a combination of allogeneic stem cells promotes cardiac ... · c ell-based therapy reduces scar...

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ª 2 0 1 7 B Y T H E A M E R I C A N CO L L E G E O F C A R D I O L O G Y F O U N DA T I O N

P U B L I S H E D B Y E L S E V I E R

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A Combination of Allogeneic Stem CellsPromotes Cardiac Regeneration
Makoto Natsumeda, MD,a Victoria Florea, MD,a Angela C. Rieger, MD, MSC,a Bryon A. Tompkins, MD,a,b
Monisha N. Banerjee, MD,a,b Samuel Golpanian, MD,a,b Julia Fritsch, BS,a Ana Marie Landin, PHD,a

Nilesh D. Kashikar, MD, PHD,c Vasileios Karantalis, MD, PHD,a Viky Y. Loescher, MD,a Kostas E. Hatzistergos, PHD,a

Luiza Bagno, DVM, PHD,a Cristina Sanina, MD,a Muzammil Mushtaq, MD,a Jose Rodriguez, BS,a Marcos Rosado, BS,a

Ariel Wolf, BS,a Kevin Collon, BS,a Louis Vincent, BS,a Anthony J. Kanelidis, BS,a Ivonne H. Schulman, MD,a,d

Raul Mitrani, MD,d Alan W. Heldman, MD,d Wayne Balkan, PHD,a,d Joshua M. Hare, MDa,d

ABSTRACT

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BACKGROUND The combination of autologous mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs)

synergistically reduces scar size and improves cardiac function in ischemic cardiomyopathy. Whereas allogeneic (allo-)

MSCs are immunoevasive, the capacity of CSCs to similarly elude the immune system remains controversial, potentially

limiting the success of allogeneic cell combination therapy (ACCT).

OBJECTIVES This study sought to test the hypothesis that ACCT synergistically promotes cardiac regeneration without

provoking immunologic reactions.

METHODS Göttingen swine with experimental ischemic cardiomyopathy were randomized to receive transendocardial

injections of allo-MSCs þ allo-CSCs (ACCT: 200 million MSCs/1 million CSCs, n ¼ 7), 200 million allo-MSCs (n ¼ 8),

1 million allo-CSCs (n ¼ 4), or placebo (Plasma-Lyte A, n ¼ 6). Swine were assessed by cardiac magnetic resonance

imaging and pressure volume catheterization. Immune response was tested by histologic analyses.

RESULTS Both ACCT and allo-MSCs reduced scar size by �11.1 � 4.8% (p ¼ 0.012) and �9.5 � 4.8% (p ¼ 0.047),

respectively. Only ACCT, but not MSCs or CSCs, prevented ongoing negative remodeling by offsetting increases in chamber

volumes. Importantly, ACCT exerted the greatest effect on systolic function, improving the end-systolic pressure-volume

relation (þ0.98 � 0.41 mm Hg/ml; p ¼ 0.016). The ACCT group had more phospho-histone H3þ (a marker of mitosis)

cardiomyocytes (p¼0.04), and noncardiomyocytes (p¼0.0002) than did the placebo group in some regions of the heart.

Inflammatory sites in ACCT and MSC-treated swine contained immunotolerant CD3þ/CD25þ/FoxP3þ regulatory T cells

(p < 0.0001). Histologic analysis showed absent to low-grade inflammatory infiltrates without cardiomyocyte necrosis.

CONCLUSIONS ACCT demonstrates synergistic effects to enhance cardiac regeneration and left ventricular

functional recovery in a swine model of chronic ischemic cardiomyopathy without adverse immunologic

reaction. Clinical translation to humans is warranted. (J Am Coll Cardiol 2017;70:2504–15)

© 2017 by the American College of Cardiology Foundation.

m the aInterdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida; bDepartment of

rgery, University of Miami Miller School of Medicine, Miami, Florida; cDepartment of Pathology, University of Miami Miller

ool of Medicine, Miami, Florida; and the dDepartment of Medicine, University of Miami Miller School of Medicine, Miami,

rida. This study was funded by National Institutes of Health/National Heart, Lung, and Blood Institute grant R01 HL084275 to

. Hare and an award from Vestion Inc. to Dr. Balkan. Dr. Landin has served as a consultant to Longeveron. Dr. Hatzistergos has

closed a relationship with Vestion Inc. that includes equity. Dr. Heldman has a patent for cardiac cell-based therapy; has

eived research support from Biocardia; reports being a board member and consultant of Vestion Inc.; and has equity interest in

stion Inc. Dr. Hare has a patent for cardiac cell-based therapy; holds equity in Vestion Inc. and Longeveron; serves as a

nsultant andmember of the Board of Directors and Scientific Advisory Board of Vestion Inc.; serves as the chief scientific officer,

ompensated consultant, and advisory board member for Longeveron; is the co-inventor of intellectual property licensed to

ngeveron; and owns equity in Heart Genomics and Biscayne Pharmaceuticals. Vestion Inc. did not play a role in the design and

nduct of the study. All other authors have reported that they have no relationships relevant to the contents of this paper to

close.

nuscript received March 15, 2017; revised manuscript received September 15, 2017, accepted September 15, 2017.

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ABB R E V I A T I O N S

AND ACRONYMS

ACCT = allogeneic cell

combination therapy

BZ = border zone

CMR = cardiac magnetic

resonance

CSCs = cardiac stem cells

EDV = end-diastolic volume

EF = ejection fraction

ESPVR = end-systolic pressure-

volume relationship

ESV = end-systolic volume

ICM = ischemic cardiomyopathy

IZ = ischemic zone

J A C C V O L . 7 0 , N O . 2 0 , 2 0 1 7 Natsumeda et al.N O V E M B E R 1 4 / 2 1 , 2 0 1 7 : 2 5 0 4 – 1 5 Combination of Allogeneic Stem Cells Promotes Cardiac Regeneration

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C ell-based therapy reduces scar size and re-presses adverse remodeling secondary tomyocardial infarction (MI) in preclinical

models (1–4) and in clinical trials (5,6) of patientswith ischemic cardiomyopathy (ICM). We previouslyshowed that in a swine model of chronic MI (4),both autologous (7) and allogeneic (1,2) mesenchymalstem cells (MSCs) improved cardiac function byreducing infarct scar size, reversing negative remod-eling, and enhancing endogenous cardiomyocyte pro-liferation (3). Administration of xenogeneic human c-Kitþ cardiac stem cells (CSCs) to immunosuppressedpigs (8), and autologous CSCs to pigs (9) and humans(10) produces significant improvements in cardiacfunction. Furthermore, when CSCs are combinedwith MSCs, regeneration is enhanced (8,11).

SEE PAGE 2516

LV = left ventricular

MI = myocardial infarction

MSCs = mesenchymal stem cells

pHH3 = phospho-histone H3

PV = pressure-volume

RZ = remote zone

Treg = regulatory T cell

TESI = transendocardial stem

cell injection

Allogeneic MSCs (allo-MSCs) have distinct advan-tages over autologous cells in terms of potency andavailability (12). MSCs evade and suppress immuno-logic responses secondary to their lack of class IImajor histocompatibility complex (13), and theirrelease of immunomodulatory soluble factors (14–18).However, the immunoevasive abilities of other allo-geneic cells, in particular CSCs, is unclear and may bedependent on the proportion of allogeneic cellsexpressing major histocompatibility complex class Iand II (12,19). The POSEIDON (Percutaneous Stem CellInjection Delivery Effects on Neomyogenesis) (6,20)and LVAD (Effect of Intramyocardial Injection ofMesenchymal Precursor Cells on Myocardial Functionin Patients Undergoing LVAD Implantation) clinicaltrials (21) demonstrated that allo-MSCs did not inducesignificant immunologic responses in patients withICM up to 12 months post–cell treatment, supportingtheir safety. Furthermore, there is also evidence fromclinical trials that allogeneic cell-based therapy maybe superior to autologous cell therapy (6,20,22). Theimmunomodulatory effects of MSCs have driven theirclinical use as a primary therapeutic and even as anadjunct to allograft transplant (23). However, whetherallogeneic CSCs (allo-CSCs) can be used successfullyas an allograft, alone, or in combination with MSCshas never been tested. The objective of this study wasto examine the safety and regenerative efficacy ofallogeneic cell combination therapy (ACCT), a com-bination of allo-MSCs and allo-CSCs in a 200:1 ratio.

In this study, Göttingen swine were subjected toMI via ischemia/reperfusion injury, whichwas allowedto develop into chronic ICM (4). Threemonths post-MI,swine were administered placebo, allo-MSCs, allo-CSCs, or ACCT to test the hypotheses that: 1) ACCT is

safe and does not elicit an immunologicresponse; and 2) ACCT has greater thera-peutic efficacy than either cell type alone.

METHODS

STUDY DESIGN. All animal protocols werereviewed and approved by the University ofMiami Institutional Animal Care and UseCommittee. Female Göttingen swine weresubjected to catheter-induced ischemicreperfusion MI as previously described (4).Swine were randomized for transendocardialstem cell injections (TESI) of 1 � 106 allo-CSCsand 2 � 108 allo-MSCs (ACCT, n ¼ 7), 2 � 108

MSCs (n ¼ 8), 1 � 106 CSCs (n ¼ 4), or placebo(Plasma-Lyte A, Baxter, Illinois) (n ¼ 6).Continuous cardiac monitoring devices wereimplanted subsequent to stem cell therapy toassess arrhythmogenic events. Measure-ments of cardiac structure and function wereobtained using cardiac magnetic resonance(CMR) imaging, pressure-volume (PV) loops,and histologic analyses of phospho-histoneH3 (pHH3)þ cells and CD3þ/CD25þ/FoxP3þ

regulatory T cells (Tregs) in the extracted
hearts (Online Appendix, Online Figure 1).
CELL MANUFACTURING PROCESS AND TESI. Heart(right atrial appendage) and iliac crest bone marrowbiopsies were attained from male Yorkshire swine.Cells were cultured, amplified, characterized (24–26),and cryopreserved at passage 3. On the day of injec-tion, cells were thawed and aliquoted (total 5.1 ml)(Online Appendix).

TESI were performed at 3 months post-MI using theNOGA injection-catheter system (Johnson & Johnson,New Brunswick, New Jersey). An electromechanicalmap of the left ventricle (LV) endocardium wasgenerated. The viable border zone of dense scar wasdetermined as a unipolar voltage range of 6 to 12 mV.Cells were injected into 10 sites within the borderzone using the Myostar injection catheter (B type,Johnson & Johnson) (Online Appendix).

STATISTICAL ANALYSIS. Parametric values andstatistics are presented as mean � SEM. Normallydistributed parameterswere evaluatedwith a repeatedmeasures analysis of variance model includingbetween-group comparisons as well as time andgroup � time interaction terms. Bonferroni correctionwas applied for post hoc tests. One pig from the MSCgroup was excluded from analysis of MRI-derived datadue to multiple missing time points. The 1-monthpost-MI time point was excluded from analysis of




CENTRAL ILLUSTRATION Scar Mass and Scar Mass as Percentage of LV Mass Post-Treatment

Scar

/ LV

Mas

s %ΔΔ

(%)

10

0

******

–10

TESI

–20

Scar/LV Mass from 3m Post-MI to 3m Post-TESIM

3m Post MI

1m Post

Inj

2m Post Inj

3m Post Inj

Placebo MSC CSC ACCT

Scar

Mas

s %Δ

(%)

30

10

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* *0

TESI

–10

–20

Scar Mass from 3m Post-MI to 3m Post-TESIN

3m Post MI

1m Post

Inj

2m Post Inj

3m Post Inj

A B C G H I

D E F J K

Natsumeda, M. et al. J Am Coll Cardiol. 2017;70(20):2504–15.

Delayed enhancement cardiac magnetic resonance short-axis images show the chronologic change of scar size (scar ¼ red) before (A, D, G, J) and 3 months after

(B, E, H, K) treatment and, as seen in comparable gross histologic sections (C, F, I, L). (M) Scar mass as a percentage of left ventricular (LV) mass decreased significantly

after transendocardial stem cell injection (TESI) in allogenic cell combination therapy (ACCT) andmesenchymal stem cells (MSC). (N) Percentage decrease in scarmasswas

greater in ACCT and MSC groups than in the placebo group. *p < 0.05, ***p < 0.0001. CSC ¼ cardiac stem cell(s); Inj ¼ injection; MI ¼ myocardial infarction.

Natsumeda et al. J A C C V O L . 7 0 , N O . 2 0 , 2 0 1 7

Combination of Allogeneic Stem Cells Promotes Cardiac Regeneration N O V E M B E R 1 4 / 2 1 , 2 0 1 7 : 2 5 0 4 – 1 5

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end-diastolic volume (EDV) and end-systolic volume(ESV) for multiple missing values. Nonparametric data(pHH3 staining only) were assessed for between-groupdifferences using the Kruskall-Wallis test, and theDunn post hoc test was conducted to evaluate pairwisedifferences among groups. For categorical variables,the Pearson chi-squared test was used. We consideredp < 0.05, 2-sided, statistically significant. All analyseswere done using GraphPad Prism 6 (GraphPad Soft-ware, Inc., La Jolla, California).

RESULTS

Baseline characteristics (age, weight) (Online Table 1)and severity of induced MI as measured by scar size(p ¼ 0.83) were evenly distributed between treatmentgroups. Myocardial damage evaluated by cardiacenzyme deviation (troponin I [p ¼ 0.45], creatinephosphokinase [p ¼ 0.89], creatine kinase-myocardialband [p ¼ 0.45], creatine kinase-MM [p ¼ 0.83], andcreatine kinase-BB [p ¼ 0.67]) due to MI and injection


TABLE 1 LV Anatomy and Global Function Derived From CMR

Baseline 3 Months Post-MI 3 Months Post-TESI

End-diastolic volume, ml Placebo 42.5 � 3.3 55.1 � 6.2* 71.5 � 4.9†

MSC 37.6 � 3.1 55.3 � 5.1* 65.0 � 2.8†

CSC 45.5 � 5.5 69.1 � 5.0* 85.6 � 1.8†

ACCT 39.9 � 2.1 61.8 � 5.6* 70.4 � 4.4

End-systolic volume, ml Placebo 18.1 � 1.8 32.1 � 3.8* 42.8 � 4.1†

MSC 16.6 � 1.4 34.8 � 3.3* 41.4 � 1.8†

CSC 20.5 � 2.7 39.5 � 2.8* 48.2 � 2.7†

ACCT 18.3 � 0.8 37.6 � 3.9* 42.7 � 3.7

Ejection fraction, % Placebo 57.5 � 2.1 41.7 � 2.2* 40.7 � 2.5

MSC 55.3 � 2.5 37.1 � 2.0* 36.5 � 2.7

CSC 55.2 � 0.8 43.3 � 0.4* 43.1 � 2.6

ACCT 53.8 � 1.3 39.7 � 1.5* 41.6 � 2.8

Scar/LV mass, % Placebo 0 17.5 � 2.1 19.9 � 3.1

MSC 0 16.9 � 1.2 13.7 � 1.5†

CSC 0 12.8 � 0.2 12.4 � 0.9

ACCT 0 15.5 � 1.4 12.5 � 1.6†

Scar mass, g Placebo 0 6.2 � 0.6 7.5 � 1.4

MSC 0 7.2 � 0.6 6.9 � 0.9†

CSC 0 5.9 � 0.5 6.4 � 0.3

ACCT 0 7.1 � 0.9 6.4 � 0.9†

Values are mean � SD. Placebo, n ¼ 6; MSC, n ¼ 8; CSC, n ¼ 4; ACCT, n ¼ 7. *p < 0.05 compared with baseline.†p < 0.05 compared with 3 months post-MI.

ACCT¼ allogeneic combination cell therapy; CMR¼ cardiac magnetic resonance; CSC¼ cardiac stem cell; LV¼ leftventricular; MI¼ myocardial infarction; MSC¼ mesenchymal stem cell; TESI¼ transendocardial stem cell injection.


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insult was similar (Online Figures 2A to 2E). MSCs werecharacterized as CD105þ/CD90þ/CD44þ/CD45�; CSCswere characterized as CD117þ/CD45� (Online Figure 3).

ACCT AND MSC TREATMENT PRODUCES SIMILAR

REDUCTIONS IN SCAR SIZE. Scar mass and scar massas a percentage of LV mass were determined bydelayed enhancement CMR (Online Figure 4). High-intensity signals of the myocardium depicting scarobtained pre-treatment and at 3 months post-treatment were compared and are shown with corre-sponding histologic sections (Central Illustration,A to L). The scar mass as a percentage of LV massfor the entire study was þ15.44 � 0.72%. There was nodifference in scar size between the groups prior toTESI (p ¼ 0.45) (Table 1). In response to treatment, theACCT and MSC groups exhibited significant decreasesin scar/LV mass from 3 months post-MI to 3 monthspost-TESI by �20.7 � 7.1% (p ¼ 0.0009) and �22.3 �7.1% (p ¼ 0.0003), respectively, whereas the CSC andplacebo groups remained unchanged: �3.23 � 9.2%(p > 0.99) and �2.7 � 7.7% (p > 0.99), respectively(Central Illustration, M). The percentage reduction ofscar mass was significantly greater in the ACCT(�11.1 � 4.8%; p ¼ 0.012) and MSC (�9.5 � 4.8%,p ¼ 0.047) groups compared to the placebo group,whereas the CSC group was not different from theplacebo group (�1.5 � 5.6%, p > 0.99) (CentralIllustration, N).

ACCT PREVENTS PROGRESSIVE NEGATIVE

REMODELING. ACCT therapy suppressed the devel-opment of negative remodeling as indicated by LVvolumes measured by CMR. EDV and ESV increased inall of the treatment groups from baseline to 3 monthspost-treatment, illustrating progressive negativeremodeling (Table 1). Between 3 months post-MI and3 months post-TESI, both EDV (Figure 1A) and ESV(Figure 1B) continued to increase in the placebo(EDV: þ16.4 � 5.9 ml, p ¼ 0.001; ESV: þ10.6 � 3.5 ml,p ¼ 0.0002), the CSC (EDV: þ15.9 � 7.2 ml,p ¼ 0.018; ESV: þ8.7 � 4.2 ml, p ¼ 0.035), and MSC(EDV: þ11.5 � 5.5 ml, p ¼ 0.027; ESV: þ7.3 � 3.2 ml,p ¼ 0.013) groups. Importantly, increases in ven-tricular volumes were prevented by ACCT (EDV: þ8.7� 5.5 ml, p ¼ 0.22; ESV: þ5.2 � 3.2 ml, p ¼ 0.21),indicating that treatment with ACCT blocked theongoing process of negative chamber remodeling.

SIGNIFICANT RECOVERY OF CARDIAC FUNCTION IS

ASSOCIATED WITH ACCT. Stem cell effects on car-diac function were analyzed using CMR to assessejection fraction (EF) and by PV loops. PV loopsdisplayed a decreased end-systolic pressure-volumerelationship (ESPVR), and increased end-diastolicPV relationship (EDPVR) in placebo-, MSC-, and CSC-

treated swine (Figure 2A). In contrast, ACCT treat-ment produced the opposite effect, improving systolicfunction (Figure 2B). Hemodynamic measurementsderived from PV loops are summarized in OnlineTable 2. ESPVR decreased significantly over the studyperiod in placebo-treated swine (�1.3� 0.5 mmHg/ml,p ¼ 0.03) and improved in response to TESI only inACCT-treated swine (þ1.0 � 0.4 mm Hg/ml, p ¼ 0.016)whereas it remained unchanged in MSC- and CSC-treated swine (p ¼ 0.52 and p > 0.99, respectively).Furthermore, only ACCT produced a significantimprovement in percentage change of ESPVR inresponse to TESI compared with placebo (between-group difference ¼ þ52.8 � 26.0%, p ¼ 0.04), whereasthe other groups were not significantly different fromplacebo, demonstrating the recovery of contractility inresponse to ACCT treatment (Figure 2C). There was atrend to improved diastolic function evident in theACCT that did not reach statistical significance(Figure 2D).

All animals experienced a similar, significantreduction in EF as measured by CMR following MI(p < 0.0001). However, cell treatment did not signif-icantly improve EF in any group (Figure 2E).

MYOCARDIAL PERFUSION IS ENHANCED BY ACCT

AND MSC THERAPY. Measurement of myocardialperfusion in all 3 cardiac zones—ischemic zone (IZ),border zone (BZ), remote zone (RZ)—was derived from






FIGURE 1 Suppressed Negative Remodeling of LV Volumes in Response to ACCT Treatment

EDV

(mL)

0

25

20

15

10

5

End-Diastolic Volume Change Post-TESI

Placebo

*

*

*

MSC CSC ACCT

A

ESV

(mL)

0

25

20

15

10

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End-Systolic Volume Change Post-TESI

Placebo

*

*

*

MSC CSC ACCT

B

From transendocardial stem cell injection (TESI) at 3 months post-myocardial infarction to the end of the study, (A) end-diastolic volume

(EDV) increased in the placebo, cardiac stem cell (CSC), and mesenchymal stem cell (MSC) groups but remained unchanged in the allogenic

cell combination therapy (ACCT) group. (B) End-systolic volume (ESV) increased in the placebo, CSC, and MSC groups but remained

unchanged in ACCT, indicating that ACCT halts the progression of negative remodeling. *p < 0.05.



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CMR using the regional signal upslope method. From3 months post-MI onward, myocardial perfusionincreased in the IZ with ACCT by þ41.8 � 12.6%(p ¼ 0.0001). The percentage change in IZ perfusionfrom 3 months post-MI to 3 months post-TESIimproved in ACCT compared with placebo byþ53.8 � 30.7% (p ¼ 0.0005) and was not significantlydifferent from placebo in the other groups (Figure 3).There was no within or between treatment differencein myocardial perfusion following TESI in the BZ andRZ (data not shown). These results indirectly portraythe vasculogenic characteristics of ACCT in the scar-red areas of the heart that are undergoingregeneration.

THE EVALUATION OF MITOSIS IN RESPONSE TO

CELL THERAPY. To understand the mechanism un-derlying cell therapy-mediated scar reduction andreduced remodeling, tissue sections were evaluatedby staining for pHH3, indicative of mitosis, andmyosin light chain 2, a marker of cardiomyocytes.Figures 4A and 4B show confocal microscopy imagesof pHH3þ cardiomyocytes and pHH3þ non-cardiomyocytes. Within the IZ, scattered pHH3þ car-diomyocytes were observed in the cell-treatedgroups, whereas none were seen in the placebo group(p ¼ 0.34) (Figure 4C). Compared with placebo, theACCT and MSC hearts contained 2-fold more pHH3þ

cardiomyocytes; in the BZ (p ¼ 0.31) (Figure 4D)and in the RZ, there was a significant differencebetween ACCT and placebo (p ¼ 0.025) (Figure 4E).

There was w6-fold increase of pHH3þ non-cardiomyocytes in the BZ of ACCT compared withplacebo (p ¼ 0.0002) (Figures 4F to 4H).

ARRHYTHMIC EVENTS OBSERVED AFTER STEM CELL

INJECTION. Remarkable arrhythmic events aresummarized in Online Table 3. None of the cell-treated swine showed evidence of either sustainedor nonsustained ventricular tachycardia or ventric-ular fibrillation, demonstrating the low proar-rhythmic profile of the injected cells (OnlineAppendix).

IMMUNE RESPONSE TO ALLOGENEIC STEM CELLS.

There was no clinical indication of hyperacute oracute immune rejection in any of the swine throughthe course of the study. Ten slides from each zone (IZ,BZ, RZ) of the heart were stained with hematoxylinand eosin or Masson trichrome and analyzed by apathologist blinded to treatment. Calcification, fatinfiltration, fibrosis, collagen, inflammatory cells,granulomas, and/or foreign bodies constitute themajor histologic observations of this study. Calcifi-cation, fat infiltration, fibrosis, and collagen arerepresentative findings compatible with the chronicphase of post-infarct myocardial necrosis (27) andwere evenly presented in all groups (data not shown).

Although inflammatory cells were detected occa-sionally in all treatment groups, this observation isconsistent with findings of chronic phase MI (28).Occasional focal lymphocyte aggregates in the peri-vascular areas (Figures 5A and 5B) were seen in a small




FIGURE 2 Cardiac Function Improved by Combination Stem Cell Therapy

LV P

ress

ure

(mm

Hg)

3 Months Post TESI

LV Volume (mL)

LV P

ress

ure

(mm

Hg)

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ress

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(mm

Hg)

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ress

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LV Volume (mL)20 40 60 80 100 120

LV Volume (mL)20 40 60 80 100 120

LV Volume (mL)20 40 60 80 100 120

3 Months Post MI

Pes = 1.7xVes–26.7,r2 = 0.98Ped = 0.2xVed–9.0,r2 = 0.97

Pes = 1.4xVes–42.8,r2 = 0.93Ped = 0.4xVed29.1,r2 = 0.97

Pes = 3.4xVes–106.4,r2 = 0.99Ped = 0.3xVed–9.0,r2 = 0.93

Pes = 1.7xVes+11.8,r2 = 0.99Ped = 0.3xVed–9.7,r2 = 0.97

A

3 Months Post MI 3 Months Post TESIB

EC D

–30

0

30

60

90

ESPV

R %

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nge

ESPVR % Change Post TESI *

Placebo MSC CSCs ACCTPlacebo MSC CSC ACCTPlacebo MSC CSC ACCT–10

–5

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Δ (%

)

Ejection Fraction Change Post TESI

–40

–30

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R %

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Representative pressure-volume loops display the (A) decrease in end-systolic pressure-volume relationship (ESPVR) (in blue) and increase in end-diastolic pressure-

volume relationship (EDPVR) (in red) in placebo. In contrast, (B) ACCT produced the opposite effect. In response to TESI, (C) the percentage increase in ESPVR was

significant only in ACCT, both within group and compared with placebo. (D) EDPVR and (E) ejection fraction (EF) did not improve significantly in any group. *p < 0.05.

LV ¼ left ventricular; MI ¼ myocardial infarction; other abbreviations as in Figure 1.


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fraction of IZs (between-groups, p ¼ 0.98) and BZs(between groups, p ¼ 0.99) of the cell-treated groups(ACCT: 20.0% of IZ, 21.4% of BZ; MSC: 25.0% of IZ,12.5% of BZ; CSC: 0% of IZ, 2.5% of BZ; placebo, 0%)(Figures 5C to 5E). Inflammatory cells were dispersedwithin the interstitium without evidence of myocar-dial necrosis or disruption of normal architecture.This lack of obvious immunoreaction to the

allogeneic cells is consistent with absent to low-gradeimmune rejection as defined by the grading systemestablished by the International Society for Heart andLung Transplantation (29). Three swine receivingACCT exhibited foreign bodies in the IZ (between-group, p ¼ 0.95) and BZ (between-group, p ¼ 0.93).

Considering that the acute phase of allotransplantimmune rejection is predominantly mediated by T

FIGURE 3 Myocardial Perfusion of Ischemic Zone Improved With Cell Treatment

Rela

tive

Upsl

ope

%Δ

(%)

–20

60

40

20

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Perfusion IZ Change Post-TESI


***

***

Perfusion cardiac magnetic resonance imaging according to upslope analyses showed

improvement of myocardial tissue perfusion in the ischemic zone of ACCT compared with

placebo-treated animals. ***p < 0.001. IZ ¼ ischemic zone; other abbreviations as in

Figure 1.



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lymphocytes, lymphoid aggregates were stained forTregs, which act to promote immunotolerance (30) inorder to elucidate the significance of the observedfocal inflammation. All 46 of the lymphoid aggregatesand granulomas contained CD3þ/CD25þ/FoxP3þ cellsindicative of Tregs (p < 0.0001) (Figures 6A to 6D).Finally, fluorescent in situ hybridization for theY chromosome, although not quantitative, revealed alow number of engrafted cells in the ACCT and MSCgroups at 3 months post-TESI (Online Figure 5).

NECROPSIES SHOW NO EVIDENCE OF REMOTE

INFLAMMATION. No remote inflammation, tumori-genicity, or ectopic tissue formation were observedduring whole body necropsies (Online Appendix).

DISCUSSION

This blinded, placebo-controlled preclinical studyinvestigated the safety and efficacy of the combina-tion allo-MSCs and allo-CSCs for the treatment ofchronic ICM and is the first study directly comparingallo-MSCs, allo-CSCs, and the combination of these 2cell types (ACCT). We demonstrated 2 important andnovel outcomes of allogeneic stem cell therapy forchronic ICM. First, TESI of allo-MSCs and allo-CSCs inswine is feasible and safe with regard to proceduraltechnique, arrhythmogenicity, and immune responseup to 3 months post-TESI. Second, ACCT producessynergistic effects in improving perfusion in theinfarcted area, halting the progression of negativeremodeling, and augmenting cardiac contractility,when compared with either allogeneic cell type alone.

EFFECTS OF ACCT. Clinical trials demonstrate thatautologous MSCs and CSCs reduce scar size andregenerate lost myocardium in acute and chronic MI(6,9,31). MSCs promote myocardial regeneration viaseveral mechanisms, including direct differentiationand stimulation of endogenous cardiomyocyte cellcycling (3,32). Additionally, MSCs secrete an array ofgrowth factors, cytokines, metalloproteinases, andexosomes that trigger endogenous repair mechanismsthrough paracrine signaling (33–35). CSCs promoteproliferation and differentiation of cardiac lineagesand induce angiogenesis (9). However, the efficaciesof these adult stem cells have been questioned due totheir uncertain cardiomyogenic potency, engraft-ment, and short-term availability in situ (36). Wepreviously showed that autologous (11) and xenoge-neic (8) transplantation (in immunocompromisedswine) of a combination of MSCs and CSCs produceimproved outcomes over MSCs alone, supportingthat these 2 cell types work synergistically toboost regenerative capacity and reduce fibrosis.The mechanism underlying this interplay is regulatedby the increased proliferation, differentiation,and migration of CSCs via the stromal cell-derivedfactor-1/C-X-C motif chemokine receptor-4 andstem cell factor/c-Kit signaling pathways of MSCs(37). The increased regenerative capacity of combi-nation therapy secondary to direct cell-cellsignaling in the stem cell niche was reinforced byQuijada et al. (38) who demonstrated that chimeras ofcardiac progenitor cells and MSCs produce superiortherapeutic efficacy in a mouse model of MI. Finally,a recent rat study showed that the combination ofallo-CSCs and allo-MSCs improves angiogenesisvia paracrine signaling better than either cell typealone (39).

A major limitation of the translation of cell com-bination therapy is the increased risk of rejection ofCSCs. Here we present several findings that offset thisconcern and demonstrate the superiority of ACCTover allo-MSC and allo-CSC therapy alone, paving theway for testing ACCT in humans. In the present study,ACCT retained the ability to reduce scar mass and scarmass as a percentage of LV mass, similar to allo-MSCs.In ACCT only, this reduction was accompanied byimprovements in perfusion in the IZ as compared toplacebo. This result suggests that as scar size isdiminished, perfusion increases to areas that werepreviously infarcted, possibly through paracrinestimulation of angiogenesis (9,22,39,40). Further-more, only the ACCT treatment halted the progres-sion of negative remodeling, whereas it continued toworsen significantly in the placebo, CSC, and MSCgroups. ESPVR, a measure of contractility, was only



FIGURE 4 Increased Mitosis of Cardiomyocytes and Noncardiomyocytes in Response to ACCT

pHH3

+ Non

-Car

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FpHH3+ Non-Cardiomyocyte Staining

pHH3

+ Non

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Placebo MSC

****

CSC ACCT

G

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H

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+ Car

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pHH3+ Cardiomyocyte Staining

pHH3+ cardiomyocyte pHH3+ non-cardiomyocyte


C

pHH3

+ Car

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D

*pH

H3+ C

ardi

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E

BA

Confocal microscopy representative images of the mitosis-specific marker, phospho-histone H3 (pHH3), in (A) cardiomyocytes and (B) noncardiomyocytes. Based on

the average number of pHH3þ cardiomyocytes in the (C) ischemic zone (IZ), (D) border zone (BZ), and (E) remote zone (RZ), there was a 2-fold increase in the RZ

of ACCT, compared with placebo. pHH3þ noncardiomyocytes in the (F) IZ, (G) BZ, and (H) RZ; there was a 6-fold increase in the BZ of ACCT compared with placebo.

*p < 0.05, ****p < 0.0001. DAPI ¼ 40, 6-diamidino-2-phenylindole; other abbreviations as in Figure 1.


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FIGURE 5 Lymphocyte Aggregates Predominantly Seen in Injection Sites of Cell-Treated Animals

Inflammatory Cells -IZ

0 5 10

C

Placebo

MSC

CSC

ACCT

NoneSparseLymphocyte Aggregates

Inflammatory Cells -BZ

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Number of Slides Number of Slides Number of Slides

Inflammatory Cells -RZ

0 5 10

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A B

Hematoxylin and eosin staining of extracted heart specimens demonstrate (A) lymphocyte aggregates in the BZ of an ACCT swine. Bar ¼ 200 mm.

(B) High magnification of delineated area shows lymphocytes confined to the interstitium without cardiomyocyte damage. Bar ¼ 50 mm. Semi-

quantitative analysis of 10 slides from each swine demonstrate lymphocyte aggregates were present in 20% to 25% of ACCT and MSC groups of

(C) IZ (between groups, p ¼ 0.98) and (D) BZ (between groups, p ¼ 0.93). Lymphocyte aggregates were not seen in the (E) RZ. Abbreviations as

in Figures 1, 3, and 4.



2512

improved in ACCT, indicating that the addition ofCSCs to allo-MSCs improves cardiac function.Enhancement of cardiomyocyte and non-cardiomyocyte mitoses in ACCT, as evaluated bypHH3 staining, may contribute to the improvedcontractility. Cardiomyocyte proliferation signifi-cantly increased in the ACCT group only in the RZ,which may be secondary to the paracrine effects ofboth MSCs and CSCs (39). Furthermore, the inhibitionof progressive negative remodeling, which is global,may involve cardiomyocyte mitosis in areas distantfrom the scar and injection sites.

There are important similarities and differences toour previous study of autologous cell combinationtherapy. Similar to autologous therapy, both MSC andcombination therapy reduced infarct scar size, and thecombination group significantly improved functionalparameters as compared to MSCs alone (8,11). Incontrast to the autologous study, here we demon-strate that the functional parameter improved isESPVR, whereas the autologous combinationimproved EF and diastolic strain. Finally, ACCT

significantly improved perfusion in this study,whereas there was only a trend toward improvementby the autologous combination. The similarities be-tween autologous and allogeneic therapy in swinemay be because autologous cells are taken fromyoung, healthy animals. In humans, autologous ther-apy may not be as effective as allogeneic cells becauseautologous cells are generally obtained from patientswith advanced age and multiple comorbidities, whichlikely diminish the efficacy of the cells (12).

Finally, allo-CSCs on their own failed to reduce scarsize or improve anatomic or functional parameters.Although not definitively tested in this study, it islikely that allo-CSC retention in the myocardium ispoor, especially without the protective, immuno-modulatory effects of MSCs.

IMMUNE RESPONSE TO ALLOGENEIC STEM CELL

TRANSPLANTATION. Allogeneic stem cells harvestedfrom young healthy donors possess superior thera-peutic potency and can overcome the disadvantagesrelated to the disease and morbidity associated with

FIGURE 6 Treg Are Expressed in All Lymphocyte Aggregates and Granulomas

(A) Low magnification immunofluorescence confocal image demonstrates CD3þ/CD25þ/

FoxP3þ regulatory T cells (Treg) in a lymphocyte aggregate from an ACCT-injected

animal. (B to D) High magnification of the delineated area in A. Treg (arrows) were

confirmed in all lymphocyte aggregates and granulomas. Bars ¼ 20 mm. Abbreviations

as in Figures 1 and 4.


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autologous stem cells (12). For example, increased ageand heart failure decrease the number of functionallycompetent stem cells (41), whereas diabetes leads totelomere shortening in stem cells and expression ofsenescent and apoptotic proteins (42,43). The centralconcern with allogeneic stem cells is the potential ofimmune rejection. MSCs evade rejection due to theirlack of major histocompatibility complex class II (44),inhibit T-cell proliferation, and trigger anti-inflammatory subsets (17). Calderon et al. (45)demonstrated the immunomodulatory effects ofMSCs by combining adipose-derived stromal cellswith embryonic stem cell–derived cardiac pro-genitors. In their study, the stromal cells inhibited theproliferation of lymphocytes induced by allogeneiccardiac precursors. In preclinical studies, allo-CSC aretolerated immunologically (46,47), and it is note-worthy that the cardiospheres used in these studiesare CD105þ cardiac stromal cells. The safety of allo-geneic c-Kitþ CSCs, is yet to be demonstrated in theclinical setting.

In this study, there were no cases of hyperacuteor acute immune rejection, and no evidence ofremote tissue inflammation as assessed by wholebody necropsy. Serial blood draws showed similarfluctuations of cardiac enzymes (creatine phospho-kinase, creatine kinase-myocardial band, troponin I)regardless of treatment, supporting the absence ofsystemic reaction. Histologic analysis revealedthat <25% of cell-treated animals exhibited aggre-gates of inflammatory cells localized to the trans-endocardial injection sites. Similar examples oflocalized inflammation were seen when autologousMSCs were administered to swine via anteriorthoracotomy 12 weeks post-MI (7), suggesting thatlow levels of inflammation are a characteristic of cellinjection into the myocardium. Histology alsorevealed only absent to low-grade immune rejectionand inflammatory cells were dispersed within theinterstitium without evidence of cardiomyocyteinfiltration or damage. All inflammatory lymphoidaggregates stained positive for Tregs. Since thedepletion of Tregs in target tissues is related toallograft rejection (30), these cells are indicators oftolerance in transplant immunity. Taken together,these data support that ACCT does not produceimmune rejection.

MSCs and CSCs work in concert to create a milieufor regeneration with superior efficacy compared witha single cell therapy in animal models (8,11). TheCONCERT-HF (Combination of Mesenchymal andc-Kitþ Cardiac Stem Cells as Regenerative Therapy forHeart Failure) clinical trial a double-blind, placebo-controlled study comparing the therapeutic efficacy

of the combination of autologous MSCs and CSCscompared to each cell type alone for ICM, is currentlyrecruiting patients.

STUDY LIMITATIONS. This study is limited by arelatively small sample size and a lack of directcomparison to autologous cells. Although ACCT hadwithin-group benefits not seen in the other groupsand was the only group superior to placebo for severalparameters, between-group analysis did not show asignificant difference from MSC. Therefore, while ourdata suggest that ACCT outperforms MSCs alone,larger studies are needed to substantiate this finding.Future studies will address these issues and will alsotest for immune rejection using other methodologiessuch as formation of donor-specific antibodies andmixed lymphocyte reactions.

CONCLUSIONS

Here we demonstrate that ACCT for the treatment ofchronic ICM effectively reduces scar size, improvesmyocardial perfusion, and augments cardiac functionin a manner superior than either cell type alone.

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE:

Combining allogeneic MSCs and CSCs has additive

effects in correcting anatomic and functional deficits

following ischemic injury without inducing an adverse

immunogenic reaction.

TRANSLATIONAL OUTLOOK: Large-scale clinical

trials are needed to confirm the benefit of combined

allogeneic MSC and CSC therapy.



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Furthermore, allogeneic stem cell injections appearsafe and do not produce immune rejection up to3 months post-administration. This study providesimportant insights into the therapeutic effects ofallogeneic stem cell therapy as a favorable option forfurther applications in clinical trials.

ADDRESS FOR CORRESPONDENCE: Dr. Joshua M.Hare, Interdisciplinary Stem Cell Institute, Universityof Miami Miller School of Medicine, BiomedicalResearch Building, Room 908, PO Box 016960 (R125),1501 Northwest 10th Avenue, Miami, Florida 33101.E-mail: [email protected].

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KEY WORDS allogeneic, cardiac stem cell,ischemic cardiomyopathy, mesenchymalstem cell

APPENDIX For supplemental Material,Methods, and Results sections as well as figuresand tables, please see the online version of thisarticle.


















































































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