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Current Status of Stem Cell Therapy in Pulmonary Hypertensive Vascular

Disease6TH INTERNATIONAL CONFERENCE

Neonatal & Childhood Pulmonary Vascular DiseaseDr. Duncan Stewart

June 21-22, 2013San Francisco, California

Duncan J Stewart

Disclosures

• CEO and founding scientist of Northern Therapeutics

Modified from Thébaud and Abman Am J Respir Crit Care Med 2007

Lung vascular injury: a tale of two diseases

ALI/ARDS

PAH

Ang1/2

PAH results from functional “pruning” of the lung micro-vasculature

Normal Pulmonary Arterial Hypertension

?

MR perfusion images courtesy of Evangelos Michelakis, Edmonton, Alberta

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Nonlinear Progression of PAH

0 25 50 75 100Loss of Lung Vascular Surface area (%)

Death

Clin

ical

sym

ptom

s

threshold for symptomatic PAHmild

mod

erat

ese

vere

“Angiogenic” Gene Therapy for PAH

• VEGF– Partovian et al, Am J Respir Cell Mol Biol, 2000 – Adenoviral

vector, transtracheal delivery, chronic hypoxia “prevention” model

– Zhao et al, Circulation, 2001 – cell based (FBs), IV delivery, MCT “treatment” model (2 weeks)

• NOS– Janssens et al, J Clin Invest, 1996 – eNOS: Adenovirus, MCT

“prevention” model – Budts et al, Circulation, 2000 – iNOS: Adenovirus, Chronic

hypoxia “prevention” modell

• Angiopoietin 1– Campbell et al, Circ Res, 2003 –cell-based, MCT “prevention”

model

Angiogenic gene therapy for MCT PAH: Treatment model

Zhao et al. AJRCMB VOL 35 2006

Days

Control MCT D21 MCT-eNOS D35

Endothelial progenitor cells

Role of EPCs in pulmonary vascular regeneration?

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Early growth cells – 3-7 days “Circulating Angiogenic Cells” (CACs):

- CD14+, CD45+- express EC markers- Nonproliferative- Highly angiogenic

Culture-Specified “EPCs”

>2 weeks – cell clusters

Late outgrowth EPCs >2 wks

“Endothelial Colony Forming cells” (ECFCs):

- CD14, CD45 negative- Strong EC phenotype- Highly proliferative

Circulating angiogenic cells -CAC

Arterioscler Thromb Vasc Biol. 2008;28:1584-1595

Effect of EPCs on PAH in the MCT “prevention’ model of PAH

FMAFMA SMASMA

ControlMCT-FB

MCT-EPC

Zhao et al. Circ Res. 2005; 96(4):442-50

0

10

20

30

40

50

60

RVSP (mmHg)

Con MCT FB EPC

****

††

Preclinical Stem/Progenitor Cell Therapy Studies

EPCs Model Citation/Key findingsCells only MCT Takahashi et al, 2004

• Prevention of PAH in dogsCells only MCT Zhao et al, 2005

• Prevented PAH; no reversalCells only MCT Yip et al, 2008

• Reversal of PAH in ratsCells only MCT Mirsky et al, 2011

• No reversal of PAH in ratsMSCs Model Citation/Key findingsCells only

MCT Baber et al, 2007 • Reduced PAH in rats (2 wks post

MCT)Cells only

MCT Umar et al, 2009• Attenuated PAH in rats (2 wks

post-MCT)

Cell therapy alone

Adapted from: Mei et al. Comprehensive Physiology: in press

Potential differences:• Efficacy of EPCs >

MSCs?• But, MSCs can be

given as allogeneic cell product

Treatment models:• Both have limited

efficacy

EPC Therapy for Idiopathic Pulmonary Arterial Hypertension

• Randomized, nonblinded design• Circulating MNCs cultured on FN x 5 days – no transfection• 31 pts (16 – conventional Rx; 15 – EPCs, IV injection)

• 11 x 106 cells (range 4 – 22 x 106)

Wang et al. JACC Vol. 49, No. 14, 2007

~10% change

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Cell and gene therapy maybe more effective in treatment models of PAH

EPCs + Model Citation/Key findingsAdreno-medullin

MCT Nagaya et al, 2003• superior to EPCs alone in prevention model

CGRP L-R shunt Zhao et al, 2007 • Reduced mPAP and PVR in prevention

modeleNOS MCT Zhao et al, 2005

• reversed established PAH in ratsMSCs + Model CitationsKey findingseNOS MCT Kanki-Horimoto et al, 2006

• Reversed PAH in ratsHO-1 Hypoxia Liang et al, 2011

• Reversed MCT-induced PAHPCS MCT Takemiya et al, 2010

• Reversed established PAH in ratsAdspted from: Mei et al. Comprehensive Physiology: in press

RV

SP

(m

mH

g)

MCT EPCsEPCs/eNOS

0

20

40

60

80

Control

Day 21

Day 35

**

** p<0.001 vs. d21 MCT* p<0.01 vs. d21 MCT

Zhao et al. Circ Res. 2005; 96(4):442-50

Effect of EPCs in the reversal of MCT-induced PAH (RVSP)

*

Principle Investigators:John Granton (Toronto); David Langleben (Montreal)

Safety study– Io EP: tolerability of cell transplantation in patients with

PAH refractory to all standard therapiesCell delivery– eNOS transfected autologous early growth EPCs– Delivery via SG catheter

• Pacing port (i.e. RV delivery) – allows continuous monitoring of PA pressure

– Dose ranging for eNOS transfected cells given over 3 days in divided doses

Pulmonary Hypertension And Cell Therapy (PHACeT) Trial PHACeT Trial Cell Processing

Transfection with eNOS

Viability: >98%2.5 x106 heNOS-Tx EPCs/ml

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1 week post MCT

Pre-capillaryarteriole

Pulmonaryarteriole

Mechanism of benefit: Transdiffer-entiation vs. “paracrine” effects

15 minutes

Zhao et al. Circ Res. 2005; 96(4):442-50

Injected MSCs Retained in Lungs After 3 Days

MSCs were labeled with cell tracing dye CFDA SE (green) prior to injection. Nuclei were stained with TO-PRO-3 (blue).

20 µm

Confocal Microscope Image Flow Cytometric Analysis

LPS/MSCs-15 min

Exosome fraction mediates protective effect of MSC-CM in hypoxia-induced PH

Lee et al. (Kourembanas) Circulation 2013

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Effects of human E-EPCs versus L-EPCs in the rat MCT xenotransplant model of PAH

Ormiston et al.AJRCMB 2010

“Cocooning” to enhancing cell survival and engraftment

“Cocooned” Bone Marrow Stromal Cells

Red – CMTMR-labeled MSCs

Green – Oregon green-fibrinogen

Golnaz Karoubi

60 x 60 x

20 x 40 x

24 h

7 days

15 min

Efficient high-throughput micro-encapsulation using microfluidics

Custom microfluidic chamber designed for: A. hydrogel polymerization B. enrichment of cell containing dropletsC. serial amplification for high throughput

Oil

Cells/hydrogel

Michel Godin, Dept of Physics, uOttawa

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Summary and Conclusions• Cell therapy represents a potentially promising new

treatment strategy for PAH– EPCs appear to be more effective than MSCs in

prevention models• But, MSCs can be used as an allogeneic cell product …

– Limited clinical data suggests modest efficacy of EPCs in IPAH (Wang et al. 2007)

• Combining cell and gene therapy is more effective in preclinical “treatment” models of established PAH– Limited clinical experience to date supports safety of

eNOS-enhanced EPCs (PHACeT trial)• need for further clinical evaluation (i.e. PHACeT-2)

Summary and Conclusions• Therapeutic effects of EPC and MSCs in PAH

appear to be mediated by “paracrine” mechanisms– Self-fulfilling prophecy?

• i.e. very limited cell persistence with current delivery strategies precludes possibility of transdifferentiation and direct role in vascular repair/regeneration

• Strategies are needed to enhance cell survival and engraftment in order to assess potential role for transdifferentiation– Late outgrowth EPCs (ECFCs)?– Pluripotent stem cells?

Thank you!

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MSCs partially prevent MCT-induced PAH in the rat

Barber at al. Am J Physiol Heart Circ Physiol. 2007

1.0

0.9

0.8

0.70.60.5

0.4

0.3

Cum

ulat

ive

Sur

viva

l

353331292725

Days post MCT

Zhao et al. Circ Res. 2005; 96(4):442-50

MCT

MCT-CAC

P<0.05P<0.05

MCT-CAC

P<0.05

MCT-CAC/eNOS

P<0.02P<0.02

MCT-CAC/eNOS

P<0.02

Survival analysis following cell therapy in the treatment MCT-PAH model

N = 63