applications related to
natural vascular diseases could include stroke therapy, MI and embolization of
visceral or peripheral arteries. Another major application could be traumatic
injuries, including traumatic brain injury. Ischemic brain injury caused by cardiac
surgery could also be an indication of IV-transplanted pronase-detached cells.
109
6.9 Limitations of the study
Correlations between results gained from animal studies and clinical practice is
always a question of great importance. Generally, it is considered that a large
animal model, such as the pigs used in this study, is well comparable to a human
patient. Pigs are considered especially valid as cardiovascular models. Major
organs and their relationships to each other and body size closely resemble the
human body. However, one particular difference is brain size compared to the
whole body, as the porcine brain forms only 0,5% of its body weight. This can be
considered a major limitation of study II, when assessing cell migration to the
damaged brain.
It has been observed that mechanisms of MSC immunomodulation vary
between different species (Ren et al. 2009) and it is possible that preclinical
studies conducted with animals hold potential for bias when translating their
results to human clinical trials. It is commonly recognized that animal studies
employing more highly developed species, such as primates, dogs or pigs, have
closer relevance to human subjects. Comparability between human and porcine
MSCs and their biological properties has been studied by Noort et al. and the
authors have concluded that studies with porcine MSCs can be reliably
extrapolated to correspond with results that would be seen when using human
MSCs (Noort et al. 2012). Our results with a porcine model cannot be considered
as directly translatable to human trials, but general information provided by
Studies II and III can be considered accurate enough to act as guidance for future
trials.
The Tc-HMPAO labelling studies offer the potential to globally track the
biodistribution of the transplanted cells in a porcine model. This method,
however, has its limitations due to the limited half-life of the radionuclide isotope.
Obvious limitations of Studies I, II and III is the small sample size. The variation
seen in these studies is indicative of the need for power calculations for further
studies.
Another limitation of Study I is related to the difference in the cell types used.
Cell doses were not directly comparable, as a tenfold greater quantity of BM-
MNCs was used. This was required to reach similar input radioactivity doses, as
the labelling efficiency of BM-MSCs was also tenfold higher. The applied BM-
MSC quantity was comparable to a porcine study conducted by Wolf et al. in
which the dose effectiveness of BM-MSCs was examined in a myocardial
infarction model (Wolf et al. 2009). Although organ accumulation trends reported
110
in Study I can be compared between cell lineages after normalization, results
should be interpreted with caution, as a tenfold higher dose presumably affects
administration dynamics; larger cell doses are reported to cause more capillary
occlusion (Furlani et al. 2009). Furthermore, BM-MNC groups underwent bone
marrow harvesting, while the BM-MSC groups did not, which could bias the
presumed native biodistribution due to local mechanical disruption of the bone
marrow.
6.10 Summary
In Study I, our data indicates that intra-arterial administration appears to possess
the potential to allow more cells to reach the systemic vasculature, thereby
targeting organs of cellular therapy, such as the brain, heart and kidneys, whereas
intra-venous administration allows targeting of the lung, which can also be
clinically relevant. None of the administration combinations showed any evidence
of safety threats, but an awareness of the possibility of embolization should result
in a cautious approach to utilising intravascular cell administration. Although this
experimental model is limited to offering a descriptive insight on the subject,
future studies could provide basic mapping of BM-MSC and BM-MNC
biodistributions following systemic IA or IV administration.
In contrast to promising earlier studies on acute focal ischemia, the
transplantation of BM-MNCs in Study II did not result in the retention of cells in
the damaged brain in the porcine global ischemia model. BM-MNCs did not
migrate into brain tissue in substantial numbers during first 24 hours, as the cells
accumulated in other major organs and the bone marrow.
Study III showed that pronase detachment could be used to improve the MSC
lung clearance and targeting in vivo without compromising the therapeutic
properties of the cells. Pronase detachment could be easily incorporated into the
clinical production processes of adherent cells, since it could replace trypsin.
However, there are still many questions surrounding the clinical use of pronase,
including the safety of the product and the demonstration of the actual
improvement of functional outcome.
111
7 Conclusion
I Both the BM-MNCs and MSCs deposit mainly in the lungs, liver and spleen.
IA administration of MSCs diminishes their lung deposition. Intravascular
transplantation of these cells did not cause pulmonary emboli.
II Intra-arterially transplanted BM-MNCs do not reach the brain in significant
numbers in a porcine model of global ischemic-induced brain injury and there
is not a culmination point of migration during the first 24 hours
III Transient proteolytic modification of MSCs with pronase decreased lung
accumulation and improved migration to the site of injury without affecting
the characteristics of the cells.
112
113
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Original publications
I Mäkelä T, Takalo R, Arvola O, Haapanen H, Yannopoulos F, Blanco R, Ahvenjärvi L, Lehtonen S, Kiviluoma K, Kerkelä E, Nystedt J, Juvonen T & Lehenkari P. (2014) Safety and Biodistribution Study of Bone Marrow Derived Mesenchymal Stromal Cells and Mononuclear Cells and the Impact of the Administration Route in an Intact Porcine Model. Manuscript
II Mäkelä T, Yannopoulos F, Alestalo K, Mäkelä J, Lepola P, Anttila V, Lehtonen S, Kiviluoma K, Takalo R, Juvonen T & Lehenkari P. (2013) Intra-Arterial Bone Marrow Mononuclear Cell Distribution after Experimental Global Brain Ischemia Scand Cardiovasc J. 47(2):114-20.
III Kerkelä E, Hakkarainen T, Mäkelä T, Kilpinen L, Lehtonen S, Ritamo I, Pernu R, Pietilä M, Takalo R, Nikkilä J, Tikkanen J, Juvonen T, Laitinen S, Valmu L, Lehenkari P & Nystedt J. (2013) Transient Proteolytic Modification of Mesenchymal Stromal Cells Decreases Lung Entrapment in vivo. Stem Cells Translational Medicine. 2(7):510-20.
Reprinted with permission from Informa Healthcare (II) and AlphaMed Press (III)
Original publications are not included in the elecronic version of the dissertation.
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A C T A U N I V E R S I T A T I S O U L U E N S I S
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1257. Lahti, Anniina (2014) Epidemiological study on trends and characteristics ofsuicide among children and adolescents in Finland
1258. Nyyssönen, Virva (2014) Transvaginal mesh-augmented procedures in gynecology :outcomes after female urinary incontinence and pelvic organ prolapse surgery
1259. Kummu, Outi (2014) Humoral immune response to carbamyl-epitopes inatherosclerosis
1260. Jokinen, Elina (2014) Targeted therapy sensitivity and resistance in solidmalignancies
1261. Amegah, Adeladza Kofi (2014) Household fuel and garbage combustion, streetvending activities and adverse pregnancy outcomes : evidence from urban Ghana
1262. Roisko, Riikka (2014) Parental Communication Deviance as a risk factor forthought disorders and schizophrenia spectrum disorders in offspring : The FinnishAdoptive Family Study
1263. Åström, Pirjo (2014) Regulatory mechanisms mediating matrix metalloproteinase-8 effects in oral tissue repair and tongue cancer
1264. Haikola, Britta (2014) Oral health among Finns aged 60 years and older :edentulousness, fixed prostheses, dental infections detected from radiographsand their associating factors
1265. Manninen, Anna-Leena (2014) Clinical applications of radiophotoluminescence(RPL) dosimetry in evaluation of patient radiation exposure in radiology :Determination of absorbed and effective dose
1266. Kuusisto, Sanna (2014) Effects of heavy alcohol intake on lipoproteins,adiponectin and cardiovascular risk
1267. Kiviniemi, Marjo (2014) Mortality, disability, psychiatric treatment and medicationin first-onset schizophrenia in Finland : the register linkage study
1268. Kallio, Merja (2014) Neurally adjusted ventilatory assist in pediatric intensive care
1269. Väyrynen, Juha (2014) Immune cell infiltration and inflammatory biomarkers incolorectal cancer
1270. Silvola, Anna-Sofia (2014) Effect of treatment of severe malocclusion and relatedfactors on oral health-related quality of life
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äkelä
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Tuomas Mäkelä
SYSTEMIC TRANSPLANTATION OF BONE MARROW STROMAL CELLSAN EXPERIMENTAL ANIMAL STUDY OF BIODISTRIBUTION AND TISSUE TARGETING
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE,INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF SURGERY,DEPARTMENT OF ANAESTHESIOLOGY;INSTITUTE OF BIOMEDICINE,DEPARTMENT OF ANATOMY AND CELL BIOLOGY;INSTITUTE OF DIAGNOSTICS,DEPARTMENT OF DIAGNOSTIC RADIOLOGY;CLINICAL RESEARCH CENTER
