Validation of Contrast-Enhanced MRI to Monitor Regenerative … · DOI:...

DOI: 10.1161/CIRCULATIONAHA.113.002863

Validation of Contrast-Enhanced MRI to Monitor Regenerative Efficacy after

Cell Therapy in a Porcine Model of Convalescent Myocardial Infarction

Running title: Malliaras et al.; Validation of MRI to assess cardiac regeneration

Konstantinos Malliaras, MD1; Rachel R. Smith, PhD1,2; Hideaki Kanazawa, MD, PhD1; Kristine

Yee, DVM, DACVIM1; Jeffrey Seinfeld, BS1; Eleni Tseliou, MD1; James F. Dawkins, DVM1;

Michelle Kreke, PhD2; Ke Cheng, PhD1; Daniel Luthringer, MD1; Chak-Sum Ho, PhD3; Agnieszka

Blusztajn, BS2; Ileana Valle, BS2; Supurna Chowdhury, MS1; Raj R. Makkar, MD1; Rohan

Dharmakumar, PhD4; Debiao Li, PhD4; Linda Marbán, PhD1,2; Eduardo Marbán, MD, PhD1

1Cedars-Sinai Heart Institute, Los Angeles, CA; 2Capricor Inc., Los Angeles, CA; 3Gift of Life

Michigan, Ann Arbor, MI; 4Cedars-Sinai Biomedical Imaging Research Institute, Los Angeles, CA

Address for Correspondence:

Eduardo Marbán, MD

Cedars-Sinai Heart Institute

8700 Beverly Blvd.

Los Angeles, CA 90048

Tel: 310-423-7557

Fax: 310-423-7637

E-mail: eduardo.marban@csmc.edu.

Journal Subject Code: Treatment:[27] Other treatment

Michelle Kreke, PhD2; Ke Cheng, PhD1; Daniel Luthringer, MD1; Chak-Sum Ho,, PPPhDhDhD333;; AgAgAgnininieseszzka

Blusztajn, BS2; Ileana Valle, BS2; Supurna Chowdhury, MS1; Raj R. Makkar, MD1; Rohan

DhDhDharararmamamakukukumamaar,r,, PPhD4; Debiao Li, PhD4; Linda a a MaMM rbán, PhD1,2; ; Eduauaarddrdo o Marbán, MD, PhD1

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Abstract

Background—Magnetic Resonance Imaging (MRI) in the CADUCEUS trial revealed that

cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium post-

myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell

therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable

myocardium after cell therapy in a porcine model of convalescent MI.

Methods and Results—Yucatan minipigs underwent induction of MI and 2-3 weeks later were

randomized to receive intracoronary infusion of 12.5x106 mismatched allogeneic CDCs or

vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic

immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global

and regional function, decreased scar size and increased viable myocardium compared to placebo

2 months post-treatment. Extensive histological analysis validated quantitatively the MRI

measurements of scar size, scar mass and viable mass. CDCs neither altered gadolinium contrast

myocardial kinetics, nor induced changes in vascular density or architecture in viable and scarred

myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border

zone, consistent with the observed stimulation of endogenous regenerative mechanisms

(cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis).

Conclusions—Contrast-enhanced MRI accurately measures scarred and viable myocardium after

cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing

dynamic changes in the infarct and monitoring regenerative efficacy.

Key words: cardiosphere-derived cells, heart regeneration, allogeneic transplantation, magnetic resonance imaging

vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no syyssstemmmiicc

mmunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global

annd d d rereregigigiononnalalal ffunnctctctiioion, decreased scar size and innncrcrc eeeased viable mmyoyy caardrdrdiiuium compared to placebo

22 mmomonths post-t-trtreaeaatmmeeent.t.t. EEExtxtxtenenensisivveve hhiisstoologggicccal aananaalyssisisss vavaaliidadatteedd ququaannttiitatatititivvevelyly ttthehehe MMMRIRIRI

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Cell therapy has emerged as a potential therapeutic strategy for ischemic cardiomyopathy. While

the paradigm of administering bone marrow-derived cells in the setting of acute or recent

myocardial infarctions (MIs) has proven to be safe, efficacy has been inconsistent1,2. Early

clinical experience with autologous heart-derived progenitor cells has been more encouraging3,4.

In the CADUCEUS trial, intracoronary infusion of autologous cardiosphere-derived cells

(CDCs)5 in post-MI patients with left ventricular (LV) dysfunction decreased scar size, increased

viable myocardium and improved regional function, as measured by contrast-enhanced magnetic

resonance imaging (MRI)3. Contrast-enhanced MRI has been extensively validated (and is

considered the “gold standard” imaging modality) for the quantification of necrotic/scarred and

viable myocardium in the setting of acute or chronic MI6-8. Thus, MRI represents a potentially

useful tool for monitoring regenerative efficacy, as it affords the unique ability to quantify,

rigorously and independently, scar mass and viable myocardial mass in human subjects before

and after cell therapy interventions9. However, its validity in characterizing tissue viability after

cell administration has been called into question10. Without evidence, concerns have been raised

that cell therapy may promote changes in vessel density or architecture (e.g., increase in wall

thickness or decrease in vascular permeability) that could affect gadolinium (Gd)-contrast

myocardial kinetics (in the form of accelerated contrast wash-out [a phenomenon that has been

described in non-cardiac tissues11] or decreased contrast extravasation, respectively), therefore

compromising the ability of contrast-enhanced MRI to distinguish scar from viable

myocardium10.

Here, we sought to test the validity of contrast-enhanced MRI to distinguish, and

accurately measure, scarred and viable myocardium after cell therapy. We used a porcine model

of intracoronary infusion of allogeneic CDCs, which enabled us to concurrently investigate the

viable myocardium in the setting of acute or chronic MI6-8 MRI representstss a ppoototennentititialalallylly . Thus,

useful tool for monitoring regenerative efficacy, as it affords the unique ability to quantify,

igogoorororouususlylyly aaandndn indndndeepependently, scar mass and viababablele myocardial mamamar ss iinn n hhuhuman subjects before

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safety and efficacy of allogeneic heart-derived cells without immunosuppression in a clinically-

relevant model of convalescent MI.

Methods

All animal studies were performed in an American Association for Accreditation of Laboratory

Animal Care accredited facility with approval from the Institutional Animal Care and Use

Committee of the Cedars-Sinai Medical Center (IACUC 3661). The experimental protocol is

depicted schematically in Figure 1. A total of 26 minipigs were studied: 3 completed the 24-hour

retention study (Fig 1A), 10 completed the validation study (Fig 1B), 5 completed the 2-month

engraftment study, 4 were excluded per protocol for procedural mortality, 3 were used for

allosensitization protocols, and 1 served as CDC donor.

Cell culture

Allogeneic CDCs were grown from a freshly-explanted heart obtained from one male Sinclair

minipig (Sinclair Bioresources) (for detailed methods see Online Supplement). For 24-hour

retention analysis, CDCs were transduced with an adenoviral vector carrying the firefly

luciferase gene 3 days prior to infusion.

MI creation and CDC infusion

Infarcts were created in adult Yucatan minipigs by inflation of an angioplasty balloon in the mid-

left anterior descending artery (LAD) (distal to the 1st diagonal branch) for 2.5 h (for detailed

methods see Online Supplement). Two to 3 weeks later, pigs were randomized to receive 12.5

million CDCs (in 10 mls of CryostorTMCS10 containing 45 g/ml nitroglycerin and 180 U/ml

heparin) or vehicle. Intracoronary infusion was performed via an over-the-wire balloon catheter,

placed in the mid-LAD. CDCs or vehicle solution were infused in 3 cycles of intermittent

engraftment study, 4 were excluded per protocol for procedural mortality, 3 wereree usess dd d fofofor rr

allosensitization protocols, and 1 served as CDC donor.

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balloon inflation12. Minipigs were euthanized either 24 hours post-infusion, to measure short-

term cardiac retention of administered cells (n=3), or 2 months later (n=15; 10 completed the

validation study, 5 completed the 2-month engraftment study).

In vivo cardiac MRI

Baseline (2-3 weeks post-MI, before intracoronary infusion) and endpoint (2 months post-

infusion) contrast-enhanced cardiac MRI was performed to measure scar mass, viable

myocardial mass (i.e., total mass minus scar mass), scar size (scar mass divided by total mass),

volumes, global function, and regional function of the LV.

All MRI studies were performed on a 3.0 T clinical MRI scanner (Siemens

MAGNETOM Verio®, Erlangen, Germany). Typical in-plane resolution was 1.3 x 1.3 mm, and

slice thickness was 6 mm, with no gaps. Global LV function, regional systolic thickening and

regional end-systolic thickness were assessed using ECG-gated and breath-held cine steady-state

free precession acquisitions13. Minipigs undergoing baseline MRI subsequently received an IV

injection of Gd-based contrast agent gadoversetamide (OptiMARK®, Covidien Imaging

Solutions, Hazelwood, MO, USA; 0.2 mmol/kg body weight), and, 8 minutes later, delayed

contrast-enhanced images were acquired with an ECG-gated, breath-hold, interleaved, 2D-Turbo

FLASH sequence14. The inversion time was adjusted by the scanner operator to null signal from

non-infarcted remote myocardium.

In minipigs undergoing endpoint MRI, once the cine acquisitions were completed, a

short-axis slice with significant regional hypokinesia was selected (after review of cine images)

to study Gd-contrast kinetics. Minipigs received an IV injection of Gd-contrast (0.1 mmol/kg

body weight) and a series of dynamic delayed contrast enhancement images were acquired (pre–

and 1, 2, 3, 4, 5, 6, 10, 12, 14, 16, 18 and 20 minutes post-contrast administration). An ECG-

MAGNETOM Verio®, Erlangen, Germany). Typical in-plane resolution was 1.3.33 xx 11.333 mmmm,m,m, aannd

lice thickness was 6 mm, with no gaps. Global LV function, regional systolic thickening and

eegigigionononalalal eeendndnd--ssysstototolilic thickness were assessed usssiiingngng ECG-gated aandnn bbrereeaatath-held cine steady-state

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gated, breath-hold, interleaved 2D-Turbo FLASH sequence was used, and the inversion time was

kept constant at 270 ms15,16. Thirty minutes after the first contrast injection, a TI scout was

performed to ensure sufficient contrast clearance. Subsequently, minipigs received a second IV

injection of Gd-contrast (0.2 mmol/kg body weight) and 8 minutes later delayed contrast-

enhanced images were acquired to assess scar size as described above for baseline MRI. Thirty

minutes after the second contrast injection, a TI scout was performed to ensure sufficient contrast

clearance. Subsequently, minipigs received a third IV injection of Gd-contrast (0.2 mmol/kg

body weight) and were sacrificed 15 minutes later in order to perform ex vivo MRI of the heart.

All image analyses were performed using validated image processing software (QMass

MR®, Medis medical imaging systems). Global function and cardiac volumes were evaluated by

a researcher blinded to treatment allocation. To evaluate regional function, each short-axis slice

was divided into 6 segments, using the right ventricular insertion as a reference point. Scar size

from late Gd-enhanced cardiac MR images was defined based on the full-width half-max

(FWHM) criterion to delineate scarred myocardium7.

Ex vivo cardiac MRI & histology

Hearts from minipigs sacrificed 2 months post- infusion underwent ex vivo MRI. The heart was

removed ~30 minutes post Gd-DTPA administration, thoroughly rinsed in ice-cold saline to

remove any residual blood, and suspended by sutures in a plastic container filled with saline. 3D

Turbo FLASH images were acquired with a resolution of 1 x 1 x 1 mm 6. After image acquisition,

the heart was sectioned into 1 cm thick short-axis slices, which were incubated with 2% 2,3,5-

triphenyltetrazolium chloride (TTC) for 20 min at 37°C to stain viable myocardium. Each slice

was photographed with a digital camera and infarct size was determined as the percentage of LV

volume by manual tracing by a researcher blinded to treatment allocation (ImageJ, version 1.46,

MR®, Medis medical imaging systems). Global function and cardiac volumes wweeere ee evvvalalaluauauatetetedd byy

a researcher blinded to treatment allocation. To evaluate regional function, each short-axis slice

wawas s s dididivivividededed d d iinintoo 666 ssegments, using the right vennntrtrtricccular insertion n asaa aa rrrefefefere ence point. Scar size

ffrrommm late Gd-eenhnhnhaannceced d cacacardrdrdiaiaiaccc MRMRMR iimmamagges awaw s ddeefffineeed d babassesed d oonn tthehe fffululll-wiwiwidtddth h hahahalflflf-m-mmaxxx

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National Institutes of Health, Bethesda, MA, USA). Scar and viable myocardium volumes were

calculated by multiplying scar and viable area for each slice (in mm2) with the corresponding

slice thickness. Scar mass and viable myocardial mass were calculated by multiplying the scar

and viable myocardial volumes times a specific gravity of 1.05g/cm3.

Immune rejection in the heart was investigated by hematoxylin and eosin staining;

analysis was performed by an experienced cardiac pathologist blinded to treatment allocation

(DL), and rejection was graded according to the International Society for Heart & Lung

Transplantation (ISHLT) grading system, used in clinical practice to diagnose solid organ

transplant rejection. Morphometric analysis was performed with Masson’s trichrome staining.

Vascular density and architecture were investigated by fluorescent immunohistochemistry;

arterioles and arteries were defined as smooth-muscle-coated vessels with external vascular

diameter (lumen + vascular walls) <75 m and >75 m respectively. Myocyte cross-sectional

area was investigated by fluorescent immunohistochemistry; cardiomyocytes were accepted for

size measurement if they met the following criteria: (a) cellular cross-sections present (b) visible

nuclei located close to the cell center and (c) intact cell borders17. Cardiomyocyte cell-cycling

and activation of cardiac progenitors were investigated by fluorescent immunohistochemistry. In

all histological analyses, peri-infarct (border zone) area was defined as the region at the edges of

the scar (comprising areas of both viable and scarred myocardium). For detailed histological

methods see the Online Supplement.

Evaluation of 24-hour cardiac retention and 2-month cardiac engraftment

Evaluation of 24-hour cardiac retention was performed with the ex vivo luciferase assay12 (Supp

Fig 1a; for details see the Online Supplement). Two-month cardiac engraftment was investigated

by Fluorescence In Situ Hydridization (FISH) for the male Y chromosome (Supp Fig 2) 2

Vascular density and architecture were investigated by fluorescent immunohistococcheheemimistststryryry; ; ;

arterioles and arteries were defined as smooth-muscle-coated vessels with external vascular

diiamammeteteteerer (((lululummmen n +++ vav scular walls) <75 m and >7>7>755 m respectiveelylyl . MyMyMyooocyte cross-sectional

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months after sex-mismatch cell transplantation (male cells infused into infarcted female

recipients; for details see the Online Supplement).

Histocompatibility

To assess histocompatibility, low-resolution swine leukocyte antigen (SLA) typing was

performed on the donor and recipient minipigs as described in the Online Supplement.

Circulating donor-specific antibodies

To evaluate humoral immune response, recipient minipig serum samples (obtained at baseline, 1

day, 1 week, 2 weeks and 2 months post-infusion) were screened for circulating anti-donor IgG

antibodies by flow cytometry18.

An allosensitization protocol was performed to provide positive controls for the flow

cytometry assay. Peripheral bone marrow mononuclear cells (PBMNCs) were harvested from a

donor farm pig and injected intradermally and subcutaneously into the pinnae of recipient

Yucatan minipigs (n=2, 190 million PBMNCs injected per pig). Serum samples collected 2

weeks post PBMNC injection served as positive controls for allosensitization.

Statistical Analysis

Results are presented as mean ± SD in the text and as mean ± SEM in the figures. For continuous

measures, differences between 2 groups (controls, CDC-treated) were tested using independent

samples t-test. Comparisons of changes from baseline within groups were performed using

paired samples t-test. Histology data for grading of rejection were analyzed using a generalized

estimating equations (GEE) logistic regression model. Gd curves were analyzed with a linear

mixed effects model using random intercept to control for repeated measures and Tukey’s

adjustment for multiple comparisons. Serial Troponin I (TnI) measurements were analyzed using

a GEE model with an autoregressive correlation structure. Pearson correlations and the Bland-

An allosensitization protocol was performed to provide positive controls s foffor rr ththhe ee flflflowowow d

cytometry assay. Peripheral bone marrow mononuclear cells (PBMNCs) were harvested from a

doonononor r r fafafarmrmm ppiiig aaandndnd injected intradermally and suuubcbcb uutaneously inntotoo thee pppiininnae of recipient

YYuccacatan minipipiigggs ((n=n=2,2, 111909090 mmmililliliononon PPBMBMMNNCCsss injjeccctedd d ppeper r pipiig)g). SSeerurummm ssammpmplleles s cococollllllececteted dd 22

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Statistical AnAnnalalalysysysisisis

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Altman analysis method were used to compare MRI measurements against postmortem

histological measurements. All tests were 2-sided. No multiplicity adjustment for multiple

comparisons was performed. A p value of <0.05 was considered statistically significant.

Results

Safety of CDC infusion, 24-hour retention and 2-month engraftment

Four of 22 minipigs (18%) died during creation of MI due to ventricular arrhythmias. There was

no further mortality (0%) in minipigs that survived the MI.

CDCs are ~20 m in diameter, so that microvascular occlusion is expected at escalating

doses12. We thus looked for any infusion-related impairment of coronary flow or increase in

ischemic biomarkers. No adverse events (sustained ventricular arrhythmias, unresolved ST-

segment elevations) occurred during cell infusion, and no impairment of TIMI flow was

observed after infusion (Supp Fig 3b). No difference in serum TnI was detected between the

CDC-treated and placebo groups at 1,7,14, or 60 days post-infusion (Supp Fig 3A). The mild

TnI elevations observed 1 day post-infusion in both groups are consistent with transient LAD

occlusion by the stop-flow infusion procedure.

To assess short-term cardiac retention of infused CDCs, 3 minipigs were infused with

luciferase-labeled allogeneic CDCs. Twenty-four hours later, the ex vivo luciferase assay (Supp

Fig 1A) revealed that cardiac retention of CDCs was 4.3±2.2% (Supp Fig 1B). Long-term

cardiac engraftment was investigated by FISH for the male Y chromosome (after infusion of

male CDCs into female recipients). While presence of infused cells in the recipient myocardium

could be verified at 2 weeks post-infusion (Supp Fig 2), no allogeneic (male) cells could be

detected at 2 months post-infusion (5 animals, 4782 nuclei analyzed)

doses12. We thus looked for any infusion-related impairment of coronary flow or r inincrcc eaeaeaseese iiin n n

schemic biomarkers. No adverse events (sustained ventricular arrhythmias, unresolved ST-

eegmgmgmenenenttt eleleleveve aatiooonsnsns) ) occurred during cell infusiononon, aaand no impairmmment t ofofof TTIMI flow was

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Immune reaction to allogeneic CDCs

Low-resolution SLA genotyping revealed complete Class I (SLA-1, SLA-2, SLA-3) and Class II

(DRB1, DQB1, DQA) mismatches between the donor minipig and CDC recipients (Supp Fig 4).

Cardiac histology demonstrated a marginally significant increase in focal lymphoplasmacytic

infiltration (deemed to be unrelated to the natural inflammatory response to the ischemic insult)

in the peri-infarct area of CDC-treated minipigs compared to placebo (p=0.058 between groups,

Odds Ratio=8.61 [95% CI: 0.93-79.52]). (Supp Fig 5A). The infiltrating cells were localized

within interstitial and perivascular spaces (Grade 1R according to the ISHLT grading system)

(Supp Fig 5B); importantly, no foci of rejection-associated myocyte damage were detected. The

remote myocardium was consistently clear of inflammatory infiltrates. No circulating

alloreactive antibodies were detected in any recipients of allogeneic CDCs at any time point. In

contrast, in minipigs that were allosensitized by intradermal and subcutaneous PBMNC

injections, high titers of circulating alloreactive IgG antibodies were detected 2 weeks post-

injection (Supp Figure 5C,D).

Functional and structural benefits after infusion of allogeneic CDCs

To assess efficacy of allogeneic CDCs, minipigs underwent cardiac MRI before infusion (2-3

weeks post MI) and 2 months later. Allogeneic CDCs resulted in preservation of LVEF ( :-

0.5±3.2, p=0.73 within group), while LVEF decreased in control animals ( :-9.9±1.3%, p<0.001

within group, p<0.001 between groups) (Fig 2A-C, Supplemental Videos 1-4). In addition,

allogeneic CDCs attenuated LV remodeling: CDC-treated animals exhibited a smaller increase in

ESV ( :10.9±7.4ml) compared to controls ( :26.6±13.2ml, p=0.048 between groups) (Fig 2E).

EDV increased in both CDC-treated animals ( :17.7±10.5ml, p=0.020 within group) and

controls ( :30.5±19.8ml, p=0.026 within group, p=0.24 between groups) (Fig 2D).

emote myocardium was consistently clear of inflammatory infiltrates. No circululataatininng gf

alloreactive antibodies were detected in any recipients of allogened ic CDCs at any time point. In

coontntntrararaststst, ininin mmminipippigigigss that were allosensitized by y iininttrradermal and sususubccutututanananeous PBMNC

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Functional aandndnd sstrtrtrucucuctututuraaalll bbbenennefefefitittss s affftetet rr r inininfufufusisisiononon ooof f alalallolologegegeneneneicicic CCCDCDCDCsss

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Regional function was assessed in infarcted and non-infarcted myocardial segments, after visual

inspection of corresponding late Gd-enhanced images. Two months post-infusion, CDC-treated

infarcted myocardial segments displayed increased systolic thickening (p<0.001 between

groups), and increased end-systolic thickness (p=0.025 between groups), compared to infarcted

segments from placebo-treated animals (Fig 2F,G). In addition, regional function in the non-

infarcted myocardial segments was improved in CDC-treated animals compared to controls,

consistent with attenuation of LV remodeling (Fig 2F,G).

Fig. 3A shows representative late Gd-enhanced MRI acquisitions of hearts in short-axis

section at end-diastole. Gd-contrast agent accumulates in the infarct scar (as a result of a larger

distribution volume due to increased extracellular space in the scar compared with normal

myocardium19.20). In the CDC-treated minipig, the infarcted wall thickness was preserved 2

months post-infusion; importantly, the scar decreased in transmurality, while viable myocardial

mass increased. In contrast, the placebo-treated minipig was characterized by infarct thinning

and expansion, with no evidence of an increase in viable myocardium over the same time period

(Fig. 3A).

Two months post-infusion, MRI analysis revealed that scar size remained unchanged in

controls ( :0.4±0.9%, p=0.33 within group) but decreased in CDC-treated animals ( : -3.6

±2.4%, p=0.026 within group, p=0.007 between groups) (Fig 3B), resulting in significantly

smaller scar size in CDC-treated animals (9.2±0.8%) compared to controls (14.6±3.2%, p=0.006

between groups), despite similar scar size at baseline (12.8±2.8% [CDCs] vs 14.1±2.8%

[controls], p=0.48 between groups) (Fig 3E). While scar size is a conventional measure of

myocardial viability, cardiac MRI can quantify independently the individual components of scar

mass and viable myocardial mass9, enabling more discriminating insight into mechanism. Scar

distribution volume due to increased extracellular space in the scar compared wiwiththth nnnorrrmamamal ll

myocardium19.20). In the CDC-treated minipig, the infarcted wall thickness was preserved 2

momontntnthshshs ppososost-tt-iiinfufuusisisiono ; importantly, the scar decrerereasaseed in transmururrala ity,y, wwwhih le viable myocardial

mmasssss increaseded. InInIn cononntrrrasasastt,t, ttthehehe pplalalaccecebbo--treaaateeed mmminnnipiiig g g wawaas chchaaaraacteteriririzeeed d bybyby iinfnffaaarctctct tthhih nnnnninning

anndd d exexexpapapansnssioioion,n, wwwittth h nonno eeevividedeencncceee ofofof aan n innncrcrcreeeasesese iinnn vvviai bblbleee mymymyocococararrdidiiumumum oovvever r ththt e ee sasaamememe tiimime e e pepeeriiodod

Fig. 3A).

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mass decreased in CDC-treated animals ( :-1.4±1.4g), but not in controls ( :0.8±0.6g, p=0.012

between groups) (Fig 3C), resulting in a trend towards smaller endpoint scar mass in CDC-

treated animals (6.6±0.7g) compared to controls (9.7.3±3.5g, p:0.093 between groups) (Fig 3F).

In addition, CDC-treated animals exhibited significant increases in viable myocardial mass

( :10.7±3.6g) compared to controls ( :2.7±2.1g, p=0.003 between groups) over 2 months (Fig

3D), resulting in greater endpoint viable mass (65.8.9±4.5g) compared to controls (55.5±5.0g,

p=0.010 between groups) (Fig 3G). A complete list of MRI-measured parameters for each

experimental animal is provided in Supp Table 1.

Comparison of cardiac MRI with histology for assessment of scarred and viable

myocardium after cell therapy

Post-mortem histological analysis confirmed the MRI results, not just qualitatively but also with

quantitative accuracy. Fig 4A shows representative short-axis cardiac slices after incubation with

TTC, while Fig 4B shows representative sections from the infarcted wall stained with Masson’s

trichrome. CDC-treated hearts consistently exhibited significant amounts of viable myocardium

in the infarcted wall, most often in the form of endocardial and epicardial muscular layers

surrounding the scar but also in the form of islets of viable myocardial tissue interspersed

between the collagen fibers. In contrast, in control minipigs the scar was homogeneous and

largely transmural (Fig 4A,B,F). The increased amount of viable myocardium in the infarct

region after cell therapy is consistent with the improved regional contractility of infarcted

segments in CDC-treated animals compared to controls (Fig 2F,G). Histological measurement of

scar and viable myocardium demonstrated decreased scar transmurality in CDC-treated animals

(44.1±21.3% vs 79.3±25.2%, p=0.044), smaller scar size (9.1±1.2% vs 14.0±2.9%, p=0.009) and

increased viable myocardial mass (68.7±4.7g vs 55.2±4.9g, p=0.002) in CDC-treated animals

myocardium after cell therapy

Post-mortem histological analysis confirmed the MRI results, not just qualitatively but also with

ququananantitititatatatititiveveve aaaccurururaacacy. Fig 4A shows representatttivivivee short-axis cardrddiac c slslsliicices after incubation with

TTTCCC, while Fiig g 4B4B4B sshohoh wswsws rrreepeprrereseseentntntata iivveee seccctiiionss ffrrrommm tththe e innnfafarrccteed d wwallll sstataaininneded wwwititith hh MaMaMassssononon’s

rricicchrhrhromomome.e. CCCDCDDC---trreeatateeded hheeartrtrts s cococonsnsnsiisisteteentnttlylyly eeexhxhxhibibiiiteeded sssigiggnininiffificacaantntn aamomomoununntstss oof f f viviiababa lelele mmmyyoyoccacardrddiuiumm m

n the infarcttededed wwwalala ll,l, mmmosoo t t ofofoftett n n n ininin ttthehh ffforororm m m ofofof eeendndndococcarardididialalal aaandndnd eeepipicacacarrrdididialalal mmmususscucuculalalarr r lalalayers

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compared to infarcted controls (Fig 4C-F), yielding virtually identical results to those obtained

from contrast-enhanced MRI (Fig 3E-G). A complete list of histological measurements of scar

size, scar mass and viable mass for each experimental animal is provided in Supp Table 2.

To further evaluate the ability of contrast-enhanced MRI to accurately measure scarred

and viable myocardium after cell therapy, all TTC-stained cardiac slices were matched with their

corresponding late Gd-enhanced images (from both the in vivo and ex vivo MRI datasets). Fig

5A provides representative examples of this analysis for a CDC-treated and a control minipig. In

both cases, areas of hyperenhancement in cardiac MRIs correspond faithfully to regions of

scarred myocardium in histological slices. Importantly, MRI reveals significant amounts of

viable myocardium within the infarct region of the CDC-treated heart, a finding that is confirmed

by cardiac histology: the endocardial and epicardial rims of non-hyperenhanced tissue in the

infarcted wall in contrast-enhanced MRIs are virtually identical to the endocardial and epicardial

TTC-positive muscular layers surrounding the scar in histological slices. In contrast, the control

heart is characterized by a dense transmural scar, and no viable myocardium can be detected in

the infarct area by either MRI or histology. Figs 5B-D show quantitative correlation analysis

between the various MRI parameters and the corresponding histological values; the measures of

scar size (as % of isolated cardiac slices and as % of the LV), LV scar mass and LV viable mass

all correlate strongly, with a slope close to the line of identity. Bland-Altman analysis

demonstrated excellent agreement between MRI and post-mortem histology (Supp Fig 6).

MR imaging of Gd-contrast kinetics in cell-treated myocardium

To assess whether cell therapy fundamentally alters myocardial contrast uptake and/or wash-out,

we compared Gd-contrast kinetics in various regions infused with cells to the kinetics in the

remote (non-infarcted, non-cell treated) myocardium. A series of dynamic late Gd-enhanced

viable myocardium within the infarct region of the CDC-treated heart, a finding ttthahahatt isiss cconononfififirmrmed

by cardiac histology: the endocardial and epicardial rims of non-hyperenhanced tissue in the

nnfafaarcrcrcteteteddd wawaw lllll in n cococontn rast-enhanced MRIs are viirtrtrtuauaally identical totoo thee eeenndndocardial and epicardiald

TTTCCC-positive mmuususcuculalalar lalalayeyerrsrs ssururrororoununu didinng tthehee scaaar r in hhhiisistotolologigiccaal slsliciceees.. InIn ccoonontrtrasasa t,t,t ttheheh cccononntrrrol

heheararart t isisis cchahaarararactctererrizzzeded bby y y a a dedeensnseee trtrtranaansmsmmurururalalal scacacarr,,, annnd nonono vvviiaiablblblee e mymymyococo aararddidiumumm cccanana bbbee e deeetetectctc ededd iinnn

he infarct arreaeaea bbbyyy eieieiththhererer MMMRIRIRI ooor r r hihih stststolologogogy.y.y FiFiFigsgsgs 555B-B-B DD ssshohohow w w quququananantiiitatatatititiveveve cccororo rereelalaatititiononon aanalysis

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images were acquired with a fixed TI (Fig 6A)15,16, and signal intensity of specific areas

(collagenous scar, viable myocardium in the infarcted region, viable myocardium at the border

zone, remote myocardium) was measured at various timepoints following contrast administration,

resulting in the generation of site-specific Gd-contrast curves (Fig 6B; pooled data from all

CDC-treated minipigs). Analysis using a linear mixed effects model demonstrated that the only

site in which Gd kinetics were (predictably) different compared to remote healthy myocardium

was the infarct scar. Importantly, Gd-contrast kinetics were virtually identical in cell-treated

viable (non-hyperenhanced) areas and in the remote non-cell treated healthy myocardium.

Vascular density and architecture

To investigate whether cell therapy with CDCs can induce vascular changes in the treated

(scarred or viable) myocardium, we studied vessel density and architecture in the collagenous

scar, in viable myocardium in the infarcted wall and in the remote (non-infarcted, non-cell

treated) myocardium. As expected21,22, capillary density was low in the collagenous scar

(117.7±32.9 vessels/mm2) and in the border zone (525.3±274.3 vessels/mm2) as compared to

remote myocardium (1344.4±356.5 vessels/mm2). While total capillary density was increased in

the border zone (defined as the region at the edges of the scar comprising areas of both scarred

and viable myocardium) of CDC-treated hearts (721.9±192.5 vessels/mm2 vs 328.8±188.8

vessels/mm2, p=0.012) (Fig 7B), no differences in capillary, arteriolar or arterial density in

scarred or viable myocardium could be detected between CDC-treated animals and controls (Fig

7C). Thus, the increased total capillary density in the border zone of CDC-treated hearts is a

result of the increased viable mass and decreased scar mass observed in the infarct border zone

after CDC therapy, and cannot be attributed to differences in vascular density of scarred or viable

myocardium between CDC-treated hearts and controls. Vessel architecture (as quantified by

To investigate whether cell therapy with CDCs can induce vascular changes in ththhe trtrt eaateteted d d

scarred or viable) myocardium, we studied vessel density and architecture in the collagenous

ccararr, ininin vviaiaiablblbleee mymyyoococardium in the infarcted walll aaanndd in the remotetee (nononn-i-i-innfarcted, non-cell

rreaaateted) myocacardrdrdiuuum.m. AAss s exeexpepepectctededd2121,1,22222,, cccapiillll aaary ddedennsitttyy y wawaas lolowww iinn thehehe cccolollalalaggegenonooususus ssccar r r

11171717.7.77±3±3± 2.2..99 9 vevesssssellls/s/mmmmmm2) ) annnd d ininin ttthehehe bbbororrdedederr r zooonenen (55252555.3±3±3±227274.4.4 333 vvevesssssselelsss/mmmmm22))) asasa cccomomompapapareeed d totoo

emote myocacaardrdrdiuiuum m m (1(113433 4.4.4.4±4±4±35556.6.6 55 vevev sssssselelels/s//mmmmmm222).).) WhWhW ililile ee totototatatalll cacacapipiilllll ararary yy dededensnsn ititity y y wawawas s s ini creased innn

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lumen diameter [Fig 7D], wall thickness, endothelial layer thickness, smooth muscle layer

thickness [Fig 7E] and lumen/wall ratio [7F]) was similar in CDC-treated and control hearts.

Stimulation of endogenous cardiac regeneration by allogeneic CDCs

To investigate whether the increased viable myocardium observed after CDC-therapy was a

result of myocyte hypertrophy, we measured cardiomyocyte cross-sectional area in the infarct

and peri-infarct area. Myocyte size was significantly smaller in CDC-treated animals compared

to controls (Fig 8A), thus excluding myocyte hypertrophy as a contributor to the increase in

viable myocardium. The conjunction of increased viable mass and smaller myocyte size are

indicative of cardiomyocyte hyperplasia after cell therapy. With regard to the latter, we found

that transplantation of CDCs upregulated cardiomyocyte cycling (Fig 8B) and increased the

number of small round TnI+ cells (previously defined as putative myocyte progenitors22,23) in the

infarct and peri-infarct area (Fig 8C), confirming previous reports18,24. While cardiomyogenesis

is likely to play a role in the reduction of cardiomyocyte area, attenuation of adverse remodeling

(which involves cardiomyocyte hypertrophy) may potentiate the reduction in myocyte size in the

infarct and peri-infarct area of CDC-treated animals. Supporting this conjecture, myocyte cross-

sectional area in the remote myocardium tended to be lower as well (consistent with relief of

wall stress), although the differences did not reach statistical significance (Supp Fig 7).

Discussion

Heart-derived cells are particularly promising for cardiac repair and regeneration. In the

CADUCEUS trial, intracoronary infusion of autologous CDCs decreased scar size, increased

viable myocardium and improved regional myocardial function, as measured by MRI3. An

interim MRI analysis of the still-ongoing SCIPIO trial (where MRI was performed only in

hat transplantation of CDCs upregulated cardiomyocyte cycling (Fig 8B) and inncccreaee sseedd d thththe e e

number of small round TnI+ cells (previously defined as putative myocyte progenitors22,23) in the

a (nnfafaarcrcrcttt anandd d pepeperi-i-iinfnfnfara ct area Fig 8C), confirminnng gg pprrevious reportstss18,244.. WWWhile cardiomyogenesis

ss liikikely to plaay y aaa rrrolele in n n thththeee rerereduducctctioioionn ooff carrrddiiommyoocyyytetee aarreeaa,a, aatttteennuauatititionn oof ff aadadveveersrsr e e e rereemmomodededeliiingn

wwhihih chchch iinvnvvolololveves s caaardrdiioommymyococytyty ee hyhyhypeppertrttroroophphphyyy) mmmayayay pppotttenenntititiatatate thththee e rereedudud cccttioonon iiin nn mymymyocococyytyte e siiizeze iiinn ththt e

nfarct and pepeperirir -i-infnfnfarararctctt aaareea a a ofofo CCCDCDCDC-t-treeeatatatededed aaannnimimimalalls.s.. SSupupuppopoportrtr ininng g g ththhisisis ccconononjejejectctc ururre,e,e, mmmyoyoyocyc te cross-

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treated, primarily non-randomized patients) showed similar results4. Although late Gd-enhanced

cardiac MRI has been extensively validated for the quantification of necrotic/fibrotic and viable

myocardium in acute/chronic MI6-8, its validity to accurately characterize tissue viability after

cell therapy has been questioned10: cell administration may increase vascular wall thickness,

resulting in decreased vessel permeability and attenuation of Gd-contrast extravasation in cell-

treated myocardial regions, or stimulate angiogenesis, leading to enhanced drainage of Gd-

contrast from the cell-treated myocardium (a phenomenon that has been described in

hypervascular hepatocellular carcinomas11). Any of these confounding factors, if operative,

would result in altered Gd-contrast myocardial kinetics (in the form of decreased contrast

extravasation or accelerated wash-out) that could compromise the fidelity of contrast-enhanced

MRI10.

We sought to validate experimentally the ability of contrast-enhanced MRI to distinguish

and accurately measure scarred from viable myocardium after cell therapy. We find that CDC

infusion does not change vascular density or architecture in scarred and viable myocardium,

neither does it result in altered Gd-contrast myocardial tissue kinetics. Importantly, using post-

mortem histology as the gold standard, we demonstrate that contrast-enhanced MRI readily

distinguishes viable and scarred myocardium and provides accurate measurements of scar size,

scar mass and viable myocardial mass in cell-treated hearts.

The present work differs from previous human3 and porcine12 studies of CDCs in that the

prior studies used autologous cells. The effects of allogeneic CDC therapy reported here are

qualitatively similar, and at least as impressive quantitatively in terms of regenerative and

functional efficacy; however, a head-to-head comparison of allogeneic versus autologous cells

was beyond the scope of this study. The greater increase in EF observed in this study may be

extravasation or accelerated wash-out) that could compromise the fidelity of contnttraraststs -eenhnhnhananancecec dd

MRI10.

WeWeWe ssoooughghhttt toto validate experimentally theee aabbbility of contrasasst-ennhahahannced MRI to distinguish

anndd d aca curatelyy mmmeaasusurrre ssscacacarrrrrededed ffrororommm vvviaaable mmmyocccarrrdiuuummm afaffteeer r ceceelll ttheherrrappypy.. WeWeW ffinininddd thththatat CCCDCDCDC

nnfufufusisiionono ddoeoeoesss nonott cchchananngegee vvasscucuc lalalarrr deded nsnssitttyy y ororor aaarrrchchchiiteeectutuureree iiinnn scsccarara rerer ddd aaanndnd vvviaiabblble ee mymymyoococarrrdidiumumu ,,,

neither does iit t t rereresusuultltlt in nn alaa teeererered dd GdGdGd-c-cconono trtrtrasasast t mmmyoyoyocacac rdrdrdiaiai l tititissssssueueue kkkininineteticicicsss.. ImImImpopoportrtananantltltly,y,y, uuusis ng post-

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attributable to one of several factors: 1) increased efficacy of allogeneic cells (not evident in

previous rat studies, which showed equivalence of intramyocardially-injected allogeneic and

syngeneic CDCs18 or cardiospheres25); 2) the earlier administration of CDCs here (2-3 weeks

post-MI, versus 4-5 weeks in our autologous porcine study12 or 1.5-3 months post-MI in

humans3). A comparison of EF values in the present study versus our previous porcine study12

reveals higher baseline EF values in both groups (a finding consistent with the earlier timepoint

of baseline imaging) and a larger decrease in EF in the control group over the period of 2 months

in the present study; EF of the treated group is preserved over time in both studies. These data

may hint that administration of CDCs earlier in the remodeling process may offer increased

functional benefit; and 3) the absence of concomitant anti-remodeling therapies (which in the

human setting leave little room for EF improvement). Allogeneic CDCs induced a mild local

immune reaction in the heart with no signs of immune-related myocardial damage. Importantly,

no circulating anti-donor antibodies could be detected, predicting that no development of panel

reactive antibodies would occur in the human setting (at least after a single administration of

allogeneic cells). The efficacy of allogeneic cells is rationalized by their indirect mechanism of

benefit18, which relies on activation of endogenous reparative and regenerative pathways

(increased cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis), rather

than long-term engraftment and differentiation of transplanted cells (no donor cells could be

detected in the recipient myocardium 2 months post-administration).

While cardiac MRI cannot distinguish cardiac hypertrophy from hyperplasia, post-

mortem histological analysis ruled out myocyte hypertrophy as a contributor to the increase in

viable myocardium observed after CDC therapy; myocyte size was actually smaller in the infarct

and peri-infarct area of CDC-treated animals compared to controls, a finding consistent with

functional benefit; and 3) the absence of concomitant anti-remodeling therapies (w(w(whihih chchch iinn n thththe e

human setting leave little room for EF improvement). Allogeneic CDCs induced a mild local

mmmumumunnene rreaeaeactctctionnn ininin the heart with no signs of immmmmmuuune-related myyyoco arrdididiaalal damage. Importantly, f

nno ccciri culatingg aannnti--dodonnonorr r ananantititibobbodidieseses ccoououlld bbee ddeteeecttted, , , pprprededdiccctitinngg tthahatt nonono ddeeveveelelopoppmemementntt oof ff papaaneneel

eeacacactitiivevev aantntntibibibododiieiesss wowooululld d ococcucuc rrr ininin ttthehe hhumumumaanan sssetettttingngng (((atatt llleeaeasttt aaaffnn ttter rr a a a sisiingngglele aaadmdmdmininnisissttratattiooon n ooff ffff

allogeneic celelllslsl ).).). TTTheheh eeeffff icccacacacy yy ofofof aaalllllogogo eneneneieieiccc cececelllll ss s isiss rrrata ioioonananalililizezezed d d bybyby tttheheheiririr iiindndndirirececect t t mememechchc anism of

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attenuation of remodeling-associated cardiomyocyte hypertrophy and birth of new (smaller)

myocytes after cell therapy. The latter likely occurs through differentiation of endogenous

progenitors (visualized here as small round TnI+ cells) and induction of resident cardiomyocyte

proliferation in the border zone, in agreement with results from fate-mapping studies24.

Limitations

Our study has several limitations. First, delayed contrast enhancement images were acquired 8

minutes post-Gd administration. Even though the FWHM technique (used here) has been shown

to provide accurate measurement of infarct size as early as 6 minutes post contrast

administration7, standardized protocols issued by the Society for Cardiovascular Magnetic

Resonance advocate waiting at least 10 minutes26 before acquisition of delayed contrast

enhancement images. Second, allogeneic CDCs were derived from a single healthy donor

minipig; we did not investigate inter-donor variability in cell immunogenicity or potency. Finally,

we did not investigate the safety and efficacy of repeat administrations of allogeneic CDCs.

Careful preclinical studies of safety and efficacy will be required before repeat dosing with

allogeneic cells can be contemplated.

Conclusions

In conclusion, we validate the ability of late Gd enhancement MRI to accurately measure scarred

and viable myocardium after cell therapy, supporting the utility of contrast-enhanced MRI for

assessing dynamic changes in the infarct and monitoring therapeutic regenerative efficacy. We

also demonstrate that intracoronary infusion of allogeneic CDCs without immunosuppression is

safe, improves heart function and indirectly promotes cardiac regeneration in a clinically-

relevant porcine model of convalescent MI. The safety and efficacy of allogeneic CDCs in

Resonance advocate waiting at least 10 minutes26 before acquisition of delayed ccononontrtrt asasst tt

enhancement images. Second, allogeneic CDCs were derived from a single healthy donor

miminininipipipigg;g; wwwe e e dddid d nononot t investigate inter-donor variabababillity in cell immumum noogegegennnicity or potency. Finally

wwe ddid not invvesestit ggagatetee thehehe sssafafafetetety y ananand d d eeffffiicaccyy of rrepeppeatt t aadadmmminniniststraratitiononsss ooff aalllllloogogenenneieieic c c CDCDC CsCCs. f

CaCaarerer fufufull prprp ecececlillininicccall l ststuuudiieies s ofoff safafafetetety yy anannd d efefeffifificaaacycyc wwwiilill bebebe rrreqeqequiuiuirerer ddd bebebefofof rrre rrepeppeaeaeatt t dodoosisiingng wwwitith h

allogeneic celelllslsl ccananan bbe e e cocc ntntntemememplplplatata ededd.

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human subjects with LV dysfunction post-MI is currently being tested in the phase 1/2

randomized double-blind, placebo-controlled ALLSTAR trial, (ALLogeneic heart STem Cells to

achieve myocArdial Regeneration, NCT01458405)27, which uses infarct size assessed by MRI as

its primary efficacy endpoint.

Acknowledgments: We thank Adrian Glenn, Hao Zeng, Miguel Huerta, Claudia Anchante, Julie

Avalos and Stephen Taylor for their technical and surgical support, Laura Smith for performing

the MRI scans and analyzing MRIs, Nina Duong for blood processing, and David Elashoff and

Tristan Grogan for statistical consulting.

Funding Sources: This work was supported by a grant to RRS from the NIH (HL103356), and

by a grant to EM from the California Institute for Regenerative Medicine. Statistical analysis was

supported by NIH/National Center for Advancing Translational Science (NCATS) UCLA CTSI

Grant Number UL1TR000124.General laboratory support was provided by the Cedars-Sinai

Board of Governors Heart Stem Cell Center.

Conflict of Interest Disclosures: EM and LM own equity in Capricor, Inc. KM is a consultant

for Capricor, Inc. Other authors have no relationships to disclose.

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for Capricor, , InInInc.c.c OOOththt ererer autututhohohorsss hhhaaveveve nnno oo rererelalalatititionono shshshipipips s tooo dddisisisclcc ososose.ee

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25. Tseliou E, Pollan S, Malliaras K, Terrovitis J, Sun B, Galang G, Marbán L, Luthringer D, Marbán E. Allogeneic cardiospheres safely boost cardiac function and attenuate adverse remodeling post-myocardial infarction in immunologically-mismatched rat strains. J Am Coll Cardiol. 2013;61:1108-1119.

past, present, future. Radiology. 2011;261:358-374.

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211. . DuDuDuboboboisisis CCC,, LiLiiuu u XX, Claus P, Marsboom G, Pookkrkreeiisz P, Vandennwiww jnngagaaeerert S, Dépelteau H, StrebWW,W, CCChah othahaweww e e L,L,L MMaeaees s F,F,F, GGheheeysysysenenss O,O, DDebebysyserrr ZZZ, GiGillllllijijijnsnsn HH,,, PePeP llllenenss s M,M VVananandeded ndndririesesesscscchehehe TT,rCCChuuauah M, Colllelennn DD,D, VVVererrbebebekekekennn E,E, BBBele mamamans AAA,, Vaann dde WWWererff f FF,F, BBBogggaeaertrtt JJ,, JaJaansnsnssesensnss SSS.. DiDD ffffffererenenenttialefefffeectc s of prorogegg nnnittoor ccellll popoppuululattionsnsn ooonn lefttt vvventrrricccululararar rrremememooodelllinng annd mymyyooccara diiaala tneneoovovasasascuculalaariririzazatitiononon aaftftterer mmyooocacardrdrdiaiaialll ininnfafafarcrcrctititiononn.. J J AmAmAm CCCololollll CaCaCardrdrdioiooll. . 20202 1110;5;5;55:5::2222223232-2-2-22224333.

22. Lin YD, , LuLuLuooo CYCYCY, , HuHuH YYYN,N,N YYYeheheh MMML,L,L, HHHsusuueheheh YCYCYC,, ChChC ananangg g MYMYMY, ,, TsTssaiaia DDDC,C,C, WWWananang g g JNJNJN,,, TaT ng MJ, WeWeii EIEI SpSpriringngerer MMLL HHsisieheh PPCC IInsnstrtrucuctitiveve nananonofifibeberr scscafaffofoldldss wiwithth VVEGEGFF crcreaeatete aa

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27. Allogeneic Heart Stem Cells to Achieve Myocardial Regeneration (ALLSTAR) (NCT01458405). Available at: http://clinicaltrials.gov/ct2/show/NCT01458405?term=allstar&rank=1. Accessed February 28, 2013.

Figure Legends:

Figure 1. Study protocol. Schematic depiction of the 24-hour retention study (A) and the

validation study (B).

Figure 2. Allogeneic CDCs attenuate adverse remodeling and improve global and regional

function compared to controls. A,B: Matched cine short-axis images (at end-diastole [ED] and

end-systole [ES]) at baseline and 2 months (videos of the cine acquisitions are provided in the

Online Supplement) for a minipig treated with allogeneic CDCs (A) and a control minipig (B).

Images were obtained at similar levels (note the similar morphology of papillary muscles).

Changes in ejection fraction (C), end-diastolic volume (D) and end-systolic volume (E) from

baseline to 2 months in controls and CDC-treated minipigs. Systolic thickening (F) and end-

systolic thickness (G) in infarcted (infarct) and non-infarcted (remote) segments at 2 months

post-infusion in controls and CDC-treated minipigs (* p<0.05 vs CDC-treated group).

Figure 3. Allogeneic CDCs decrease scar size, decrease scar mass and increase viable

Figure 1. Study protocol. Schematic depiction of the 24-hour retention study (AA) ) anananddd ththheee

validation study ((B)).

FFiFiguuure 2. AlAllologeeenneeic CCDDCDCss atatttet nun atatatee adaddverrssee remomomodedelililingngng aaanndd impmpmproveee ggglolobabaall ana dd d reeegigiionnnal

funcnctitiono ccomompaparred d toto conntrtrolls.s. AA,BB: Maatctchehedd cicinen shohortrt-a-axixiss imimagageses ((ata eendnd-d-diaiaststole e [E[ D]D] aandnd

enendd-sysyststololee [E[ES]S])) atat bbasaselelininee anandd 22 momontnthshs ((vividedeosos ooff ththee cicinene aaccququisisititioionsns aarere pprorovividededd inin tthehe

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myocardial mass compared to controls, as assessed by MRI. A: Representative delayed contrast-

enhanced MRI acquisitions of hearts in short-axis section at end-diastole. Scarred myocardium

appears white while viable myocardium appears black. In the CDC-treated minipig the scar

decreased in transmurality and viable myocardial mass increased over the period of 2 months

after CDC infusion. The control minipig was characterized by scar thinning and scar expansion,

with no evidence of increase in viable myocardium over the same time period. Changes in scar

size (B), scar mass (C) and viable mass (D) from baseline to 2 months in controls and CDC-

treated minipigs. E: Scar size at baseline and at months post-infusion in control and CDC-treated

minipigs. Scar mass (F) and viable mass (G) at 2 months post-infusion in controls and CDC-

treated minipigs (* p<0.05 vs CDC-treated group, # p<0.05 vs baseline).

Figure 4. Allogeneic CDCs decrease scar size and increase viable myocardial mass compared to

controls, as assessed by post-mortem histology. A: Representative short-axis cardiac slices of 2

CDC-treated and 2 control minipigs at 2 months post-infusion after incubation with TTC. Slices

were obtained at similar levels (note the similar morphology of papillary muscles). Viable

myocardium stains brick-red, while scarred myocardium appears white (unstained). B:

Representative sections from the infarcted wall of a treated and a control minipig stained with

Masson’s trichrome. Viable myocardium stains red, while collagenous scar stains blue. CDC-

treated hearts exhibited significant amounts of viable myocardium in the infarcted wall, in the

form of endocardial and epicardial muscular layers surrounding the scar and in the form of islets

of viable myocardial tissue dispersed in-between the collagen fibers. In control minipigs the scar

was largely transmural. Scar size (C), scar mass (D), viable mass (E) and scar transmurality (F)

at 2 months post-infusion in controls and CDC-treated minipigs, as assessed by post-mortem

reated minipigs (* p<0.05 vs CDC-treated group, # p<0.05 vs baseline).

Fiigugugurerere 444. AlAlAllllogegeeneneneici CDCs decrease scar size aaanndnd iincrease viablleee myyocococaaardial mass compared to

coonntntror ls, as assssesessseed d bybyb popopoststst-m-m-morortetetemmm hhhissstoloogogyy. AAA: Repepprrereseseennttatatiivvee shshoorrt---axaxisisis ccarardidid acacc sslil cececes s ofofof 2

CDCDDC-C-C-trtrtreaeateteed d d ananddd 22 2 cocontntroror l l mmiminininipipipigsgsgs aatt t 22 momomonttthshsh popoposts --iinfnffusususioion nn afaffteeerrr inini ccucubbabatitiiononon wwititithh h TTTTCC.C. SSllliccees

were obtaineed d d atata sssimimimilillararar levevvelelels (n(n(nototo eee thhhee e sisisimimim lalalar r r momomorprprphohoololologygygy ooof f f papp pipipilllllararary y y mumumuscscclelees)s)s).. ViViV able

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histology (* p<0.05 vs CDC-treated group).

Figure 5. Comparison of cardiac MRI with histology for assessment of scarred and viable

myocardium after cell therapy. A: TTC-stained cardiac slices matched with their corresponding

delayed contrast-enhanced MRI images (from both the in vivo and ex vivo MRI datasets) for a

CDC-treated and a control minipig. Areas of hyperenhancement in cardiac MRIs correspond

excellently to regions of scarred myocardium in histological slices. The treated minipig MRI

demonstrates endocardial and epicardial rims of non-hyperenhanced tissue in the infarcted wall

which correspond excellently to endocardial and epicardial TTC-positive muscular layers

surrounding the scar in histological slices. The control heart is characterized by a transmural

scar, and no viable myocardium can be detected in the infarct area by either MRI or histology.

Correlation of MRI measurements of scar size as % of isolated cardiac slices (for in vivo [B] and

ex vivo [C] MRI), scar size as % of LV, LV scar mass and LV viable mass (D) with the

corresponding histological measurements.

Figure 6. Gd-contrast myocardial kinetics in cell-treated hearts. A: A series of dynamic delayed

contrast enhancement images with a fixed T1 were acquired and signal intensity of specific areas

(collagenous scar, viable myocardium in the infarcted region, viable myocardium at the border

zone, remote myocardium) was measured at various timepoints following contrast administration,

resulting in the generation of site-specific Gd-contrast curves in all cell-treated minipigs. B: Gd-

contrast kinetics pooled data from all treated minipigs. Analysis using a linear mixed effects

model demonstrated that Gd-contrast kinetics were identical in cell-treated viable (non-

hyperenhanced) areas and in the remote non-cell treated healthy myocardium. Image on the right

urrounding the scar in histological slices. The control heart is characterized by a aa trranaa smsmsmurururalalal

car, and no viable myocardium can be detected in the infarct area by either MRI or histology. d

CoCorrrrrrelelelatatatioioionn n ofofo MMMRRIRI measurements of scar size aaasss %%% of isolated cararardiacacc sssllilices (for in vivo [B] and

exx vvvivi o [C] MRMRI)II ,, sccaarar sssiizizeee asasas %% oooff f LVLVV,, LVVV ssccar mmassss aaandnd LLV V vvviaabablele mmmaasass s (((DDD) ) wiwiwiththh tthheh

coorrrrrresesspopopondnddinining g hihiistttolologoogiciccalal mmmeaeasususurererememementnttss.s.

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in A is the corresponding histological section stained with Masson’s trichrome of the inset in the

MRI acquisitions.

Figure 7. Vessel density and architecture. A: Vascular density and architecture were evaluated

by immunostaining for -sarcomeric actinin, -smooth muscle actin and isolectin. Image on the

right is a high-power image of inset on left. B: Vessel density in the border zone (defined as the

region at the edges of the scar comprising areas of both scarred and viable myocardium). Vessel

density (C), lumen diameter (D), total wall thickness, endothelial layer thickness, smooth muscle

layer thickness (E) and lumen to wall ratio (F) in viable myocardium in the infarcted wall

(viable), in the collagenous scar (scar) and in the remote non-infarcted myocardium (remote) (*

p<0.05 vs CDC-treated group).

Figure 8. Allogeneic CDCs attenuate myocyte hypertrophy and promote endogenous

regeneration in the infarct and peri-infarct area. A: Cardiomyocyte cross-sectional area in the

infarct and peri-infarct area. B: Cycling cardiomyocytes (arrows) in the infarct and peri-infarct

area. C: Small TnI+ cells (arrows; image on the right is a magnification of the inset on left) in

the infarct and peri-infarct area (* p<0.05 vs CDC-treated group).

viable), in the collagenous scar (scar) and in the remote non-infarcted myocarddiuiuum mm (r(rrememmototote)e)e) ((*

p<0.05 vs CDC-treated group).

FFiguugure 8. Alloogegeenneeicc CCDCDCDCs s atatatttetenunuuaatate e mymyoc ytytee hyyyppeertrroropphphyyy aanandd prromomoooteee enenndododogegennonoususu

eegegegenenenerarar titiononon iin n tthhee e ininfffarrcrct t annnd dd pepepeririri-i-i-infnffarara ctctct aaarerea.aa. AAA: CaCaardrddioioiomymymyococo yyyteee crcrc ooosss--sesectctc ioioionanaall l aaareaeaa iinn n ththhe

nfarct and pepeperirir -i-infnfnfarararctctt aaareea.a.a BBB: CyCyCyclclcliningg g cacacardrdrdioioiomymymyocococytyty esess ((arararroror wswsws)) innn t ttthehehe iiinfnfnfarara ctctct aaandndnd pppere i-infarct

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Figure 6

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Figure 7

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Figure 8

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Marbán and Eduardo MarbánBlusztajn, Ileana Valle, Supurna Chowdhury, Raj R. Makkar, Rohan Dharmakumar, Debiao Li, LindaTseliou, James F. Dawkins, Michelle Kreke, Ke Cheng, Daniel Luthringer, Chak-Sum Ho, Agnieszka

Konstantinos Malliaras, Rachel R. Smith, Hideaki Kanazawa, Kristine Yee, Jeffrey Seinfeld, Elenia Porcine Model of Convalescent Myocardial Infarction

Validation of Contrast-Enhanced MRI to Monitor Regenerative Efficacy after Cell Therapy in

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online September 23, 2013;Circulation.

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The online version of this article, along with updated information and services, is located on the

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SUPPLEMENTAL MATERIAL

SUPPLEMENTAL METHODS

Culture of porcine CDCs

A Sinclair mini-pig heart from a ~4 months old male was obtained from Sinclair BioResources to create

a master cell bank (MCB). The heart arrived on ice submerged in cardioplegic solution. Hearts were grossly

dissected and biopsy-sized pieces (~25mg) were seeded to create explant derived cells (EDCs). After ~14

days, the EDCs were harvested to create a MCB. MCB vials were thawed and cultured as cardiospheres

(CSps) in suspension culture. CDCs were grown by seeding CSps on tissue culture treated flasks, and

passaged when confluent. Passage 5 CDCs were resuspended in CryoStor™CS10 (BioLife Solutions) with

100 U/mL heparin in cryobags (for a total dose of 12.5 M cells/10 mL), placed directly in a CryoMed controlled-

rate freezer, and then transferred to liquid nitrogen. CDCs were thawed at the day of the infusion. Upon

thawing, 100 U/mL heparin and 50 µg/mL nitroglycerin were added as excipients to each 10 mL dose.

MI creation and CDC infusion

On day 0, animals were premedicated with ketamine 20mg/kg IM, atropine 0.05mg/kg IM, and

acepromazine 0.25mg/kg IM. Animals were subsequently induced with propofol 2-4mg/kg IV to effect,

intubated, and maintained on isoflurane 2-3%. Amiodarone 10mg/kg IV loading dose, then 0.5mg/kg IV as

needed and lidocaine 0.03µg/kg/min were given for ventricular arrhythmias. Heparin 100IU/kg IV was given for

anticoagulation. Minipigs were subjected to an anteroseptal MI by inflation of an angioplasty balloon in the mid-

left anterior descending artery (LAD) (distal to the 1st diagonal brach) for 2.5 h., followed by coronary

reperfusion. Two to 3 weeks after MI creation, infarcted minipigs were re-anesthetised using the same protocol

and drug regimen. Pigs were randomized to receive 12.5 million CDCs (in 10 mls of CryostorTMCS10

containing 45 μg/ml nitroglycerin and 180 U/ml heparin) or vehicle. Intracoronary infusion was performed via

an over-the-wire balloon catheter, placed in the mid LAD. CDCs or vehicle solution were infused in 3 cycles of

intermittent balloon inflation. Minipigs were euthanized either 24 hours post-infusion, to measure short-term

cardiac retention of administered cells, or 2 months later.

Histocompatibility

To assess histocompatibility, low-resolution swine leukocyte antigen (SLA) typing was performed on the

donor Sinclair minipig and all recipient Yucatan minipigs as described elsewhere (1,2). Divergent pig strains

were used to simulate the typical allogeneic conditions in human transplantation where donor and recipient are

frequently mismatched with regard to human leukocyte antigen. Complete SLA mismatch was confirmed if

there was at least one class I and one class II antigen mismatch at the allele group level between donor and

recipient.

Ex vivo luciferase assay

The ex vivo luciferase assay, a method with high sensitivity and reproducibility (3) (depicted

schematically in Supplemental Figure 1a) was performed as described (4). In brief, CDCs were transduced

with an adenoviral vector carrying the firefly luciferase gene 3 days prior to infusion. For each animal a

separate standard curve was constructed, as transduction efficiency and transgene copy number per cell vary

by experiment. Specifically, once the cell dose was allocated for infusion, an aliquot of cells (from the same

batch of adenovirally-transduced cells) was cultured for an additional 24 hours. Twenty-four hours after cell

delivery, the explanted LV was cut into 1g samples that were subsequently homogenized and analyzed for

luciferase signal. The animal-specific standard curve was prepared by measuring the luciferase signal from pig

myocardial samples from the left atrium (not infused with cells) spiked with known numbers of transduced

Histology

To evaluate immune rejection in the heart, hematoxylin and eosin-stained sections from myocardial

samples (fixed in 10% formalin, paraffin-embedded [FFPE]) obtained from the infarct and peri-infarct area

(n=4-6 slides/heart) and the remote myocardium (n=2 slides/heart) were analyzed by an experienced cardiac

pathologist blinded to treatment allocation (DL). Analysis was graded according to the International Society for

Heart & Lung Transplantation (ISHLT) grading system, used in clinical practice to diagnose solid organ

transplant rejection. Morphometric analysis with Masson’s trichrome staining was performed on 8 μm sections

from myocardial samples (FFPE) extending from the viable myocardium at the edges of the scar to the core of

the collagenous scar (n=4 slides/heart). To study vascular density and architecture, 8 μm sections from

samples (FFPE) obtained from the infarct, peri-infarct (n=4-6 slides/heart) and remote (n=2 slides/heart) areas

underwent immunostaining for α-sarcomeric actinin (Sigma), α-smooth muscle actin (Abcam) and isolectin

(Molecular probes). Arterioles and arteries were defined as smooth-muscle-coated vessels with external

vascular diameter (lumen + vascular walls) <75 μm and >75μm respectively. To measure myocyte cross-

sectional area, 8 μm sections from myocardial samples (FFPE) obtained from the infarct, peri-infarct (n=4-6

slides/heart) and remote (n=2 slides/heart) areas underwent immunostaining for α-sarcomeric actinin and

wheat-germ agglutinin (Molecular probes) (to visualize cell borders). Cardiomyocytes were accepted for size

measurement if they met the following criteria: (a) cellular cross-sections present (b) visible nuclei located

close to the cell center and (c) intact cell borders (17). To measure cardiomyocyte cell-cycling, 8 μm sections

from myocardial samples (FFPE) obtained from the infarct and peri-infarct area (n=4-6 slides/heart) underwent

immunostaining for α-sarcomeric actinin and Ki67 (Thermo). To visualize myocyte progenitors, 8 μm sections

from myocardial samples (FFPE) obtained from the infarct & peri-infarct area (n=4-6 slides/heart) underwent

immunostaining for Troponin I (Sigma). Alexa Fluor-conjugated secondary antibodies (Molecular probes) were

used and slides were counterstained for DAPI (Molecular probes). Sections were imaged using a confocal

laser scan microscope (Leica Microsystems) and images were processed by Leica Application Suite software.

Peri-infarct (border zone) area was defined as the region at the edges of the scar (comprising areas of both

viable and scarred myocardium).

Fluorescence In Situ Hybridization

Two to 3 weeks post-MI infarcted female mini-pigs were intracoronarily infused with allogeneic CDCs

derived from a male mini-pig donor to enable cell detection Fluorescence In Situ Hydridization (FISH).

ZytoLight porcine X/Y Dual Color Probe and ZytoLight FISH-Tissue Implementation Kit (ZytoVision GmbH)

were used to prepare slides and test for the porcine Y and X chromosomes. Sections obtained from 3 sites

were used for FISH analysis: central infarct region, infarct border zone and normal myocardium Three to 6

slides (1 or 2 from each of the 3 sites) were selected for each of the animals for analysis. A simple preparation

of male CDCs served as a positive control for the method (Supp Fig 2A).

On day one, slides were incubated for 10 minutes on a hot plate (VWR, Digital Heatblock) at 70°C,

followed by two 10 minute incubations in xylene (Sigma). Next, the slides were incubated for 5 minutes each in

100%, 100%, 90%, and 70% ethanol. In order to keep the Heat Pretreatment Solution Citric Solution at 98°C, a

coplin jar was kept in a hot water bath at boiling temperature. Immediately before placing slides in the jar, the

Heat Pretreatment Solution Citric Solution was brought up to boiling temperatures in a microwave. The solution

was placed inside the warmed up coplin jar along with the slides, slides were incubated at 98°C for 15 minutes.

Slides were then washed twice for 2 minutes in deionized water and then water was blotted off. Pepsin solution

was added drop-wise to slides and incubated for 10 minutes at 37°C in a humidity chamber. Slides were

washed for 5 minutes in Wash Buffer SSC and for 1 minute in deionized water. Slides were dehydrated by

placing in a series of ethanol concentrations, 70%, 90%, and 100% for 1 minute each. Slides were allowed to

air dry. The remaining steps were performed in the dark to protect the probe. ZytoLight FISH probe (10µL) was

pipetted onto the slide, a coverslip was placed over the probe and sealed with rubber cement. The slide was

placed on a heat block at 72°C for 10 minutes. Finally, slides were incubated overnight in a humidity chamber

at 37°C. On day two, slides continued to be protected from light. Rubber cement was carefully removed and

the slides were placed in 1 X Wash Buffer at 37°C for 3 minutes to ensure coverslip removal. Slides were then

washed twice in 1 X Wash Buffer at 37°C for 5 minutes. Slides were incubated in a series of ethanol

concentrations, 70%, 90%, and 100% for 1 minute each. After being allowed to air dry, 1 drop (~30µL) of

VECTASHIELD Mounting Medium with DAPI (Vector Laboratories) was added to the slide. A coverslip was

placed over the mounting medium and sealed with nail polish. Slides were stored in the dark at 4°C until ready

to analyze. Slides were analyzed using a confocal microscope (Leica TCS SP-X, Leica Microsystems CMS

GmbH) with three filters, red, blue, and green. Images were analyzed using LAS AF Lite (Leica Microsystems

CMS GmbH) and Image J.

References

1. Ho CS, Lunney JK, Franzo-Romain MH, Lee JH, Smith DM.. Molecular characterization of swine leucocyte

antigen class I genes in outbred pig populations. Anim Genet. 2009;40:468-78.

2. Ho CS, Lunney JK, Lee JH, Franzo-Romain MH, Martens GW, Rowland RR, Smith DM.. Molecular

characterization of swine leucocyte antigen class II genes in outbred pig populations. Anim Genet.

2010;41:428-32.

3. Terrovitis JV, Smith RR, Marbán E. Assessment and optimization of cell engraftment after transplantation into

the heart. Circ Res. 2010;106:479-494.

4. Johnston PV, Sasano T, Mills K, Evers R, Lee ST, Smith RR, Lardo AC, Lai S, Steenbergen C, Gerstenblith G,

Lange R, Marbán E. Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived

cells in porcine ischemic cardiomyopathy. Circulation. 2009;120:1075-1083.

SUPPLEMENTAL FIGURES AND FIGURE LEGENDS

Supp Fig 1. Twenty-four-hour cardiac retention of IC infused CDCs. A: Schematic protocol for

transduction of CDCs with adenoviral luciferase and ex vivo luciferase assay. B: Twenty-four-hour cardiac

retention of IC infused CDCs

Supp Fig 2. Long-term engraftment of IC infused CDCs. A: Control preparation of male donor CDCs show

both red and green fluorescence signals in each nucleus, corresponding to one X and one Y chromosome,

respectively, in each cell. B: A male CDC (box) in the recipient myocardium at 2 weeks post-infusion.

Supp Fig 3. Safety of CDC IC infusion. A: Serial TnI measurements. B: TIMI flow in the LAD before and after

IC infusion.

Supp Fig 4. Low resolution SLA genotyping. Low resolution SLA genotyping revealed complete Class I

(SLA-1, SLA-2, SLA-3) and Class II (DRB1, DQB1, DQA) mismatches between the donor minipig and CDC

recipients.

Supp Fig 5. Immune reaction to allogeneic CDCs. A: Increased focal lymphoplasmacytic infiltration (Grade

1R), deemed to be unrelated to the natural inflammatory response to the ischemic insult, was detected in the

peri-infarct area of CDC-treated minipigs (16/22 slides) compared to placebo. B: Representative image of

H&E-stained heart section revealing infiltrating cells localized within the interstitial space. C, D: Recipient

minipig serum samples were screened for circulating anti-donor IgG antibodies by flow cytometry. No

alloreactive antibodies were detected in any recipients of allogeneic CDCs at any time point. In contrast, in

minipigs that were allosensitized by intradermal and subcutaneous PBMNC injections, high titers of circulating

alloreactive IgG antibodies were detected at two weeks post-injection.

Supp Fig 6. Bland-Altman analysis. Bland-Altman analyses showing the level of agreement between MRI

and post-mortem histology for measurement of scar size as % of isolated cardiac slices (A [in vivo MRI], B [ex

vivo MRI]), scar size as % of the LV (C), scar mass (D) and viable myocardial mass (E). Dotted lines denote

95% limits of agreement.

Supp Fig 7. Cardiomyocyte cross-sectional area in the remote myocardium.

SUPPLEMENTAL TABLES

ID group

EF base (%)

EDV base (ml)

ESV base (ml)

scar mass base (g)

scar size base

(% LV)

viable mass base (g)

EF 2mos (%)

EDV 2mos (ml)

ESV 2mos (ml)

scar mass 2mos

scar size

2mos (%)

viable mass 2mos (m)

11P183 CDCs 47.76 63.49 33.17 5.94 8.84 61.25 46.56 83.76 44.76 5.72 7.92 66.5211P185 CDCs 49.79 76.44 33.38 8.27 12.88 55.93 44.83 99.24 54.75 7.61 9.79 70.0911P186 CDCs 43.23 70.29 39.90 7.61 11.69 57.48 45.96 93.12 50.32 6.86 9.28 67.0311P188 CDCs 46.08 69.08 37.25 10.39 16.28 53.45 48.40 92.50 47.73 6.75 9.14 67.0811P198 CDCs 49.91 74.21 37.18 7.87 14.36 46.93 48.37 73.27 37.59 6.26 9.74 58.0311P182 controls 49.65 81.05 40.81 6.35 11.50 48.88 37.68 88.89 55.39 7.39 12.73 50.6711P187 controls 46.62 67.16 35.85 8.34 13.69 52.59 38.03 89.85 55.68 8.76 14.35 52.2712P03 controls 42.92 71.54 40.84 13.99 18.93 59.92 32.70 132.97 89.50 15.81 20.10 62.8412P58 controls 49.79 71.26 35.78 7.23 13.16 47.69 40.11 97.61 58.46 7.45 12.30 53.1412P59 controls 48.08 75.19 39.04 8.51 13.40 54.96 39.15 109.15 66.42 9.03 13.38 58.46

Supp Table 1. A complete list of MRI-measured parameters for each experimental animal (EF: ejection fraction; EDV: End-diastolic volume; ESV: End-systolic volume; LV: left ventricle; base: baseline; mos: months).

ID group

scar size TTC

(% LV)

scar mass

TTC (g)

viable mass

TTC (g) 11P183 CDCs 7.52 5.91 72.6411P185 CDCs 10.47 8.44 72.211P186 CDCs 8.04 5.84 66.811P188 CDCs 9.61 7.51 70.611P198 CDCs 9.77 6.64 61.3211P182 controls 9.69 5.85 54.511P187 controls 14.71 9.65 55.9412P03 controls 17.93 13.4 61.3412P58 controls 13.51 7.45 47.6812P59 controls 14.25 9.36 56.33

Supp Table 2. A complete list of histological measurements of scar size, scar mass and viable mass for each experimental animal (TTC: 2,3,5-triphenyltetrazolium chloride).

LEGENDS FOR VIDEO FILES

Supplemental Video 1. Video of short-axis cine acquisition of a CDC-treated pig at baseline (before CDC

infusion).

Supplemental Video 2. Video of short-axis cine acquisition of a CDC-treated pig at 2 months post-CDC

infusion.

Supplemental Video 3. Video of short-axis cine acquisition of a control pig at baseline (before vehicle infusion).

Supplemental Video 4. Video of short-axis cine acquisition of a control pig at 2 months post-vehicle infusion.

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