Clinical Track

Intravenous Allogeneic Mesenchymal Stem Cells

for Nonischemic Cardiomyopathy
Safety and Efficacy Results of a Phase II-A Randomized Trial
Javed Butler,* Stephen E. Epstein,* Stephen J. Greene, Arshed A. Quyyumi, Sergey Sikora,
Raymond J. Kim, Allen S. Anderson, Jane E. Wilcox, Nikolai I. Tankovich, Michael J. Lipinski,
Yi-An Ko, Kenneth B. Margulies, Robert T. Cole, Hal A. Skopicki, Mihai Gheorghiade

Rationale: Potential benefits of mesenchymal stem cell (MSC) therapy in heart failure may be related to paracrine
properties and systemic effects, including anti-inflammatory activities. If this hypothesis is valid, intravenous
administration of MSCs should improve outcomes in heart failure, an entity in which excessive chronic
inflammation may play a pivotal role.
Objective: To assess the safety and preliminary efficacy of intravenously administered ischemia-tolerant MSCs
(itMSCs) in patients with nonischemic cardiomyopathy.
Methods and Results: This was a single-blind, placebo-controlled, crossover, randomized phase II-a trial of
nonischemic cardiomyopathy patients with left ventricular ejection fraction ≤40% and absent hyperenhancement
on cardiac magnetic resonance imaging. Patients were randomized to intravenously administered itMSCs (1.5×106
cells/kg) or placebo; at 90 days, each group received the alternative treatment. Overall, 22 patients were randomized
to itMSC (n=10) and placebo (n=12) at baseline. After crossover, data were available for 22 itMSC patients.
No major differences in death, hospitalization, or serious adverse events were noted between the 2 treatments.
Change from baseline in left ventricular ejection fraction and ventricular volumes was not significantly different
between therapies. Compared with placebo, itMSC therapy increased 6-minute walk distance (+36.47 m, 95%
confidence interval 5.98–66.97; P=0.02) and improved Kansas City Cardiomyopathy clinical summary (+5.22,
95% confidence interval 0.70–9.74; P=0.02) and functional status scores (+5.65, 95% confidence interval −0.11
to 11.41; P=0.06). The data demonstrated MSC-induced immunomodulatory effects, the magnitude of which
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correlated with improvement in left ventricular ejection fraction.

Conclusions: In this pilot study of patients with nonischemic cardiomyopathy, itMSC therapy was safe, caused
immunomodulatory effects, and was associated with improvements in health status and functional capacity.
Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02467387.   
(Circ Res. 2017;120:332-340. DOI: 10.1161/CIRCRESAHA.116.309717.)
Key Words: cardiomyopathy ■ clinical trial ■ heart failure ■ nonischemic ■ stem cells ■ viable myocardium

D espite available therapies, patients with heart failure

(HF) with reduced ejection fraction (HFrEF) continue
to experience high rates of mortality and hospitalization.1
Accordingly, there has been increasing focus on alternative
treatment strategies, including stem cell therapy.2,3 Despite in-
termittent positive signals in phase I and phase II trials, major
reviews of available stem cell data have failed to show a de-
Editorial, see p 256 finitive benefit in HF.4 However, virtually all published studies
In This Issue, see p 245 were centered on the concept that therapeutic efficacy depends

Original received August 3, 2016; revision received October 23, 2016; accepted November 11, 2016. In October 2016, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 15.7 days.
From the Division of Cardiology, Department of Medicine, Stony Brook University, NY (J.B., H.A.S.); MedStar Heart and Vascular Institute, MedStar
Washington Hospital Center, Washington, DC (S.E.E., M.J.L.); Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham,
NC (S.J.G., R.J.K.); Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA (A.A.Q., R.T.C.); CardioCell LLC, San Diego, CA
(S.S.); Division of Cardiology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL (A.S.A., J.E.W.); Stemedica
Cell Technologies Inc, San Diego, CA (N.I.T.); Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA (Y.-A.K.); Division of
Cardiology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia (K.B.M.); and Center for Cardiovascular
Innovation, Northwestern University Feinberg School of Medicine, Chicago, IL (M.G.).
*These authors are joint first authors.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
116.309717/-/DC1.
Correspondence to Javed Butler, Division of Cardiology, Department of Medicine, Stony Brook University, Health Sciences Center, T-16, Room 080,
SUNY at Stony Brook, NY 11794. E-mail [email protected]
© 2016 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.116.309717

332
 Butler et al   Mesenchymal Stem Cells for Heart Failure   333

Novelty and Significance

What Is Known? impractical strategy for repeated administrations. Data suggest
• The therapeutic efficacy of stem cells is thought to depend primarily on
that chronic inflammation contributes to progressive HF. Because
local myocardial effects, necessitating efficient myocardial engraftment. MSCs secrete factors with multiple activities, including anti-in-
• Intravenous delivery of stem cells results in low cardiac engraftment, flammatory, we postulated that intravenously administered MSCs
leading to a focus on catheter-based delivery of cells. could improve clinical outcomes, in part, by systemic anti-inflam-
• Continuing inflammation may contribute to heart failure (HF) progression. matory effects. Patients with nonischemic HF underwent intra-
venous injection of either saline or MSCs that were grown under
What New Information Does This Article Contribute? chronic hypoxic conditions (itMSC). Patients treated with itMSCs
demonstrated significant clinical improvement. Administration of
• Intravenous administration of ischemia-tolerant mesenchymal stem itMSCs was associated with a reduced number of natural killer
cells (itMSCs) improved patient clinical status.
cells, and the magnitude of this reduction was inversely corre-
• Administration of itMSCs caused systemic anti-inflammatory effects
lated with improved left ventricular ejection fraction. This study
(reduced number of CD4-positive and natural killer cells)
shows that despite low myocardial engraftment, intravenously
• The magnitude of itMSC-induced natural killer cell reduction was inverse-
ly associated with the magnitude of the improvement in cardiac function.
administered MSCs improve clinical end points, effects possi-
bly caused, in part, by systemic anti-inflammatory effects. The
results of this investigation, if confirmed, could impact clinical
It is generally assumed that if stem cells are to be an effective
applicability of stem cell therapeutics and could provide insights
HF therapy, they have to be delivered in large numbers to the tar-
into novel mechanisms underlying HF progression that could lead
get tissue—the heart. This requires catheter delivery of cells, an
to identification of new therapeutic targets.

In addition, it has been shown that MSCs grown under

Nonstandard Abbreviations and Acronyms
chronic hypoxic conditions (ie, ischemia-tolerant MSC [it-
CI confidence interval MSCs]) exhibit characteristics suggesting that they would be
CMR cardiac magnetic resonance therapeutically more effective in the setting of chronic car-
HF heart failure diomyopathy than MSCs grown under normoxic conditions.7
HFrEF heart failure with reduced ejection fraction Specifically, in an in vitro study comparing MSCs grown un-
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LVEDV left ventricular end-diastolic volume der normoxia versus those grown under hypoxic conditions,
LVEF left ventricular ejection fraction hypoxia-grown MSCs migrated better toward wound healing–
LVESV left ventricular end-systolic volume related cytokines and cytokines found in ischemic tissues and
MSC mesenchymal stem cell expressed higher levels of HIF-1 (hypoxia inducible factor 1;
itMSC ischemia-tolerant MSC ie, the cell’s master switch transcription factor responsible for
expressing gene products involved in the cellular response to
on injected stem cells engrafting in the myocardium and either hypoxia). Based on these results, we decided to administer
transdifferentiating into healthy cardiac myocytes or stimu- MSCs grown under chronic hypoxic conditions in the present
lating resident cardiac stem cells to expand and organize into study.
Patients with HFrEF, especially those with an ischemic
functional myocardium.5 Because the intravenous delivery of
pathogenesis, may have areas of myocyte replaced with fi-
stem cells results in low cardiac engraftment, existing clinical
brosis, which is unlikely to respond to therapy.8 Moreover,
studies have been largely limited to the direct delivery of cells
previous HF studies with interventions like β-blockers and
to the myocardium via intracoronary, transendocardial, or in-
cardiac resynchronization therapy suggest that patients with
tramyocardial methods.
nonischemic cardiomyopathy generally demonstrate more ro-
Of particular relevance, recent data suggest that an ex-
bust response to therapy, potentially related to the more likely
cessive and continuing inflammatory response constitutes a presence of dysfunctional but viable myocardium as opposed
primary mechanism in HFrEF progression.2 Interestingly, mes- to myocardial segments replaced by fibrosis.8–11 We, there-
enchymal stem cells (MSCs) secrete a broad array of molecules fore, used delayed-enhancement cardiac magnetic resonance
with potential therapeutic effects, including anti-inflammatory (CMR) imaging to define patients with nonischemic cardiomy-
and immunomodulatory activities.2,6 These properties may ben- opathy with absent hyperenhancement and to increase sensitiv-
efit cardiac function not only through local effects of cardiac- ity in detecting structural and functional cardiac responses to
engrafted cells, but also through systemic anti-inflammatory treatment.12
effects. The paracrine anti-inflammatory benefit from stem cell The present study is a phase II-a randomized trial designed
therapy also opens the possibility of a systemic effect via cells to assess the safety, as well as cardiac and systemic effects,
injected intravenously such that direct cardiac delivery may not of intravenously administered MSCs in patients with chronic
be necessary to repair and stimulate the dysfunctional viable nonischemic cardiomyopathy. To our knowledge, this trial
myocardium. Intravenous therapy also presents major logisti- represents the first published experience with intravenously
cal, cost, and safety advantages over direct cardiac delivery. administered stem cells in patients with any type of chronic
 334  Circulation Research  January 20, 2017

Figure 1. Flow of patients

through the trial.
CMR indicates cardiac
magnetic resonance
imaging; eGFR, estimated
glomerular filtration rate;
itMSC, ischemia-tolerant
mesenchymal stem cells.
a
Patient was hospitalized
for worsening heart failure
between time of screening
and randomization.
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cardiomyopathy and the first to test the hypothesis that the inotropic agents within 1 month of randomization and patients with
systemic effects of MSCs can lead to clinical improvements. severe valvular, renal (estimated glomerular filtration rate <30 mL/
min), or liver disease (alanine transaminase or aspartate transaminase
>3× normal, alkaline phosphatase or bilirubin >2× normal).
Methods
This study was a single-blind, placebo-controlled, crossover, multicenter, Study Design
randomized study with blinded end point assessment that enrolled pa- Patients who met eligibility criteria were blinded to treatment al-
tients with nonischemic cardiomyopathy with left ventricular (LV) ejec- location and randomized 1:1 to receive intravenous itMSC therapy
tion fraction (LVEF) ≤40%. The study design and rationale have been or placebo. At 90 days post-initial infusion, the 2 groups received
previously described, and the protocol is available in the Online Data the alternative treatment in the crossover phase, thus, resulting in
Supplement.13 Institutional review board approval was obtained at each each patient receiving 2 infusions (ie, 1 itMSC and 1 placebo) during
study site, and all patients provided written informed consent. the study. For reference, the 2 groups were labeled by the order in
which they received treatment: placebo–itMSC and itMSC–placebo.
Study Patients Additionally, the term control pertains to data from baseline to 90
Full inclusion and exclusion criteria for this study are detailed in the days among the placebo–itMSC group. The itMSC group refers to
trial protocol (Online Data Supplement). Briefly, eligible patients data from baseline to 90 days among the itMSC–placebo group, as
were ambulatory with chronic nonischemic cardiomyopathy with well as data from 90 days to 180 days among the placebo–itMSC
LVEF ≤40% on baseline CMR, were aged ≥18 years, and had New group. A 90-day follow-up was felt a reasonable duration to evaluate
York Heart Association (NYHA) class II/III symptoms. Nonischemic response to study treatment based on (1) prior experiences with ven-
cardiomyopathy was defined by the following: (1) no history of myo- tricular remodeling from existing HF therapeutics14 and (2) clinical
cardial infarction; (2) absent or nonobstructive coronary artery disease practice guidelines supporting delay of primary prevention implant-
on invasive or computed tomography coronary angiography within 3 able-cardioverter defibrillator ≥90 days after initiation of guideline-
years prior to randomization, and (3) no evidence of hyperenhance- directed medical therapy to allow for potential ventricular recovery.14
ment on screening delayed-enhancement CMR. Patients were receiv- The investigational therapy was an intravenous infusion of isch-
ing stable maximally tolerated guideline-directed medical therapy for emia-tolerant human donor allogeneic MSCs dosed at 1.5 million
HFrEF for ≥6 months prior to randomization. Because of the use of cells/kg. All cells expressed CD105, CD73, and CD90 surface mark-
CMR, patients with current or planned implantation of an implantable- ers and were extracted from the bone marrow of a young healthy vol-
cardioverter defibrillator, permanent pacemaker, or cardiac resynchro- unteer. Cells were grown under hypoxic conditions from the moment
nization therapy devices were excluded, as where patients treated with of extraction. For purposes of cryopreservation, cells were suspended
 Butler et al   Mesenchymal Stem Cells for Heart Failure   335

Table 1. Baseline Characteristics of Study Population thawed within the pharmacy of the local study site and suspended in
Lactated Ringer’s solution at a concentration of 1×106 cells/mL. The
All Patients (n=22)* placebo therapy was an intravenous infusion of Lactate Ringer’s solu-
Age, y 47.3 (12.8) tion at a volume of 1 mL/kg.

Male sex 13 (59.1%) Study End Points

Weight, kg 92.50 (19.96) The primary safety end points were assessed at days 30, 60, 90,
120, 150, 180, 270, and 450 post-initial infusion. At each time
Body mass index 32.24 (7.56) point, all-cause mortality, all-cause hospitalization, and adverse
Race events (including severity of event and perceived relationship
to the study products as related or not related) were reported by
 White 15 (68.2%) study investigators. Vital signs, 12-lead electrocardiograms, and
 Black 7 (31.8%) laboratory tests (including complete blood counts, comprehensive
chemistry panels, troponin I, and creatinine kinase) were collected
 Other 0 (0%) at each time point. Twenty-four-hour Holter monitoring was done
NYHA class at baseline at all time points through 270 days post-initial infusion. An inde-
pendent data and safety monitoring board monitored patient safety
 Class II 21 (95.5%) during the trial.
 Class III 1 (4.5%) Multiple secondary efficacy end points were prespecified. CMR
testing was prespecified at baseline, day 90, and day 180 for all
Systolic blood pressure, mm Hg 120 (17.1) patients. CMR end points included change in LVEF, wall motion
Diastolic blood pressure, mm Hg 74 (9.6)
summary score, LV end-systolic volume (LVESV), and LV end-
diastolic volume (LVEDV) from the time of infusion to 90 days
Heart rate, bpm 78 (14.2) post-infusion between itMSC and control groups. Six-minute walk
distance (6MWD), NYHA class, and Kansas City Cardiomyopathy
NT-proBNP, pg/mL 212 (IQR 841)
Questionnaire data were collected at day 30 and day 90 after initial
Troponin I, μg/mL 0.009 (0.003) and second infusions. Protocol-prespecified serum biomarkers (in-
cluding N-terminal pro-B-type natriuretic peptide, troponin I, fibro-
Serum sodium, mmol/L 138 (2)
blast growth factor 23, and vascular endothelial growth factor) and
Serum creatinine, mg/dL 0.96 (0.23) lymphocyte proliferation panels (including levels of CD3, CD4, and
natural killer [NK] cells) were assessed at these same time points.
AST, IU/L 21.86 (8.64)
ALT, IU/L 21.82 (9.57) Statistical Analysis
Because of the exploratory nature of this early-phase study, a sample
Total bilirubin, mg/dL 0.80 (0.40)
size of 23 patients were targeted as appropriate for a phase II-a pri-
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Albumin, g/dL 4.2 (0.36) marily safety trial. Summary data are presented as mean or median as
appropriate, and all analyses used a 2-sided 0.05 significance level.
Medical history
Comparative analyses for the primary safety end points of adverse
 Hypertension 5 (22.7%) and serious adverse events are presented in entirety and also by at-
tributing adverse events to either the placebo or the itMSC therapy
 Diabetes mellitus 3 (13.6%) for the 90-day period after the administration of either intervention.
 Atrial fibrillation 2 (9.1%) To investigate the effects of itMSCs on efficacy end points, the
changes in these parameters during the 2 treatment periods were
 Chronic kidney disease 1 (4.5%) combined. To assess changes in the placebo group, only data from
Baseline cardiac magnetic resonance data those who received placebo first (0–90 days) were used. Those re-
ceiving placebo during the 90- to 180-day period were excluded to
 LVEF, % 31.6 (9.8) avoid any confounding from a possible carryover effect because of
 LVESV, mL 189.6 (114.2) the cell therapy received during the first 90-day period. Improvement
in NYHA classification was analyzed using logistic regression
 LVEDV, mL 264.9 (120.4) with treatment and group assignment (itMSC–placebo versus
Baseline medications
 Loop diuretic 16 (72.7%)
Table 2. Adverse Events During the 90 Days After Each
 ACEI or ARB 22 (100.0%)
Study Intervention (Combined Precrossover and Postcrossover
 Mineralocorticoid receptor antagonist 18 (81.8%) Periods)
 Beta-blocker 22(100.0%) Placebo
Data reported as n (%) or mean (standard deviation) unless otherwise (n=22) itMSC (n=22)
noted. ACEI indicates angiotensin-converting enzyme inhibitor; ALT,
Adverse events 34 35
alanine transaminase; ARB, angiotensin II receptor blocker; AST, aspartate
transaminase; IQR, interquartile range; LVEDV, left ventricular end-diastolic Serious adverse events 0 0
volume; LVEF, left ventricular ejection fraction; LVESV, left ventricular end-
Cell-related adverse events* 0 1
systolic volume; NT-proBNP, N-terminal pro-B-type natriuretic peptide; and
NYHA, New York Heart Association. All-cause hospitalization 0† 0
*N reflects the randomized patients who received any study therapy.
All-cause death 0 0
itMSC indicates ischemia-tolerant mesenchymal stem cells.
in Cryostar CS10 freezing medium (BioLife Solutions, Bothell, WA), *Infusion related (bruising at intravenous infusion site).
frozen in a freezer at a controlled rate, and stored in the vapor phase †There was 1 hospitalization for atrial fibrillation in the itMSC–placebo group
of liquid nitrogen. Within 8 hours prior to patient infusion, cells were that occurred 1 y after placebo infusion.
 336  Circulation Research  January 20, 2017

Table 3. Differences in Change From Baseline in Study End 10 itMSC–placebo patients who completed study treatment
Points Between itMSC and Control Treatment Periods at 90 in 90 days plus 12 placebo–itMSC patients who completed
Days study treatment in 180 days. Thus, the itMSC-treated group
Odds Ratio included 22 patients who completed treatment in 90 days after
Estimated Difference, (95% CI) itMSC infusion.
itMSC Minus Placebo [itMSC vs P Value Baseline characteristics of the population are presented
End Point (95% CI) Placebo] (2-Sided) in Table 1. Mean age was 47.3 years, and over two thirds of
CMR End Points the patients were white. All but 1 patient had NYHA class II
symptoms, and median N-terminal pro-B-type natriuretic pep-
 LV wall motion
0.50 (−1.02 to 2.02) 0.52 tide was 212 pg/mL. Rates of comorbidities were generally
summary score
low, with <25% of patients having hypertension, diabetes mel-
 LVEF, % 0.01 (−1.50 to 1.52) 0.99
litus, atrial fibrillation, and chronic kidney disease. At baseline,
 LVEDV, mL 1.67 (−8.60 to 11.93) 0.75 patients were receiving loop diuretics (72.7%), angiotensin-
 LVESV, mL 0.67 (−7.28 to 8.62) 0.87 converting enzyme inhibitors/angiotensin II receptor blockers
(100.0%), β-blockers (100.0%), and mineralocorticoid recep-
6-min walk test
tor antagonists (81.8%).
 Distance, m 36.47 (5.98–66.97) 0.02
Safety
 Distance
(% change from 15.94 (1.63–30.24) 0.03
Safety data are displayed in Table 2, and the values reflect
baseline) events that occurred in the 90 days after placebo or itMSC
infusion. During these 90-day periods, no patients experi-
Kansas City Cardiomyopathy Questionnaire
enced death, hospitalization, or serious adverse events. Rates
 Functional of adverse events were generally balanced between the 2
5.65 (−0.11 to 11.41) 0.06
status score treatments at 90 days. There was 1 cell-related adverse event
 Clinical related to bruising at the intravenous infusion site. Over the
5.22 (0.70–9.74) 0.02
summary score course of the study, there were no deaths, and there was a total
New York Heart Association Classification of 1 hospitalization (which was for atrial fibrillation that oc-
curred within the itMSC–placebo group 1 year after placebo
 Lower NYHA
functional class
3.67
0.33 infusion). Holter monitoring showed clinically significant ab-
(0.27–50.03) normalities in 1 patient over the course of the study. This pa-
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at 90 days
CMR indicates cardiac magnetic resonance imaging; itMSC, ischemia-
tient, randomized to the placebo–itMSC group, had an event
tolerant mesenchymal stem cells; LV, left ventricular; LVEDV, left ventricular defined as other on day 60 and an episode of ventricular tachy-
end-diastolic volume; LVEF, left ventricular ejection fraction; LVESV, left cardia on day 120. Online Table I displays safety data for the
ventricular end-systolic volume; and NYHA, New York Heart Association. 90 days after original treatment (ie, precrossover period only).

placebo–itMSC) as covariates. All other continuous secondary effi- Cardiac Remodeling

cacy end points related to change from baseline were analyzed using There were no significant differences between itMSC and
linear regression (with treatment, randomization group assignment, placebo treatment periods in any 90-day CMR end point, in-
and the baseline value as covariates) to determine estimated mean, cluding LV wall motion summary score, LVEF, LVEDV, and
the differences compared with placebo, and 95% confidence inter-
LVESV (all P≥0.5; Table 3). In ad hoc exploratory analyses
vals (CI). Given that the data from patients assigned to the placebo–
itMSC group were used in both the itMSC group and the control that examined changes from baseline after initial randomiza-
group, correlations within the same patient were accounted for using tion (ie, first 90 days, precrossover), there were significant re-
generalized estimating equation in all models. As exploratory analy- ductions in LVEDV (estimated mean difference −17.86 mL;
ses, we also compared these end points in subjects during the initial P=0.04) and LVESV (estimated mean difference −16.60 mL;
randomization (0–90 days) period between itMSC and placebo using P=0.02) and increases in LVEF (estimated mean difference
the same models but adjusted only for baseline values for continuous +2.31; P=0.02) within the itMSC group. During this same
variables and Fisher exact test for NYHA classification. Available
case analysis was used to handle missing data.15 Data were analyzed time period, changes in those patients who were receiving
using SAS version 9.3. placebo were not significant (LVEDV, estimated mean differ-
ence −10.56 mL, P=0.22; LVESV, estimated mean difference
Results −8.90 mL, P=0.27; LVEF, estimated mean difference +1.62,
Of the 34 patients screened, 23 were randomized and 22 re- P=0.13). However, the differences in these parameters be-
ceived the study intervention (Figure 1). Patients were ran- tween the 2 treatments during this period were not significant.
domized from 4 sites across the United States between June Health Status and Functional Capacity
2014 and April 2016. Overall, 12 patients randomized to pla- Treatment with itMSCs resulted in statistically significant im-
cebo–itMSC completed the treatment in 90 days and, thus, provements in health status and functional capacity end points
constituted the control treatment. Those receiving placebo (Table 3). When the change from baseline to 90 days was com-
after receiving itMSCs were not included in the control treat- pared after itMSC therapy with the change during the control
ment because there may have been carryover effects from the period, 6MWD was significantly greater by 36.47 m (95%
initial cell therapy period. The itMSC-treated group included CI 5.98–66.97; P=0.02) or 15.9% (Figure 2A). Specifically,
 Butler et al   Mesenchymal Stem Cells for Heart Failure   337
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Figure 2 . Change from baseline to 90 days in 6-minute walk distance (A), KCCQ Clinical Summary Score (B), and KCCG
Functional Status Score (C) for experimental and placebo groups. Pre within the itMSC group represents values prior to itMSC
infusion, whether occurring at day 0 or day 90. Post within the itMSC group represents values measured 90 days after itMSC infusion,
whether occurring at day 90 or day 180. Pre and post within the placebo group represent values measured at day 0 and day 90,
respectively. KCCQ indicates Kansas City Cardiomyopathy Questionnaire.

within the itMSC group, 6MWD increased by an average of 0.27–50.03; P=0.33) of lower NYHA classification at 90 days.
27.40 m (95% CI 0.28–54.52; P=0.05), but it decreased by an Specifically, 90 days after itMSC therapy, NYHA classifica-
average of 10.83 m (95% CI −38.66 to 17.00, P=0.45) among tion was significantly improved compared with that at baseline
control patients. (P≤0.01), whereas there was no significant change within the
Compared with the change from baseline during the con- control group (only 1 out of 12 patients had improvement). In
trol period, itMSC therapy resulted in greater improvements ad hoc analysis restricted only to precrossover data, there were
in Kansas City Cardiomyopathy Questionnaire clinical sum- no significant differences at 90 days for any health status end
mary (+5.22, 95% CI 0.70–9.74; P=0.02) and functional sta- point between the control and itMSC groups (Online Table II).
tus scores (+5.65, 95% CI −0.11 to 11.41; P=0.06; Figure 2B
and 2C). In addition, compared with the change from base- Biomarker
line during the control period, itMSC therapy was associated Within the itMSC group, compared with baseline, there were
with a nonsignificant greater odds (odds ratio 3.67, 95% CI significant changes in immunomodulatory markers at 30 days
 338  Circulation Research  January 20, 2017

Table 4. Baseline and Change From Baseline With itMSC Discussion

Therapy This phase II-a trial was designed to evaluate the safety and
P Value* preliminary efficacy of intravenous administration of itM-
Biomarker N Mean (SD) (2-Sided) SCs in a relatively homogenous population of patients with
NT-proBNP, pg/mL nonischemic cardiomyopathy. Overall, this study found a
single dose of intravenous itMSCs to be safe, to be well-tol-
 Baseline 22 806.27 (1387.85) …
erated, and to provide clinically relevant signals for efficacy.
 Change from baseline to 30 d 22 −102.00 (528.30) 0.375 itMSC therapy resulted in improvements in patient health
 Change from baseline to 90 d 20 768.25 (2945.53) 0.258 status. Although study sample size was small, concordance in
Troponin I, ng/mL
improvement in Kansas City Cardiomyopathy Questionnaire
scores and 6MWD decreases the likelihood of a chance finding.
 Baseline 22 0.009 (0.003) … Both of these metrics are routinely used in the early- and late-
 Change from baseline to 30 d 22 −0.001 (0.004) 0.083 phase HFrEF trials, and baseline values have typically shown
 Change from baseline to 90 d 20 0.000 (0.006) 1.00 good correlation with survival and quality of life.16–18 6MWD, in
particular, is strongly associated with peak exercise oxygen con-
VEGF, pg/mL
sumption, which is among the most powerful and objective pre-
 Baseline 22 119.41 (106.26) … dictors of functional status and short-term event-free survival.16
 Change from baseline to 30 d 19 −17.05 (59.13) 0.225 Most prior stem cell studies have been designed under the
 Change from baseline to 90 d 11 4.55 (40.59) 0.718
assumption that the mechanism of benefit of stem cell therapy
accrues only from activities derived from cells engrafted in
FGF 23, RU/mL
the dysfunctional myocardium, whether these activities reflect
 Baseline 21 187.57 (248.50) … cells differentiating into cardiac myocytes, stimulating resident
 Change from baseline to 30 d 19 −7.47 (110.57) 0.772 cardiac stem cells to expand and form more functioning myo-
cytes, or reflect broad paracrine and systemic activities that al-
 Change from baseline to 90 d 11 −58.55 (281.58) 0.506
low, for example, favorable cardiac remodeling, enhancement
CD3 cells (total T-cells), % of angiogenesis, and decreased apoptosis.2,5 Thus, studies have
 Baseline 22 73.31 (7.66) … focused on delivery methods that maximize myocardial en-
 Change from baseline to 30 d 22 1.59 (3.50) 0.045 graftment, including intracoronary, transendocardial, or intra-
myocardial routes. However, such strategies involve catheter
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 Change from baseline to 90 d 20 0.65 (6.81) 0.674

or surgical techniques and suffer major practical limitations, in
CD4 cells (helper T cells), % that a single injection of cells is unlikely to be curative and that
 Baseline 22 49.45 (7.28) … multiple invasive cell administrations would likely be required
over the course of the condition.
 Change from baseline to 30 d 22 2.09 (3.18) 0.006
Because intravenous cell delivery results in low rates of
 Change from baseline to 90 d 20 1.30 (6.73) 0.399 cardiac engraftment, to our knowledge, there has been only
NK cells, % a single clinical trial assessing the efficacy of intravenous
 Baseline 22 10.45 (4.92) … administration, with that study limited to patients with acute
myocardial infarction.19 Interestingly, that study still oper-
 Change from baseline to 30 d 22 −1.32 (2.57) 0.025
ated under the belief that MSCs need robust engraftment in
 Change from baseline to 90 d 20 −1.05 (4.32) 0.090 ischemic myocardium to show benefit, and no data on po-
FGF indicates fibroblast growth factor; itMSC, ischemia-tolerant tential systemic immunomodulatory effects of cell therapy
mesenchymal stem cells; NK, natural killer; NT-proBNP, N-terminal pro-B-type were obtained.
natriuretic peptide; and VEGF, vascular endothelial growth factor. In the present study, clinical improvements with intravenous
*P values represent change from baseline to the indicated time point (ie, 30
d or 90 d).
delivery of itMSCs were associated with statistically significant
alterations in the numbers of several circulating inflammatory
cells, suggesting that intravenous delivery of MSCs does convey
systemic signals, in particular, a modulatory effect on the inflam-
post-infusion, including an increase in the percentage of CD3 matory system. Importantly, we found a statistically significant
(P=0.045) and CD4 cells (P=0.006) and a decrease in absolute and inverse relationship between change in NK cell number and
percent NK cells (P=0.025 and P=0.006, respectively; Table 4). LVEF, whereby the degree of NK cell reduction correlated with
Changes from baseline in other biomarkers, including N-terminal the magnitude of improvement in LVEF at 90 days (Figure 3).
pro-B-type natriuretic peptide and troponin I, during the itMSC Two preclinical studies in murine models of acute myocar-
phase were nonsignificant at 30 and 90 days post-infusion. dial infarction and ischemic cardiomyopathy in the laboratory
In ad hoc analysis, there was an inverse relationship be- of 2 authors (S.E.E. and M.J.L., manuscripts submitted and in
tween the degree of reduction in NK cells from baseline to 90 review) support the concept that an MSC-induced decrease
days and the magnitude of improvement in LVEF (R2=0.31, in the NK cell population mechanistically contributes to the
P=0.01; Figure 3) but not with LVEDV (R2=0.06, P=0.29) or therapeutic effect of itMSCs. The studies demonstrated that
LVESV (R2=0.12, P=0.14). intravenous injection of the same human itMSCs used in this
 Butler et al   Mesenchymal Stem Cells for Heart Failure   339

that significant improvements in LVEDV (P=0.04), LVESV

(P=0.05), and LVEF (P=0.02) by CMR occurred over the
first 3 months of the trial (ie, precrossover) within the itMSC
group, but not in the control group. Because these findings
were identified as part of an ad hoc analysis, they must be
considered exploratory only. Future studies will be necessary
to conclusively determine whether intravenous administration
of itMSCs enhances cardiac function in HF patients.
Novel features of the present study design deserve men-
tion. The wide heterogeneity of the HFrEF population has
been increasingly recognized as a challenge in successful
therapy development.20 HFrEF is a clinical syndrome and not
a specific disease, representing various combinations of car-
diac and noncardiac abnormalities. Accordingly, it has been
postulated that different subgroups may respond differently to
various investigational therapies and, thus, dilute efficacy sig-
nals within a given clinical trial.21,22 The present study aimed
to isolate a specific homogenous patient subset using a rigor-
ous definition of nonischemic cardiomyopathy through a com-
bination of clinical, angiographic, and CMR parameters. The
goal of such criteria was to maximize presence of dysfunc-
tional viable myocardium and, thus, the chances of reverse
remodeling.8,11 To our knowledge, no prior HF study has so
Figure 3. Change in NK cells over time by study intervention specifically defined its patient population, and this trial may
and relationship between itMSC-induced reductions in NK represent a model for future early-phase HF trials.
cells and change in left ventricular ejection fraction. itMSC Limitations of the present study must be acknowledged.
indicates ischemia-tolerant mesenchymal stem cells; LVEF, left
ventricular ejection fraction; MSC, mesenchymal stem cells; and The sample size for this study was small and, thus, precludes
NK, natural killer. definitive conclusions regarding the efficacy of itMSCs in this
population. However, such a sample size is typical of an early-
Downloaded from http://ahajournals.org by on November 15, 2024

trial significantly improves LV function and adverse remodel- phase study with the primary goal of assessing safety. Like all
ing, and most importantly, that a major mechanism responsible ad hoc analyses, the analysis correlating NK cell reduction
for these beneficial effects is through their immunomodulatory with change in LVEF was exploratory and requires prospec-
effects. Notably, it was demonstrated in the acute myocardial tive validation. Moreover, inherent to a crossover study de-
infarction model that the MSC-induced decrease in NK cells sign, we cannot definitively determine if any adverse event
was causally related to the beneficial LV functional outcomes occurring >90 days after itMSC infusion within the itMSC–
seen with MSC administration. In aggregate with the current placebo group represents placebo effect, delayed consequence
study results, these data support the importance of immuno- of cell therapy, or random chance. Finally, this study used
modulatory and anti-inflammatory mechanisms of itMSCs and MSCs grown under chronic hypoxia because prior in vitro
suggest that suppression of NK cells in nonischemic HFrEF studies demonstrated that such cells have greater activity to-
facilitates ventricular recovery, thereby, supporting NK cells as ward tissue healing than cells grown under normoxic condi-
a potential target for therapy.2 Moreover, in the present study, tions. Although there were signals of efficacy in this study,
the finding of an itMSC-induced 30-day reduction in NK cell the trial was not designed to test whether the administration of
number that attenuated with time from itMSC infusion sup- MSCs grown under chronic hypoxia versus those grown under
ports further study of serial repeated administrations of MSCs normoxic conditions accounted for these positive signals.
to examine the hypothesis that sustained reductions in NK cells In conclusion, intravenous administration of itMSCs in pa-
further enhance potential favorable cardiac effects. tients with nonischemic cardiomyopathy with reduced LVEF
It is unclear why itMSC therapy resulted in significant was safe and well-tolerated. At 90 days post-infusion, compared
improvements in health status and functional capacity pa- with control, a single dose of study therapy consistently im-
rameters compared with placebo without an effect on cardiac proved multiple measurements of patient health status, but was
function or N-terminal pro-B-type natriuretic peptide. It is not associated with significant cardiac structural or functional
plausible that this discordance could be explained by noncar- improvements on CMR compared with placebo. Ad hoc explor-
diac systemic effects of itMSC therapy, perhaps mediated via atory analysis of data limited to changes from baseline after ini-
anti-inflammatory properties. One could speculate that such tial study therapy found significant reductions in LVEDV and
systemic effects could influence processes such as skeletal LVESV and increases in LVEF within the itMSC group. These
muscle performance and oxygen delivery, thereby, improving changes were not observed in those patients who were receiving
functional status independent of the heart. Importantly, how- placebo. Moreover, of mechanistic relevance, intravenously ad-
ever, this study was not powered to definitively evaluate car- ministered itMSCs caused systemic immunomodulatory effects
diac-specific effects of itMSC. In this regard, it is of interest that correlated with improvement in LVEF. Further studies are
 340  Circulation Research  January 20, 2017

needed to explore the efficacy of serial dosing of intravenously 7. Vertelov G, Kharazi L, Muralidhar MG, Sanati G, Tankovich T, Kharazi
A. High targeted migration of human mesenchymal stem cells grown in
administered itMSCs to produce more sustained immunomodu-
hypoxia is associated with enhanced activation of RhoA. Stem Cell Res
latory effects and, thereby, perhaps facilitate improvement in Ther. 2013;4:5. doi: 10.1186/scrt153.
LV structure and function and clinical outcomes. 8. Bayeva M, Sawicki KT, Butler J, Gheorghiade M, Ardehali H. Molecular
and cellular basis of viable dysfunctional myocardium. Circ Heart Fail.
2014;7:680–691. doi: 10.1161/CIRCHEARTFAILURE.113.000912.
Acknowledgments 9. Cleland JG, Pennell DJ, Ray SG, Coats AJ, Macfarlane PW, Murray GD,
We thank Ms Shelah Mendoza for editorial assistance in the prepara- Mule JD, Vered Z, Lahiri A; Carvedilol hibernating reversible ischaemia
tion of this article. We thank Ms Samantha M. Noreen for statistical trial: marker of success investigators. Myocardial viability as a determinant
support. The sponsor participated in the design, conduct, and man- of the ejection fraction response to carvedilol in patients with heart failure
agement of the study; the collection and analysis of the data; and the (CHRISTMAS trial): randomised controlled trial. Lancet. 2003;362:14–21.
preparation, review, and approval of this article. The sponsor had no 10. Barsheshet A, Goldenberg I, Moss AJ, Eldar M, Huang DT, McNitt S, Klein
role in the interpretation of the data. HU, Hall WJ, Brown MW, Goldberger JJ, Goldstein RE, Schuger C, Zareba
W, Daubert JP. Response to preventive cardiac resynchronization therapy in
patients with ischaemic and nonischaemic cardiomyopathy in MADIT-CRT.
Sources of Funding Eur Heart J. 2011;32:1622–1630. doi: 10.1093/eurheartj/ehq407.
This study was funded by CardioCell LLC (San Diego, CA). 11. Wilcox JE, Fonarow GC, Ardehali H, Bonow RO, Butler J, Sauer AJ,
Epstein SE, Khan SS, Kim RJ, Sabbah HN, Díez J, Gheorghiade M.
“Targeting the heart” in heart failure: myocardial recovery in heart failure
Disclosures with reduced ejection fraction. JACC Heart Fail. 2015;3:661–669. doi:
Dr Butler reports research support from the National Institutes of 10.1016/j.jchf.2015.04.011.
Health and European Union and is a consultant to Amgen, Bayer, 12. Kim RJ, Wu E, Rafael A, Chen EL, Parker MA, Simonetti O, Klocke FJ,
CardioCell, Novartis, Boehringer-Ingelheim, Trevena, Relypsa, Bonow RO, Judd RM. The use of contrast-enhanced magnetic resonance
Z Pharma, Pharmain, and Zensun. Dr Epstein is a consultant to imaging to identify reversible myocardial dysfunction. N Engl J Med.
and holds equity interest in CardioCell. Dr Quyyumi has research 2000;343:1445–1453. doi: 10.1056/NEJM200011163432003.
funding from the National Institutes of Health and American 13. Greene SJ, Epstein SE, Kim RJ, Quyyumi AA, Cole RT, Anderson AS,
Heart Association and is a consultant and holds equity interest in Wilcox JE, Skopicki HA, Sikora S, Verkh L, Tankovich NI, Gheorghiade
M, Butler J. Rationale and design of a randomized controlled trial of al-
CardioCell Inc. Drs Sikora is an employee of CardioCell LLC.
logeneic mesenchymal stem cells in patients with nonischemic cardiomy-
Dr Tankovich is employed by Stemedica Cell Technologies. Dr opathy. J Cardiovasc Med (Hagerstown). 2015. [Epub ahead of print].
Gheorghiade reports relationships with Abbott Laboratories, 14. Yancy CW, Jessup M, Bozkurt B, et al. 2013 ACCF/AHA guideline for
Astellas, AstraZeneca, Bayer Schering Pharmarama AG, CardioCell the management of heart failure: executive summary: a report of the
LLC, Cardiorentis Ltd, Corthera, Cytokinetics, CytoPherx, Inc., American College of Cardiology Foundation/American Heart Association
DebioPharm S.A., Errekappa Terapeutici, GlaxoSmithKline, Task Force on practice guidelines. Circulation. 2013;128:1810–1852. doi:
Ikaria, Intersection Medical, Inc., Johnson & Johnson, Medtronic, 10.1161/CIR.0b013e31829e8807.
Merck, Novartis Pharma AG, Ono Pharmaceuticals USA, Otsuka 15. Little RJA, Rubin DB. Statistical Analysis With Missing Data, 2nd ed.
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Pharmaceuticals, Palatin Technologies, PeriCor Therapeutics, Hoboken, NJ: John Wiley & Sons, Inc.; 2002.
Protein Design Laboratories, Sanofi-Aventis, Sigma Tau, Solvay 16. Cahalin LP, Mathier MA, Semigran MJ, Dec GW, DiSalvo TG. The six-
Pharmaceuticals, Sticares InterACT, Takeda Pharmaceuticals, and minute walk test predicts peak oxygen uptake and survival in patients with
Trevena Therapeutics. The other authors report no conflicts. advanced heart failure. Chest. 1996;110:325–332.
17. Bittner V, Weiner DH, Yusuf S, Rogers WJ, McIntyre KM, Bangdiwala SI,
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