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C O 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 . T H I S I S A N O P E N A C C E S S A R T I C L E U N D E R

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Randomized Comparison of AllogeneicVersus Autologous MesenchymalStem Cells for NonischemicDilated CardiomyopathyPOSEIDON-DCM Trial

Joshua M. Hare, MD,a,b Darcy L. DiFede, RN, BSN,a Angela C. Rieger, MD, MSC,a Victoria Florea, MD,a

Ana M. Landin, PHD,a Jill El-Khorazaty, MSC,c Aisha Khan, MSC, MBA,a Muzammil Mushtaq, MD,b

Maureen H. Lowery, MD,b John J. Byrnes, MD,b Robert C. Hendel, MD,b Mauricio G. Cohen, MD,b

Carlos E. Alfonso, MD,b Krystalenia Valasaki, MSC,a Marietsy V. Pujol, MBA,a Samuel Golpanian, MD,d

Eduard Ghersin, MD,e Joel E. Fishman, MD, PHD,e Pradip Pattany, PHD,e Samirah A. Gomes, MD, PHD,a

Cindy Delgado, MA,a Roberto Miki, MD,b Fouad Abuzeid, MD,a Mayra Vidro-Casiano, MPH,a Courtney Premer, BSC,a

Audrey Medina, BSC,a Valeria Porras, BSC,a Konstantinos E. Hatzistergos, PHD,a Erica Anderson, MSC,c

Adam Mendizabal, PHD,c Raul Mitrani, MD,b Alan W. Heldman, MDb

ABSTRACT

Fro

MedD

of

BACKGROUND Although human mesenchymal stem cells (hMSCs) have been tested in ischemic cardiomyopathy, few

studies exist in chronic nonischemic dilated cardiomyopathy (NIDCM).

OBJECTIVES The authors conducted a randomized comparison of safety and efficacy of autologous (auto) versus

allogeneic (allo) bone marrow-derived hMSCs in NIDCM.

METHODS Thirty-seven patients were randomized to either allo- or auto-hMSCs in a 1:1 ratio. Patients were recruited

between December 2011 and July 2015 at the University of Miami Hospital. Patients received hMSCs (100 million) by

transendocardial stem cell injection in 10 left ventricular sites. Treated patients were evaluated at baseline, 30 days,

and 3-, 6-, and 12-months for safety (serious adverse events [SAE]), and efficacy endpoints: ejection fraction, Minnesota

Living with Heart Failure Questionnaire, 6-min walk test, major adverse cardiac events, and immune biomarkers.

RESULTS There were no 30-day treatment-emergent SAEs. Twelve-month SAE incidence was 28.2% with allo-hMSCs

versus 63.5% with auto-hMSCs (p ¼ 0.1004 for the comparison). One allo-hMSC patient developed an elevated (>80%)

donor-specific calculated panel reactive antibody level. The ejection fraction increased in allo-hMSC patients by 8.0

percentage points (p¼ 0.004) compared with 5.4 with auto-hMSCs (p¼ 0.116; allo vs. auto p ¼ 0.4887). The 6-min walk

test increased with allo-hMSCs by 37.0 m (p ¼ 0.04), but not auto-hMSCs at 7.3 m (p ¼ 0.71; auto vs. allo p ¼ 0.0168).

MLHFQ score decreased in allo-hMSC (p¼0.0022) and auto-hMSC patients (p¼0.463; auto vs. allo p¼0.172). Themajor

adverse cardiac event rate was lower, too, in the allo group (p ¼ 0.0186 vs. auto). Tumor necrosis factor-a decreased

(p ¼ 0.0001 for each), to a greater extent with allo-hMSCs versus auto-hMSCs at 6 months (p ¼ 0.05).

CONCLUSIONS These findings demonstrated safety and clinically meaningful efficacy of allo-hMSC versus auto-hMSC

in NIDCM patients. Pivotal trials of allo-hMSCs are warranted based on these results. (Percutaneous Stem Cell Injection

Delivery Effects on Neomyogenesis in Dilated Cardiomyopathy [PoseidonDCM]; NCT01392625) (J Am Coll Cardiol

2017;69:526–37) © 2017 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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

dicine, University of Miami Miller School of Medicine, Miami, Florida; cThe Emmes Corporation, Rockville, Maryland;

epartmentof Surgery,UniversityofMiamiMiller SchoolofMedicine,Miami, Florida; and the eDepartmentofRadiology,University

Miami Miller School of Medicine, Miami, Florida. This study was funded by the National Heart, Lung, and Blood Institute with

AB BR E V I A T I O N S

AND ACRONYM S

6MWT = 6-min walk test

allo = allogeneic

auto = autologous

CI = confidence interval

cPRA = calculated panel

reactive antibody

EDV = end-diastolic volume

EF = ejection fraction

EPC-CFU = endothelial

progenitor cell colony forming

unit

FMD = flow-mediated

vasodilation

HF = heart failure

hMSC = human mesenchymal

stem cell

IQR = interquartile range

LV = left ventricular

J A C C V O L . 6 9 , N O . 5 , 2 0 1 7 Hare et al.F E B R U A R Y 7 , 2 0 1 7 : 5 2 6 – 3 7 Cell Therapy for Dilated Cardiomyopathy

527

N onischemic dilated cardiomyopathy (NIDCM)is a progressive disorder with no currentcure, often culminating in heart transplan-

tation (1,2). Cell-based therapy for heart disease isa promising new treatment strategy undergoing eval-uation (3–8), with a major challenge and opportunityin developing allogeneic (allo) therapy (9). Bonemarrow-derived human mesenchymal stem cells(hMSCs) may be a viable source of allo-cells,because they lack major histocompatibility class IIand costimulatory molecules, rendering themimmune-evasive (8,10). Allo-hMSCs also have immu-nomodulatory effects (8), which could have therapeu-tic importance in NIDCM, a disorder with a majorcomponent of immune dysregulation as an underly-ing etiology (11,12). Although allo-hMSCs offera major opportunity as an off-the-shelf therapeutic,they may lack the efficacy of autologous (auto)-hMSC therapy because some preclinical data indi-cated a higher risk of immunological clearance (13).

SEE PAGE 538 MACE = major adverse cardiac

event(s)

MLHFQ = Minnesota Living

with Heart Failure

Questionnaire

NIDCM = nonischemic dilated

cardiomyopathy

NYHA = New York Heart

Association

QOL = quality of life

SAE = serious adverse event

TEMRA = terminally

differentiated effector memory

CD45RAD

TE-SAE = treatment-emergent

serious adverse event

TESI = transendocardial stem

cell injection

TNF-a = tumor necrosis factor-a

Compared with auto-hMSCs, allo-hMSC therapy hasgreat potential for developing readily available,disease-free cell products in a cost-effective manner,an important issue for disorders with high incidence.In the field of heart failure (HF), hMSCs exert anti-fibrotic and proregenerative effects leading toimproved ventricular function and architecture in pa-tients with antecedent myocardial infarction (6,8,9).Because MSCs have powerful and sustained anti-inflammatory effects (8,14) and stimulate restorationof endothelial health (15), they could be of substantialtherapeutic importance in conditions such as NIDCM.

METHODS

The POSEIDON-DCM (Percutaneous Stem Cell Injec-tion Delivery Effects on Neomyogenesis in DilatedCardiomyopathy) trial was a randomized trial testingthe hypothesis that allo-hMSCs represent a safe andefficacious alternative to auto-hMSCs in patientswith NIDCM. The study design was previously pub-lished (16).

Patients provided written informed consent. Allpatients were recruited between December 2011 and

grant number RO1 HL RO110737. Dr. Hare is a board member, a consultan

member, a consultant, and holds equity in Longeveron. Ms. DiFede is a consu

Dr. Landin andMs. Khan are consultants for Longeveron LLC. Dr. Hendel is a

Valasaki hold equity in Vestion Inc. Dr. Heldman has received research sup

tems; and is a board member, a consultant, and holds equity in Vestion Inc

study. The other authors have reported that they have no relationships rele

Manuscript received October 11, 2016; revised manuscript received October

July 2015 at the University of Miami Hospital(Figure 1). Thirty-seven patients were ran-domized to either auto- or allo-hMSCs in a 1:1ratio. Following cardiac catheterization andcell injections, patients remained hospital-ized for a minimum of 2 days, with in-clinicfollow-up at 2 weeks post-catheterization,and in-person/in-clinic follow-up at 2, 3, 6,and 12 months for safety and efficacy as-sessments. An electronic data entry systemwas used for randomization and data collec-tion. Although this was an open-label study,all data analysis was masked to those assess-ing all study endpoints, and statistical analysiswas performed by a third party for unmasking.Detailed methods are explained in the OnlineAppendix. The National Heart, Lung, andBlood Institute Gene and Cell Therapy Dataand Safety Monitoring Board provided safetyoversight of the trial.

PATIENTS, PROCEDURES, AND CELLS

USED. Patient eligibility was determined af-ter confirmation of NIDCM diagnosis with anejection fraction (EF) <40% and either a leftventricular (LV) end-diastolic diameter >5.9cm in male subjects or >5.6 cm in femalesubjects, or an LV end-diastolic volume index>125 ml/m2, as previously described (16).

Baseline assessments included chemistryand hematology laboratory tests and echo-cardiography, plus chest, abdominal, andpelvic computed tomography scans. Cardiaccomputerized tomography (CT) or magneticresonance imaging (MRI) was performed (9).

All allo-hMSCs and auto-hMSCs were man-ufactured at the University of Miami Interdis-ciplinary StemCell Institute (8,16). Allo-hMSCswere derived from Caucasian male donors,

mean age 25.4� 3.3 years, and the samples were 80% to90% viable at time of transendocardial stem cell in-jection (TESI). The auto-hMSCs were from 11 men witha mean age of 58.0 � 9.9 years and 6 women with amean age of 55.0 � 12.4 years.

Injection sites were selected to prioritize TESIsafety and to distribute sites throughout the

t, and holds equity in Vestion Inc.; and is a board

ltant for Biologics Delivery Systems and Longeveron.

consultant for Astellas Pharma. Drs. Hatzistergos and

port from Biosense Webster Biologics Delivery Sys-

. Longeveron LLC and Vestion Inc. did not fund this

vant to the contents of this paper to disclose.

26, 2016, accepted November 1, 2016.

FIGURE 1 Study Flow

57 Patients assessed for eligibility

20 Excluded: 14 Did not meet inclusion criteria 4 Declined to participate 2 Other reasons

37 Randomized 1:1

18 Included in the primary analysis (30-dayTE-SAEs)

16 Included in the primary analysis (30-dayTE-SAEs)

12 completed one year follow-up • 2 withdrawn or lost to follow-up • 2 deaths

• 16 Received treatment as randomized• 2 Did not receive treatment as randomized: • 1 withdrew consent • 1 underwent AICD placement and could no longer be injected

• 18 Received treatment as randomized• 1 Did not receive treatment as randomized: • 1 death pre-injection

18 Randomized to receive autologousmesenchymal stem cell treatment

19 Randomized to receive allogeneicmesenchymal stem cell treatment

15 completed one year follow-up • 3 withdrawn or lost to follow-up

Following screening, 37 patients were enrolled and randomized, and included for analysis of the primary endpoint, 30-day

treatment-emergent serious adverse events (TE-SAEs). AICD ¼ automatic implantable cardioverter-defibrillator.

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528

accessible myocardial territories. Considerations for siteselection included avoidance of the ventricular apex andoptimization of catheter stability before needleextension.

STUDY ENDPOINTS. The primary safety endpoint wasthe incidence of any treatment-emergent seriousadverse events (TE-SAEs) occurring within 30 daysafter treatment (16). Secondary safety endpointsincluded other adverse events, ectopic tissue forma-tion, and forced expiratory volume in 1 s (FEV1). Sec-ondary efficacy endpoints included incidence of majoradverse cardiac events (MACE), LV structure andfunction, patient quality of life (QOL) measured byNew York Heart Association (NYHA) functional classand Minnesota Living with Heart Failure Question-naire (MLHFQ), 6-min walk test (6MWT), maximaloxygen consumption (VO2), endothelial function, andimmunologic status.

Endothelial function was assessed at baseline and3 months post–allo-hMSC or auto-hMSC. Endothelialprogenitor cell colony forming units (EPC-CFUs)

from peripheral blood samples and flow-mediatedvasodilation (FMD) brachial artery diameter mea-surements and percent of FMD were performed. Asubset of patient results for endothelial function waspreviously described (15).

Calculated panel reactive antibodies (cPRA) weremeasured at baseline and at 6 months. Serum tumornecrosis factor (TNF-a) was measured using a humanTNF-a enzyme-linked immunoadsorbent assay high-sensitivity kit. Lymphocytes were stained for T-cellmarkers of activation, late/exhausted T cells, B cellsubsets (switched memory and late/exhausted Bcells) and TNF-a by B cells. All samples were acquiredusing the LSR-Fortessa-HTS analyzer (BD BiosciencesPharmigen, San Diego, California) and analyzedwith FlowJo version 10 software (FlowJo, Ashland,Oregon).

STATISTICAL ANALYSIS. The sample size of 18 pertreatment arm was chosen to be appropriate for aphase I/II study; if the true TE-SAE event rate was25%, the probability of observing at least 1 event per

TABLE 1 Baseline Characteristics

Cell Type

Allogeneic (n ¼ 18) Autologous (n ¼ 16)

Age at injection, yrs 54.4 � 11.5 57.4 � 11.0

Sex

Male 14 (77.8) 10 (62.5)

Female 4 (22.2) 6 (37.5)

NIDCM diagnosis before TESI, yrs 6.05 � 6.2 6.93 � 7.3

History of adriamycin chemotherapy 1 (5.5) 2 (12.5)

AICD or BiV/CRT 15 (83.3) 14 (87.5)

Hispanic or Latino 4 (22.2) 8 (50.0)

White 16 (88.9) 14 (87.5)

African American 2 (11.1) 1 (6.3)

End-diastolic diameter, cm 7.2 � 1.3 7.1 � 1.7

History of coronary interventions 2 (11.1) 1 (6.3)

Previously referred for AICD placement 15 (83.3) 14 (87.5)

History of atrial or ventricular arrhythmia 5 (27.8) 1 (6.3)

History of hypertension 7 (38.9) 3 (18.8)

NYHA functional class

I 4 (22.2) 6 (37.5)

II 9 (50.0) 8 (50.0)

III 5 (27.8) 2 (12.5)

History of congestive heart failure 11 (61.1) 7 (43.8)

History of valvular heart disease 3 (16.7) 2 (12.5)

History of smoking 11 (61.1) 6 (37.5)

History of diabetes 0 (0.0) 1 (6.3)

Peak VO2, ml/kg/min 17.9 � 5.2 16.0 � 5.1

6-min walk test, m 427.2 � 67.4 416.4 � 105.7

FEV1, % 81.1 � 22.2 80.8 � 23.8

MLHFQ score 38.0 (23.0–64.0) 30.5 (16.5–60.5)

LV size and function

Ejection fraction, % 27.6 � 9.0 25.2 � 10.5

LV end-diastolic volume, ml 326.6 (259.1–348.6) 280.8 (239.2–360.4)

LV end-systolic volume, ml 239.8 (179.4–331.2) 191.1 (167.3–317.8)

End-diastolic diameter, mm 73.4 (66.1–80.0) 70.0 (61.1–80.2)

End-diastolic long-axis diameter, mm 105.6 (102.0–117.0) 98.8 (92.9–111.0)

Sphericity index 0.5 � 0.08 0.6 � 0.14

Values are mean � SD, n (%), or median (interquartile range).

AICD ¼ automatic implantable cardioverter-defibrillator; BiV ¼ biventricular pacing; CRT ¼ cardiacresynchronization therapy; FEV1 ¼ forced expiratory volume in 1 s; LV ¼ left ventricular; MLHFQ ¼ MinnesotaLiving with Heart Failure Questionnaire; NIDCM ¼ nonischemic dilated cardiomyopathy; NYHA ¼ New York HeartAssociation; TESI ¼ transendocardial stem cell injection; VO2 ¼ oxygen consumption.

J A C C V O L . 6 9 , N O . 5 , 2 0 1 7 Hare et al.F E B R U A R Y 7 , 2 0 1 7 : 5 2 6 – 3 7 Cell Therapy for Dilated Cardiomyopathy

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treatment arm would be 99%. All patients whoreceived study injection were included in analysis.

Continuous variables were summarized using thefollowing descriptive statistics: n (nonmissing samplesize), mean � SD (or median and interquartile range[IQR] as appropriate), maximum, and minimum. Thefrequency and percentages (based on the nonmissingsample size) of observed levels were reported for allcategorical measures. Outcomes, whichwere collectedat multiple follow-up visits, were analyzed using amixed model for repeated measures to comparetreatment groups, with treatment group considered aneffect as well as a group-by-time interaction.

Within-group effects were described using model-estimated contrasts. Outcomes that were highlyskewed were analyzed using ranked analysis ofcovariance adjusting for baseline at each follow-upassessment, and within-group effects were describedusing a Wilcoxon signed rank test. Categorical vari-ables were compared between groups using Fisherexact tests. Analysis of time-to-event data was doneusing a 2-sided log-rank test, censoring those who didnot experience an event at their last known follow-upday. All statistical tests were performed at a ¼ 0.05using 2-sided tests. All data analyses and statisticalcomputations were conducted with SAS version 9.3(SAS Institute, Cary, North Carolina).

RESULTS

Of the 37 patients randomized to either auto- or allo-hMSCs, 34 received study injection of either auto-(n ¼ 16) or allo-hMSCs (n ¼ 18). Three patients did notreceive the study injection: 1 withdrew consentbefore treatment; 1 was recruited, but did not receivetreatment due to automatic implantable cardioverter-defibrillator placement; and 1 died before treatment.

The mean age of injected participants was55.8 � 11.2 years, 29% were female, and 35% wereHispanic (Table 1). The mean years of NIDCM diag-nosis before TESI was 6.1 � 6.2 years for allo and6.9 � 7.3 years for auto hMSCs patients (p ¼ 0.5between groups). Fifty percent of patients had NYHAfunctional class II symptoms, mean baseline globalEF was 26.5 � 9.64%, mean 6MWT was 422 � 86.8 m,and median baseline MLHFQ score was 36 (IQR: 18.0to 64.0).

SAFETY AND LONG-TERM ADVERSE EVENTS. TESIwas technically successful in 33 (97.05%) patients.One patient experienced ventricular tachycardia afterthe ninth injection, and did not receive the last in-jection. No patients experienced significant post-procedural pericardial effusion. The interventionwas safe in all TESI recipients, with no TE-SAEs

within 30 days. Furthermore, the incidence of AEsby 30 days did not significantly differ by cell type(p ¼ 0.6117) (Table 2). Moreover, SAE rates wereinfrequent through day 30 and similar in both groups(p ¼ 0.6238) (Table 2). Accordingly, the study met theprimary safety endpoint, documenting the safety ofTESI in patients with NIDCM.

The 12-month post-TESI SAE incidence was 28.2%(95% confidence interval [CI]: 12.8 to 55.1) in allo-hMSC and 63.5% (95% CI: 40.8 to 85.7; p ¼ 0.1004)in auto-hMSC patients. Post-TESI, 2 auto-hMSCspatients and 1 allo-hMSCs patient underwent hearttransplantation; similarly, LV assist devices wereimplanted in 1 patient in each group. Two deaths

TABLE 2 Safety and Clinical Efficacy

Cell Type

Allogeneic (n ¼ 18) Autologous* (n ¼ 16)

30 days post-TESI

Incidence of AE 7 38.9 (20.8–64.7) 5 31.3 (14.4-59.5)

Incidence of SAE 2 11.1 (2.9–37.6) 1 6.3 (0.9–36.8)

Incidence of TE-SAE 0 0.0 (0.0–18.5) 0 0.0 (0.0–20.6)

Incidence of MACE 0 0.0 (0.0–18.5) 0 0.0 (0.0–20.6)

6 months post-TESI

Incidence of AE 12 66.7 (45.5–86.3) 13 81.3 (59.8-95.4)

Incidence of SAE 4 22.2 (9.0–48.9) 4 25.0 (10.2–53.7)

Incidence of MACE 1 5.6 (0.8–33.4) 3 18.8 (6.5–47.5)

Incidence of death 0 0.0 (0.0–18.5) 1 6.3 (0.9–36.8)

1 yr post-TESI

Incidence of AE 12 66.7 (45.5–86.3) 14 87.5 (67.2-97.9)

Incidence of SAE 5 28.2 (12.8–55.1) 10 63.5 (40.8–85.7)

Incidence of MACE 3 20.3 (6.8–52.1)† 9 57.1 (34.9–81.2)

Incidence of death 0 0.0 (0.0–18.5) 2 12.5 (3.3–41.4)

Values are n and % (95% CI). Percentages are Kaplan-Meier event rates; exact binomial CIs usedfor zero counts. *Two deaths occurred post-injection: 1 patient experienced a fatal subduralhematoma due to trauma on day 152 post-injection, and a second patient died of NIDCM 291 dayspost-injection. Both events were considered unrelated to auto treatment. †Between-groupp #0.05.

AE ¼ adverse event; CI ¼ confidence interval; MACE ¼ major adverse cardiac event(s);SAE ¼ serious adverse event; other abbreviations as in Table 1.

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occurred post-injection in the auto-hMSCs group: afatal subdural hematoma due to trauma on day 152post-injection and a death due to NIDCM 291 dayspost-injection. Both events were considered unre-lated to study treatment.

The 12-month all-cause rehospitalization rate waslower in the allo- versus the auto-hMSCs recipients:28.2% (95% CI: 12.8% to 55.1%) versus 70.0% (95% CI:47.0% to 89.8%), respectively (p ¼ 0.0447). Similarly,MACE over 12 months was lower in the allo-hMSCsgroup: 20.3% (95% CI: 6.8% to 52.1%) comparedwith 57.1% (95% CI: 34.9% to 81.2%) in the auto-hMSCs group (p ¼ 0.0186). No ectopic tissue forma-tion was identified in either group at 1-year follow-upby computed tomography of the chest, abdomen andpelvis.

LV FUNCTION AND OTHER TESTING RESULTS. Atbaseline, the average EF was 26.5 � 9.6%, and medianLV end-diastolic diameter was 70.4 mm (IQR: 64.1 to80.0). EF increased significantly in the allo-hMSCsgroup by 8.0 percentage points (95% CI: 2.8 to 13.2percentage points; p ¼ 0.004), but not in the auto-hMSCs cohort (5.4; 95% CI: �1.4 to 12.1; p ¼ 0.116) at12 months (p ¼ 0.49 between group) (Figures 2A and3). This resulted in the EF rising above 40% in46.7% of the allo-hMSC patients (Figure 3) versus22.2% of the auto-hMSCs patients. Stroke volume(Figure 2B), end-diastolic volume (EDV) (Figure 2C),

and end-systolic volume (Figure 2D) did not signifi-cantly decrease from baseline. End-diastolic long-axisdiameter decreased 3.5 mm (95% CI: �6.4 mm to �0.6mm; p ¼ 0.04) from baseline to 12 months in the allo-hMSCs group compared with 1.7 mm (95% CI: �7.3mm to 3.9 mm) in the auto-hMSCs arm (p ¼ 0.73)(Figure 2E). Neither sphericity index, end-diastolicdiameter, nor end-systolic diameter changed frombaseline to 12 months in either group (data notshown).

Functional capacity and QOL showed greaterimprovement with allo- compared with auto-hMSCsuse. The 6MWT distance significantly increased inpatients receiving allo-hMSCs by 37.0 m (95% CI: 2.0m to 72.0 m; p ¼ 0.04) at 12 months compared withbaseline, but did not significantly change in the auto-hMSCs group (7.3 m; 95% CI: �47.8 m to 33.3 m;p ¼ 0.71) (Central Illustration, part A). The between-group difference was 67.7 m (95% CI: 16.4 m to118.9 m; p ¼ 0.0116) and 46.5 m (95% CI: �5.5 m to98.5 m; p ¼ 0.0770) at 6 and 12 months, respectively(overall comparison between allo and autop ¼ 0.0168). At 12 months, allo-hMSC patients wasassociated with a 66.7% improvement in NYHAfunctional class, whereas only 27.3% of auto-hMSCpatients improved in NYHA functional class (betweengroup change in NYHA functional class p ¼ 0.0527).Two patients receiving auto-hMSCs worsened by12 months (Central Illustration, part B). There were nosignificant differences in the maximal VO2 at 6 or12 months compared with baseline in either group(data not shown). FEV1 improved in allo-hMSCspatients by 3.7% (95% CI: �0.08% to 0.30%;p ¼ 0.2423) compared with a decrease of 3.8%(95% CI: �0.36% to 0.06%; p ¼ 0.16) among the auto-hMSCs group at 12 months. However, the between-group difference at 12 months was 0.29 l (95% CI:0.01 l to 0.56 l; p ¼ 0.0430) (Figure 4A).

The median MLHFQ score at baseline was 38 (IQR:23 to 54) in the allo-hMSC cohort versus 30.5 (IQR:16.5 to 60.5) with auto-hMSCs. The MLHFQ improvedin both groups (Figure 4B) over 12 months (allop ¼ 0.0022; auto p ¼ 0.1719). Unlike with auto-hMSCs,EPC-CFU significantly increased with allo-hMSCs(p ¼ 0.0107) (Figure 4C) as did percentage of FMD at3 months (p ¼ 0.09). The percent of FMD increasedfrom baseline to 3 months in the allo-group, from4.5% (IQR: 2.9% to 7.4%) to 6.4% (IQR: 5.1% to 12.3%)(p ¼ 0.0005), compared with no significant change inthe auto-cohort, from 6.4% (IQR: 3.7% to 10.0%) to5.8% (IQR: 4.4% to 10.0%) (p ¼ 0.8457) (Figure 4D).

The cPRA results showed that two-thirds of allo-and nearly all auto-hMSCs recipients had no reactionto low cPRA (0% to 20% cPRA), with only 1 allo-hMSC

FIGURE 2 Changes in LV Structure and Function Over 12 Months

28

Change from Baseline in EF Change from Baseline in Stroke Volume Change from Baseline in EDV

Change from Baseline in ESV Change from Baseline in Long Axis Diameter

6050403020100

-10-20-30-40-50-60

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*1612840

-4-8

-12-16

# Allo# Auto

N=15N=9

# Allo# Auto

N=15N=9

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Ejec

tion

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olum

e (m

l)

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-300

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Axi

s Dia

met

er (m

m)

A B C

ED

(A) EF increased from baseline in allo but not in auto: 8 U (95% CI: 2.8 U to 13.2 U). Neither group showed significant improvement in (B) stroke volume,

(C) end-diastolic volume (EDV) and (D) end-systolic volume (ESV). (E) Structural remodeling was evident in the reduction in long-axis diameter in the allo-hMSCs

group from baseline to 12 months. *Within-group p # 0.05. allo-hMSC ¼ allogeneic human mesenchymal stem cell; auto-hMSC ¼ autologous human mesenchymal

stem cell; EF ¼ ejection fraction; LV ¼ left ventricular; solid circle ¼ allo-hMSC; solid triangle ¼ auto-hMSC.

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subject displaying a high cPRA response ($80% cPRA)(Table 3).

Elevated levels of TNF-a decreased from baselineto 6 months in both groups (allo �10.6 � 1.6 pg/ml;p < 0.0001; auto �6.8 � 1.4 pg/ml; p < 0.0001;between-group p ¼ 0.05) (Table 4). Terminallydifferentiated effector memory CD45RAþ (TEMRA)T cells (exhausted T-cell phenotype) also werereduced in both groups with a greater decrease inallo-hMSC (�15.9 � 5.4%; p < 0.0001) than auto-hMSC(9.3 � 3.3%; p < 0.0001; between-group p ¼ 0.0111)(Table 4). Suppressed percent switch memory Bcells (a predictive biomarker for antibody response) at

baseline were significantly increased at 6 months inboth groups but more so with allo- (10.2 � 4.9%;p < 0.0001) versus auto-hMSCs (4.3 � 3.9%;p ¼ 0.0014; between-group p < 0.0001) (Table 4).

Finally, intracellular TNF-a expression in B cellswas also decreased at 6 months relative to baselinein both groups (between-group p ¼ 0.174) (Table 4).By contrast, late/exhausted B cells decreasedsignificantly in both groups, whereas early T-cellactivation decreased to similar degrees in both groups(Table 4). However, late/chronic T-cell activationdid not significantly decrease in either group(allo �2.3 � 1.3%; p ¼ 0.4; auto �3.4 � 2.7%; p ¼ 0.7).

FIGURE 3 Change in Global Ejection Fraction

Baseline

Ejec

tion

Frac

tion

(%)

50

40

30

20

10

1 Year Baseline

Allo hMSCs Auto hMSCs

Visit1 Year

Overall, 7 of 15 allo-hMSC patients increased EF to $40% at 12 months as compared to

baseline, whereas only 2 of 9 auto-hMSC patients showed the same increase. Depicted in

this graph are the individual patient response in EF; shaded areas are 95% CI. Abbrevi-

ations as in Figure 2.

CENTRAL ILLUSTRATION Allo-hMSCs vs. Auto-hMSCs in NIDCM

B

200

160

120

80

40

0

-40

-80

-120

-160

-200

†*†

# Allo# Auto

N=16N=13

N=13N=9

6 Months 12 Months

Six

Min

ute

Wal

k Te

st (m

eter

s)

100

90

80

70

60

50

40

30

20

10

02

Perc

ent o

f Pat

ient

s

A. Change from Baseline in 6-MWT

Hare, J.M. et al. J Am Coll Cardiol. 2017;69(5):526–37.

Depicted are changes from baseline in functional capacity (A) and quality of life (B): (A) 6-min w

6 months and 12 months and (B) New York Heart Association (NYHA) functional classification impr

patients at 1 year. *Within-group p # 0.05; †between-group p # 0.05. allo-hMSC ¼ allogeneic

mesenchymal stem cell.

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DISCUSSION

The POSEIDON-DCM study was a randomized com-parison of allo-hMSCs versus auto-hMSCs in patientswith NIDCM (16). Results supported the safety andfeasibility of TESI for both types of cells, and neitherwere associated with any ectopic tissue formation.Importantly, allo-hMSCs produced a constellation ofclinically meaningful effects of greater magnitudethan auto-hMSCs, including significant improvementin EF, 6MWT, and MLHFQ scores. Endothelialfunction was improved, but only in the allo group.Similarly, TNF-a suppression was greater with allo-hMSCs, and these cells were also associated with ev-idence of clinical efficacy, including improved NYHAfunctional class, lower MACE, and lower hospitaliza-tion rates at 1 year. Together, these findings show asubstantial magnitude of clinical responsiveness inpatients with NIDCM, a group with major unmetmedical needs.

The findings here provide evidence of a clinicallyrelevant effect that was substantially larger than

. New York Heart Association

NYHA class change from Baselineimproved no change worsened

M 3M 6M 12M 2MVisit

3M 6M 12M

Allo hMSCs Auto hMSCs

alk test (6MWT) increased in allo-hMSCs, but not in auto-hMSCs at

oved 66.7% in the allo-hMSC patients, and 27.3% in the auto-hMSC

human mesenchymal stem cell; auto-hMSC ¼ autologous human

FIGURE 4 Pulmonary Function, Patient Quality of Life, and Endothelial Function

15 87654321

0-1-2-3-4-5

13

11

9

7

5

3

1

-1

-3

-5

60

50

40

30

20

10

0

#Allo#Auto

N=17N=16

N=14N=16

N=18N=16

N=14N=15

N=15N=9

Baseline Day 60 Day 90 Day 180 Day 365

A B

C D

†

# Allo# Auto

N=12N=11

Change from Baseline in EPC-CFU Change from Baseline in FMD

MLHFQ Total Score

3 Months# Allo# Auto

N=12N=11

3 Months

EPC-

CFU

(CFU

)

FMD

(%)

MLH

FQ T

otal

Sco

re

1.41.21.00.80.60.40.20.0

-0.2-0.4-0.6-0.8-1.0

# Allo# Auto

*

*

*

**

*

N=17N=14

N=14N=10

6 Months 12 Months

FEV1

(Lite

rs)

Change from Baseline in Forced ExpiratoryVolume in One Second

allo-hMSC, but not auto-hMSC, therapy showed an improvement in (A) forced expiratory volume in 1 s (FEV1) by 0.11 l; (B) 12-months

Minnesota Living with Heart Failure Questionnaire (MLHFQ) score. (C) The production of endothelial progenitor cell colony forming units

(EPC-CFU) and (D) 3-month flow-mediated vasodilation (FMD) was significantly greater in allo-hMSCs compared to auto-hMSCs. Solid circle¼allo-hMSC; solid triangle ¼ auto-hMSC. *Within-group p # 0.05; †between-group p # 0.05. Abbreviations as in Figure 2.

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previous trials of cell therapy in patients withischemic cardiomyopathy (4–7,17). Whereas celltherapy in ischemic cardiomyopathy produces areduction in infarct scar size (6,9), increased EFhas been difficult to show in this population (6,17).In the present study, EF increased by 8 percentagepoints in the allo-hMSC group, with nearly one-halfof these patients increasing EF to levels above 40%(Figure 3 and Figures 5A and 5B), an accepted

cutoff for the diagnosis of NIDCM (1,18). HF withrecovered EF is a recognized syndrome that carriesan improved prognosis relative to HF with persis-tently low EF (19). As such, if the present resultsare replicated in a larger trial, they represent amajor clinical advance for NIDCM, a conditionaffecting individuals of all ages and accounting forapproximately one-half of all heart transplants(2,20).

TABLE 3 Recipient cPRA Change 6 Months Post-TESI

cPRA Risk

Cell Type

Allogeneic(n ¼ 15)

Autologous(n ¼ 13)

No reaction to low risk (0%–20% cPRA) 10 (66.7) 12 (92.3)*

Moderate risk (21%–79% cPRA) 4 (26.7) 1 (7.7)*

High risk (þ80% cPRA) 1 (6.7) 0 (0)

Values are n (%). *Between-group p #0.05.

cPRA ¼ calculated panel reactive antibodies; TESI ¼ transendocardial stem cellinjection.

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In this study, 2 pathophysiological features wereidentified that could underlie the effects of allo-hMSCs. First, hMSCs exerted a significant restora-tion of endothelial dysfunction, implicated as anunderlying contributor to the failing circulation inischemic cardiomyopathy and NIDCM (15,21). Second,hMSCs reduced the elevated TNF-a levels in the studypopulation. Elevated levels of TNF-a, a crucialproinflammatory cytokine, are tied to heart diseaseprogression and are implicated in modulating bothcardiac contractility and peripheral resistance (22,23).

The use of entanercept to inhibit TNF-a in HF didnot improve mortality and hospitalization rates (24);however, hMSC therapy has the advantage ofreducing several proinflammatory cytokines, favoringan anti-inflammatory profile. In our trial, TNF-alevels were significantly reduced at 6 months post-TESI by both cell types, but allo-hMSCs were moreeffective.

Several reasons might account for allogeneicMSCs providing greater efficacy relative to auto-hMSCs. These include age of the donors (mean agein the allo-hMSC group was roughly one-half that ofthe auto-hMSC group) and possible adverse impact ofthe disease milieu (e.g., the proinflammatory pheno-type) (25). Alternatively, preferential response ofallo- versus auto-hMSCs might reflect enhanced

TABLE 4 6-Month Effects of hMSCs on Immunity

Immune Biomarkers Baseline

Serum TNF-a, pg/ml 13.5 � 1.5

Early T-cell activation (CD3þ, CD69þ), % 13.4 � 4.4

Late/chronic T-cell activation (CD3þ, CD25þ), % 8.9 (5.9–10.4)

TEMRA (CD3þ, CD45RAþ, CCR7�), % 33.7 � 7.6

Late/exhausted B cells (CD19þ, CD27�, IgD�), % 19.3 (17.9–28.6

Switched memory B cells (CD19þ, CD27high, IgD�), % 10.0 � 3.6

B cells expressing intracellular TNF-a, % 32.1 � 7.3

Values are mean � SD or median (interquartile range). *Within-group p #0.001. †Betw

hMSC ¼ human mesenchymal stem cell; TEMRA ¼ terminally differentiated effector m

endogenous repair, an important mechanism under-lying hMSC cardio-repair (26). Further studies todelineate potency differences between auto andallo hMSCs in NIDCM are underway.

In addition, our present findings included a detailedevaluation of humoral lymphocytes following hMSCtherapy. These cells favorably altered several immu-nologic markers typically elevated in chronic inflam-mation, such as TEMRA T cells (exhausted phenotype)and late/exhausted B cells (27,28). As with TNF-a,hMSCs reversed the exhausted immune phenotype(TEMRA T cells and late/exhausted memory B cells)(29). In contrast to previous studies, we showed theimportant finding that hMSC therapy (allo-cells inparticular) increased switched memory B cells (a pre-dictive biomarker for a protective antibody response)(28). Restoration of immune competence may haveclinical relevance in these patients who are of higherrisk for comorbid infectious disease.

The cPRA yielded mostly no-to-low response withvery few moderate responses for either allo or auto-hMSCs. Only 1 patient in the allo group mounted anelevated cPRA response that included donor-specificantibodies. This incidence was similar to 2 previousstudies employing allo MSCs or mesenchymal pre-cursor cells (9,30). This cPRA response did not causeclinical immunologic rejection in the patient.Ongoing immunologic monitoring is warranted dur-ing larger pivotal trials.

There are limited previous cell-based therapy trialsin NIDCM showing improved cardiac function and/orQOL. TOPCARE-DCM (Transplantation of ProgenitorCells and Functional Regeneration EnhancementPilot Trial in Patients With Nonischemic DilatedCardiomyopathy) showed that intracoronary deliveryof auto bone marrow cells improved LV EF 3 monthsafter cell administration (31). Similarly, another studydemonstrated an increased EF and sustainedimprovement in QOL 3 years post-treatment with

Allogeneic Autologous

6 Months Baseline 6 Months

2.3 � 0.2*† 11.8 � 0.9 4.8 � 0.8*

7.9 � 3.3* 13.3 � 4.8 10.6 � 5.4‡

3.4 (3.0–5.0)‡ 8.1 (5.2–14.1) 4.4 (3.6–11.1)

17.9 � 5.2*† 30.1 � 10.8 21.3 � 7.5*

) 14.7 (14.2–17.3)*† 20.0 (17.7–32.0) 15.5 (14.0–19.0)‡

20.2 � 3.1*§ 9.6 � 3.1 14.1 � 4.5‡

20.4 � 6.3* 28.2 � 6.8 19.7 � 4.8*

een-group p #0.05. ‡Within-group p #0.05. §Between-group p #0.001.

emory CD45RAþ; TNF-a ¼ tumor necrosis factor-a.

FIGURE 5 Allogeneic Mesenchymal Stem Cell Therapy for NIDCM

Representative example of cardiac computed tomography images from a patient with an EF response exceeding 40%. As shown, global ejection fraction

(EF) was depressed at baseline (A). At 12 months after transendocardial stem cell injection (B), global EF improved as had measures of end-diastolic (ED)

and end-systolic (ES) volume (EDV and ESV, respectively).

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auto bone marrow cells (32,33). Although severalother groups have shown that cell-based therapyappears to reduce the incidence of heart trans-plantation and/or mortality in NIDCM, there is

variability in the selected cells used for therapy andtheir distinct cell surface markers, such as CD34þ

(34). Perin et al. (32), in a study including bothischemic cardiomyopathy and NIDCM patients,

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: In a

pilot study involving patients with NIDCM,

transendocardial injections of bone marrow-derived

allogeneic hMSCs were well tolerated and associated

with favorable effects on the immune system and

myocardial and endothelial function.

TRANSLATIONAL OUTLOOK: Additional studies in

larger cohorts comparing allogenic with autologous

mesenchymal stem cells are needed to assess the

relative risks and benefits of these therapeutic

strategies in patients with NIDCM.

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delivered bone marrow-derived mesenchymal pre-cursor cells, characterized by surface antigenexpression of STRO-1, STRO-3, CC-9, and HLA class Iand II antigens. This study, however, did not addressdifferences in responses between ischemic cardio-myopathy and NIDCM.

To date, no previous study to our knowledge hascompared bone marrow-derived allo-hMSC and auto-hMSC in patients with NIDCM. In the POSEIDON pilotstudy, we showed that allo- and auto-hMSCs weresafe and did not increase SAEs or immunologic re-actions with allo-hMSCs therapy. Furthermore, weshowed reverse remodeling of the LV chamberdimension (shown by reduction of the long axisdiameter), decreased scar size, improved EDV andsphericity index, and increased EF with low-doseallo-hMSC (9).

STUDY LIMITATIONS. The limitations of our studyinclude the lack of a placebo group, which was by trialdesign. Another limitation is the loss of patients dueto withdrawal of consent or loss to follow-up. Thistrial also was limited by small sample size, which wasprospectively determined based upon a pre-setthreshold for 30-day TE-SAE rate. The sample sizelimits interpretation of efficacy results; nevertheless,the findings are of significant value in designing anddetermining sample size for future pivotal trials.Future phase II/III studies will incorporate a placebo-controlled, double-blind design.

CONCLUSIONS

This study tested the safety and efficacy of allo- versusauto-hMSCs in NIDCM patients. This study revealed ahighly acceptable safety profile at 30 days in bothgroups, similar to previously published trials usingTESI (6,9,32). Importantly, several lines of evidencesupported superiority for allo-hMSCs versus auto-hMSCs in regard to efficacy, including EF, 6MWT,

MLHFQ, and endothelial function. In addition, theproinflammatory/exhausted immune phenotype inpatients receiving allo-hMSCs for NIDCM had a dra-matic remodeling of the immune cells toward a lessinflammatory or exhausted phenotype. These dataprovided important and clinically relevant insightsinto the therapeutic basis and effects of allo-hMSCsand auto-hMSCs for NIDCM patients. Clinical benefitsof the magnitude shown here support the develop-ment of allo-hMSCs for treating NIDCM.

ACKNOWLEDGMENTS The authors thank Dr. Huw S.Kruger Gray and Patricia Guevara, MS, at the Uni-versity of Miami, Sylvester Comprehensive CancerCenter Flow Cytometric Core, for their assistance.Alina Gutierrez, BS, MT, and Dr. Phillip Ruiz from theUniversity of Miami Transplant Department for cPRAassay and interpretation.

ADDRESS FOR CORRESPONDENCE: Dr. Joshua M.Hare, Interdisciplinary Stem Cell Institute, Universityof Miami Miller School of Medicine, 1501 Northwest10th Avenue, 9th Floor, Miami, Florida 33136. E-mail:JHare@med.miami.edu.

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KEY WORDS endothelial function, heartfailure, idiopathic dilated cardiomyopathy,immune biomarker, stem cell therapy

APPENDIX For an expanded Methodssection, please see the online version of thisarticle.