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� The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. 1513
Journals of Gerontology: Medical Sciences
cite as: J Gerontol A Biol Sci Med Sci, 2017, Vol. 72, No. 11, 1513–1521
doi:10.1093/gerona/glx137
Advance Access publication July 17, 2017This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
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Translational Article
Stem Cell Transplantation for Frailty
Research Article
Allogeneic Mesenchymal Stem Cells Ameliorate Aging
Frailty: A Phase II Randomized, Double-Blind, Placebo-
Controlled Clinical Trial
Bryon A. Tompkins, MD,
1,2
Darcy L. DiFede, RN, BSN,
1,5
Aisha Khan, Msc, MBA,
1
Ana Marie Landin, PhD,
1
Ivonne Hernandez Schulman MD,
1,3
Marietsy V. Pujol, MBA,
1
Alan W. Heldman, MD,
1
Roberto Miki, MD,
3
Pascal J. Goldschmidt-Clermont, MD,
3
Bradley J. Goldstein, MD,
1,2
Muzammil Mushtaq, MD,
3
Silvina Levis-Dusseau, MD,
3
John J. Byrnes, MD,
3
Maureen Lowery MD,
3
Makoto Natsumeda, MD,
1
Cindy Delgado, MA, CCRC,
1
Russell Saltzman, BS.Ed,
1
Mayra Vidro-Casiano, MPH,
1
Moisaniel Da Fonseca, AA,
1
Samuel Golpanian, MD,
2
Courtney Premer, PhD,
1
Audrey Medina, BSc,
5
Krystalenia Valasaki, MSc,
1
Victoria Florea, MD,
1
Erica Anderson, MA,
4
Jill El-Khorazaty, MS,
4
Adam Mendizabal, PhD,
4
Geoff Green, BA, MBA,
5
Anthony A. Oliva, PhD,
5
and Joshua M. Hare, MD
1,3
1
The Interdisciplinary Stem Cell Institute,
2
Department of Surgery, and
3
Department of Medicine, University of Miami Miller School of
Medicine, Florida.
4
EMMES Corporation, Rockville, Maryland.
5
Longeveron LLC, Miami, Florida.
Address correspondence to: Joshua M. Hare, MD, Louis Lemberg Professor of Medicine, Director, Interdisciplinary Stem Cell Institute, University
of Miami Miller School of Medicine, Biomedical Research Building, 1501 N.W. 10
th
Ave., Room 824, P.O. Box 016960 (R125), Miami, FL 33101. E-mail:
jhare@med.miami.edu
Received: April 14, 2017; Editorial Decision Date: June 15, 2017
Decision Editor: Anne Newman, MD, MPH
Abstract
Background: Aging frailty, characterized by decreased physical and immunological functioning, is associated with stem cell depletion. Human
allogeneic mesenchymal stem cells (allo-hMSCs) exert immunomodulatory effects and promote tissue repair.
Methods: This is a randomized, double-blinded, dose-finding study of intravenous allo-hMSCs (100 or 200-million [M]) vs placebo delivered
to patients (n = 30, mean age 75.5 ± 7.3) with frailty. The primary endpoint was incidence of treatment-emergent serious adverse events (TE-
SAEs) at 1-month postinfusion. Secondary endpoints included physical performance, patient-reported outcomes, and immune markers of
frailty measured at 6 months postinfusion.
Results: No therapy-related TE-SAEs occurred at 1 month. Physical performance improved preferentially in the 100M-group; immunologic
improvement occurred in both the 100M- and 200M-groups. The 6-minute walk test, short physical performance exam, and forced expiratory
volume in 1 second improved in the 100M-group (p = .01), not in the 200M- or placebo groups. The female sexual quality of life questionnaire
improved in the 100M-group (p = .03). Serum TNF-α levels decreased in the 100M-group (p = .03). B cell intracellular TNF-α improved in
both the 100M- (p < .0001) and 200M-groups (p = .002) as well as between groups compared to placebo (p = .003 and p = .039, respectively).
Early and late activated T-cells were also reduced by MSC therapy.
Translational
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Conclusion: Intravenous allo-hMSCs were safe in individuals with aging frailty. Treated groups had remarkable improvements in physical
performance measures and inflammatory biomarkers, both of which characterize the frailty syndrome. Given the excellent safety and efficacy
profiles demonstrated in this study, larger clinical trials are warranted to establish the efficacy of hMSCs in this multisystem disorder.
Clinical Trial Registration: www.clinicaltrials.gov: CRATUS (#NCT02065245).
Keywords: Immunomodulation, Tumor necrosis factor-α, Regenerative medicine
There is increasing recognition of the health burden of frailty, a syn-
drome that increases in incidence with aging. Frailty confers an increased
vulnerability to adverse health outcomes and mortality in response to
stressors (1 ,2). Of note, the frailty syndrome is driven mostly by bio-
logical aging processes that include inflammation and stem cell dysfunc-
tion, as opposed to chronological aging (2–5). Early intervention may
improve quality of life, reduce hospitalizations, and nursing home costs
(6,7). Therefore, it is increasingly important to recognize the clinical
onset of frailty, and to develop effective therapeutic strategies.
There are two main models used to define frailty (7): The defi-
cit and the physical phenotype model. The deficit model accounts
for a person’s geriatric syndromes, diseases, psychosocial, physical,
and cognitive impairments, and combines them to create a “Frailty
Index” (8). The physical phenotype model consists of the identifi-
cation of at least three factors: weight loss, exhaustion, weakness,
slowness, and decreased physical activity, which together comprise
an underlying state of multisystem dysregulation (9,10). Despite the
use of different criteria for evaluating frailty, both models show evi-
dence that the prevalence of the syndrome increases with age and is
higher among women (9.6%) than men (5.2%) (11). In a study of
over 44,000 community-dwelling elderly adults, the overall preva-
lence of frailty was found to be 10.7% (11).
Currently, several multimodal interventions are employed to man-
age frailty, namely resistance/aerobic exercise, caloric support, vita-
min D, and optimization of polypharmacy (7 ). However, there are
no specific medical or biologic treatments that ameliorate or reverse
frailty (12, 13). Stem cell depletion is a key mechanism postulated to
contribute to frailty (14–16). In this regard, we recently conducted a
phase I open label study of human allogeneic mesenchymal stem cells
(allo-hMSCs) intravenously infused for frailty, which showed that the
cells could be safely administered, improved measures of functional
capacity, and reduced inflammation (17). Therefore, we conducted the
current phase II double-blinded and placebo-controlled study in order
to test the hypothesis that exogenous allo-hMSCs could reverse signs
and symptoms of frailty in older individuals. Similar approaches have
been shown to exert beneficial effects on the cardiovascular system,
with functional improvements on various types of heart disease (18–
20), endothelial function (21), and systemic inflammation (22). Given
their pleiotropic mechanisms of action, which include antifibrotic,
anti-inflammatory, proangiogenic properties (23), and their ability to
stimulate endogenous progenitor cells (21, 24), we hypothesize that
their use may offer a novel treatment strategy in frail patients.
Methods
The AllogeneiC Human Mesenchymal Stem Cells in Patients
with Aging FRAilTy via IntravenoUS Delivery (CRATUS) study
(#NCT02065245) is a phase II, randomized, double-blinded, pla-
cebo-controlled study of allo-hMSCs delivered intravenously (IV)
in frail individuals to test the safety and efficacy of allo-hMSCs in
reducing markers of inflammation and improving markers of physi-
cal and mental functioning and quality of life (15,25).
Study Design, Stem Cell Procurement and
Randomization
The study design and phase I of the CRATUS study have been
recently published (17,26). Screening and patient randomization
are outlined in Figure 1 (26), and available in the Supplementary
Material.
Patient Inclusion Criteria and Timeline
The inclusion criteria were as follows: (i) Patients were pro-
vided written informed consent, (ii) patients were aged ≥60 and
≤95 years at the time of signing the Informed Consent Form, and
(iii) they showed the signs of frailty based on physician assessment,
apart from a concomitant condition, by a score between 4 and 7
as denoted by the Canadian Study on Health Aging (25, 27,28).
Major exclusion criteria and a detailed timeline have been pub-
lished (26) .
Study Endpoints
The primary endpoint was the safety of allo-hMSCs at 1 month,
assessed by treatment emergent-serious adverse events (TE-SAE).
TE-SAEs were defined by the following: death, nonfatal pulmonary
embolism, stroke, hospitalization for worsening dyspnea, and clini-
cally significant laboratory abnormalities.
The secondary endpoints assessed the efficacy of the therapy.
Efficacy was demonstrated by differences in the rate of change of
frailty markers as defined by: reduced activity (Community Healthy
Activities Model Program for Seniors (CHAMPS) questionnaire),
slowing of mobility (6-minute walk test (6MWT), 4-m gait speed test
(4MGST), and the short physical performance battery (SPPB) score,
comprised of balance tests, gait speed tests, and chair stand tests),
weight loss, diminished hand grip strength (dynamometry), exhaus-
tion-multidimensional fatigue inventory (MFI), quality of life assess-
ments (Sexual Quality of Life-Female (SQOL-F) and International
Index of Erectile Dysfunction (IIEF) Questionnaires), dobutamine-
induced ejection fraction (EF) via echocardiography, C-reactive pro-
tein (CRP), IL-6, D-dimer, complete blood cell count (CBC) with
differential, and TNF-α.
Immune Monitoring
Immune biomarkers were measured at baseline and 6 months as
described previously (17) and in the Supplementary Material.
Statistical Analysis
No formal statistical justification was performed to determine sam-
ple size for this study. Sample size was determined to be appro-
priate for an early phase study to assess safety in this population.
Due to the early phase nature of this study, no adjustments were
made for multiple analyses (26). Statistical analysis was completed
by statisticians at the Emmes Corporation and is available in the
Supplementary Material.
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Figure 1. Study flow chart. Patient screening, follow-up, and randomization in a 1:1:1 fashion to either the 100M-group, 200M-group, or placebo. M = Million.
Results
Patient Population
Table 1 shows the baseline characteristics of the enrolled patients.
Sixty percent of the patients were White males and the mean age was
75.5 ± 7.3 years.
Safety
No TE-SAEs occurred in any of the three groups in the first 30 days.
Similarly, there were no cumulative treatment-related SAEs in either
group throughout the duration of the study (Table 2). None of the
patients showed any signs of adverse cardiopulmonary reaction
following the intravenous infusion. There were no clinically signifi-
cant changes in basic hematologic and chemistry laboratory tests
throughout the duration of the study.
Long-term Adverse Events
One patient in the 200M-group died of an unrelated event prior
to the 12-month follow-up. Additionally, one patient in the placebo
had an unrelated stroke 307 days postinfusion. The proportion of
patients with adverse events at 12 months did not differ between
groups at the 6- and 12-month time points (p = .300 and p = .141,
respectively).
Hospitalization
There were four patients who required hospitalization within the
12-month follow-up. Two of the hospitalizations were reported in
one patient in the 100M-group, both of which were moderate in
severity; however, none of the hospitalizations were secondary to the
procedure. No patients in the 200M-group were hospitalized. The
remaining three patients belonged to the placebo group; one patient
had two moderate hospitalizations and one severe, another had a
hospitalization that was moderate in severity, and another had one
severe hospitalization. None of the hospitalizations were related to
the procedure.
Functional Status, Quality of Life, and Pulmonary
Function
Quality of life and functional status were monitored throughout the
study. These outcomes preferentially improved in patients randomized
to receive 100M allo-hMSCs. The 6MWT increased in the 100M-group
from baseline to 6 months (345.9 ± 103.4 to 410.7 ± 155.4 m, p = .011;
Figure 2A). There was no significant change at 6 months (p = .263) in
either the 200M-group or placebo (p = .112). The 4MGST showed no
significant differences among groups (p = .659) at 6 months. Consistent
with the improvement in 6MWT, the SPPB total score was significantly
improved in the 100M-group from baseline to 6 months (median
10.5, IQR 9.0, 12.0 to 12.0, IQR 11.0, 12.0; p = .031; Figure 2B).
However, there were no significant differences in the 200M-group
(p = .812) or placebo (p = .875). The CHAMPS-questionnaire showed
a reduced total caloric expenditure per week at moderate intensity in
the 200M-group from baseline to 6 months (median 5,118.8, IQR
1,470.0, 1,4542 to 1,509.4, IQR 472.5, 6,090.0; p = .008) and pla-
cebo (median 3,386.3, IQR 1,286.3, 4,042.0 to 2,021.3, IQR 682.5,
3,150.0; p = .039; Figure 2C). Conversely, there was no significant
reduction in the 100M-group at 6 months (p = .641). There were no
differences between groups in weight loss (p = .7599), MFI, which
assessed mental fatigue (p = .548), and handgrip strength as assessed
via the average of dominant hand scores (p = .676). Ejection fraction,
assessed by dobutamine stress echo, remained stable throughout the
study in all patients. FEV1 improved in the 100M-group from base-
line to 6 months (2.5 ± 0.66 to 2.6 ± 0.77 L/min, p = .025) without
significant changes in the 200M-group (p = .259) or placebo (p = .883;
Figure 2D).
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Immune Biomarkers
Immunotolerability was assessed using a calculated PRA (cPRA)
measured at baseline and 6 months postinfusion on each patient.
Three patients had a mild/moderate increase in donor specific anti-
bodies (one mild in the 100M- and two moderate in the 200M-group).
There were two other patients in the 200M-group that had a mild/
moderate increase in cPRA but were not donor-specific reactions
(Table 3). There were no clinically significant immune reactions
reported. Both the 100M and 200M doses were effective in modu-
lating immune parameters whereas placebo was not. Reduction
in the early-activation CD69 cells was noted in the 200M-group
(27.0 ± 4.30 to 16.4 ± 7.25%, p = .004; Figure 3A) at 6 months.
There were no reductions in the 100M-group (p = .269) or placebo
(p = .0797; Figure 3A). There was a reduction in the late-activation
CD25 cells in both the 100M-group (12.6 ± 6.87 to 6.9 ± 3.30%,
p = .007) and 200M-groups (12.0 ± 6.68 to 8.0 ± 4.64%, p = .048)
from baseline to 6 months. No significant reduction was noted in the
placebo (p = .119; Figure 3B). The CD8 T-cell marker decreased signif-
icantly in the 200M-group from baseline to 6 months (28.7 ± 15.04
to 19.9 ± 10.03%, p = .022; Figure 3C). This is a crucial finding
as aging is marked by an expansion of CD8 cells (29). There were
no significant changes in the 100M-group (p = .978) or placebo at
6 months (p = .0797; Figure 3C). There were no significant changes
in CD4 cells in the 200M-group (p = .052), 100M-group (p = .135),
or placebo (p = .540). The CD4/CD8 ratio appropriately increased
in the 200M-group at 6 months (1.2 ± 1.05 to 1.9 ± 1.17, p = .014),
however there were no changes in the 100M-group (p = .609) or
placebo (p = .104; Figure 3D).
Serum TNF-α decreased in the 100M-group at 6 months (median
3.2, IQR 2.8, 3.8 to 1.2, IQR 1.0, 2.8, p = .031), whereas it did
not significantly change in the 200M-group (p = .129) or placebo
(p = .094; Figure 3E). Similarly, B cell intracellular TNF-α signifi-
cantly decreased in both the 100M- and 200M-groups (17.3 ± 1.8 to
7.0 ± 1.0, p < .0001, and 17.1 ± 2.0 to 8.4 ± 1.1, p = .001, respectively;
Figure 3F) with no improvement in placebo at 6 months (p = .69).
The reductions in both the 100M- and 200M-groups were significant
compared to placebo (p < .00001 and p = .00002; Figure 3F). Finally,
there were no significant changes noted in IL-6, CRP, D-dimer, CBC,
or fibrinogen at 6 months in any group (data not shown).
Sexual Quality of Life
Among female patients, the SQOL-F exhibited a remarkable increase
in the 100M-group at 6 months (59.8 ± 15.3 to 76.0 ± 12.9, p = .035),
but no changes were observed in the 200M-group (p = .882) or pla-
cebo (p = .941; Figure 4). Conversely, there were no differences among
male participants in the IIEF from baseline to 6 months (p = .666).
Discussion
The CRATUS trial is a randomized, double-blind, placebo-con-
trolled evaluation of allo-hMCSs to treat the signs and symptoms of
Table 1. Baseline Characteristics
Treatment Group
Total (N = 30) N (%)Characteristics Allo-100M (N = 10) N (%) Allo-200M (N = 10) N (%) Placebo (N = 10) N (%)
Gender
Male 6 (60%) 6 (60%) 6 (60%) 18 (60%)
Female 4 (40%) 4 (40%) 4 (40%) 12 (40%)
Ethnicity
Hispanic or Latino 1 (10%) 1 (10%) 2 (20%) 4 (13%)
Not Hispanic or Latino 9 (90%) 9 (90%) 8 (80%) 26 (87%)
Race
American Indian/Alaskan Native0 (0%) 1 (10%) 0 (0%) 1 (3%)
White American 10 (100%) 9 (90%) 10 (100%) 29 (97%)
Age at infusion (years) 75.0 ± 7.4 76.3 ± 8.4 75.3 ± 6.8 75.5 ± 7.3
Infusion status
Yes 10 (100%) 10 (100%) 10 (100%) 30 (100%)
No 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Unknown 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Canadian Clinical Frailty Score
4 5 (50%) 7 (70%) 5 (50%) 17 (57%)
5 3 (30%) 1 (10%) 5 (50%) 9 (30%)
6 2 (20%) 2 (20%) 0 (0%) 4 (13%)
7 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Mini-mental state examination 29.3 ± 0.8 28.5 ± 1.1 29.5 ± 1.0 29.1 ± 1.0
Hemoglobin level (g/dL) 14.1 ± 1.2 13.5 ± 1.3 14.3 ± 1.2 14.0 ± 1.3
WBC count (cells/mm
3
) 7,160 ± 2,438 6,600 ± 1,304 7,070 ± 2,215 6,943 ± 1,989
Platelet count (cells/mm
3
) 207,000 ± 64,389 194,500 ± 37,936 194,500 ± 57,880 198,667 ± 52,999
AST (U/L) 24.5 ± 7.6 20.7 ± 3.6 29.3 ± 11.1 24.8 ± 8.5
ALT (U/L) 23.0 ± 16.2 16.5 ± 6.0 31.9 ± 15.6 23.8 ± 14.5
Six-min walk test (m) 345.9 ± 103.4 390.6 ± 148.9 385.8 ± 83.1 374.1 ± 112.9
FEV1 (L) 2.5 ± 0.7 2.3 ± 0.9 2.3 ± 0.5 2.4 ± 0.7
FEV1 (percent predicted) 90.6 ± 10.4 86.9 ± 25.4 87.9 ± 15.2 88.5 ± 17.6
Tumor necrosis factor-α (pg/mL) 3.2 (2.8, 3.8) 3.2 (2.6, 3.4) 2.4 (1.1, 3.1) 3.1 (2.1, 3.4)
Note: Values are mean ± SD, N (%), or median (interquartile range [IQR]). FEV1 (Liters) = Forced Expiratory Volume in one second. Hemoglobin (grams/
deciliter). WBC (cells/millimeters = White blood cells. AST (U/L) = Aspartate Aminotransferase (units/liter). ALT = Alanine Aminotransferase. Six-min walk test
distance (m, meters). Tumor necrosis factor-α (pg/mL, picogram/milliliter).
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Table 2. Safety Summary
System Organ Class
MedDRA Preferred
Term
Treatment Group
Allo-100M (N = 10) Allo-200M (N = 10) Placebo (N = 10) Total (N = 30)
Events PatientsEvents PatientsEvents PatientsEvents Patients
N (%) N (%) N (%) N (%) N (%) N (%) N (%) N (%)
General disorders and administration site conditions
Death 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Hepatobiliary disordersCholecystitis 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Infections and infestations
Gastroenteritis 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Musculoskeletal and connective tissue disorders
Flank pain 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Spinal column stenosis 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Spondylolisthesis 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Neoplasms benign,
malignant and
unspecified (incl cysts
and polyps)
Glioblastoma 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Renal and urinary
disorders
Ureteric stenosis 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Vascular disorders Aneurysm 1 (16%) 1 (10%) 1 (10%) 1 (3%)
Hypotension 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Total 2 (100%)1 (10%)2 (100%)2 (20%)6 (100%)4 (40%)10 (100%)7 (23%)
Note: There were no TE-SAEs at 1 month or SAEs in either group. One patient died in the 200M-group unrelated to the treatment. There were seven hospitaliza-
tions, two in the 100M-group, and five in the placebo. None were related to the procedure. Details of the hospitalizations are located in the supplementary material.
SAE = Serious adverse events; TE-SAE = Treatment-emergent serious adverse events.
frailty. The results support the safety and feasibility of administering
allo-hMSCs in this population. With regard to efficacy, there was a
preferential effect towards improvement of functional capacity and
patient reported outcome measures in patients receiving lower dose
MSCs, although immunologic bioactivity was evident with both
doses. Together, these findings suggest that allo-hMSCs may be an
effective biological modifier of aging frailty, and support ongoing
investigation of allo-hMSCs alone or as an adjunct to current physi-
cal training strategies for aging frailty.
These findings are in agreement with a recently completed
dose-finding phase I safety study (17). In that study, two impor -
tant sets of observations were made. First, a constellation of physi-
cal performance measures improved with cell therapy, and second,
100M cells represented the peak responsiveness dose, with a pla-
teau and/or reduction in efficacy being noted with 200M cells.
Accordingly, this study was based, in part, on phase I. Importantly,
randomizing patients in a double-blind fashion to 100M cells, 200M
cells, or placebo was performed to validate the results of the earlier
study and to confirm both the constellation of physical performance
findings and the dose–response.
The findings here replicate in large part the results of the earlier
open label study, support the concept that MSCs have bioactivity
against aging frailty, and confirm the fact that 100M represents a
superior dose level compared to 200M. The reasons underlying the
inverse dose relationship noted here remain incompletely understood.
The 100M dose group produced significant improvements in both
physiologic and immunologic markers of frailty, while the high dose
group solely demonstrated positive immunomodulatory effects. It is
important to note that there is a precedent for this in earlier studies,
and a number of stem cell-based clinical trials exhibit greater effects
with lower doses (19, 30). However, the available preclinical and clin-
ical evidence regarding dose relationship in stem cell therapy is con-
flicting (31), with some studies reporting that lower cell dosage and/
or infusion cell concentration may provide the most benefit (19, 32),
while others finding either a direct or nonlinear relationship (33).
There are several factors that could contribute to nonlinear dose
response curves with cell-based therapy. These include variation in
functional activity of the cells rather than the absolute number of cells
infused. In this regard, higher cell concentrations could impair cell
activity through physical effects such as concentration-dependent cell
aggregation, or damage of cells due to excessive shear forces on cells
during infusion that could influence the relationship between cell dose
and clinical benefit (31). Therefore, studies have recently been focus-
ing on cell activity and or genetic modification to enhance their activ-
ity, rather than quantity (34, 35). However, as with all progenitor cell
types in various disease processes, whether modified or not, exact dos-
ing has yet to be established, and thus is a weakness of this particular
study. Given the novel use of MSCs in frailty, a patient population for
whom a successful therapy has yet to be developed, dosing was based
on safety as established by previous studies (18, 19) and phase I (17),
and was further investigated in the current study. Importantly, safety
was ultimately established in both cell-dose groups. The optimal effec-
tive dosing will be investigated in future larger randomized trials.
In the current study, we employed allo-hMSCs, which can
target two pathways implicated in the pathogenesis of aging
frailty—inflammation and stem cell depletion. The current
findings support the idea that biological modification of aging
frailty is not only feasible but has the potential to meaningfully
impact the physical performance of older individuals with mild
to moderate. It is noteworthy that the effects on inflammatory
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cytokines may have practical value by providing a clinical use-
ful biomarker. Importantly, both circulating TNF-α levels as well
as B-cell intracellular TNF-α appear to be candidate biomakers
that could be used to index the efficacy of hMSCs in this patient
population.
The 6MWT, SPPB-questionnaire, FEV1, and the CHAMPS-
questionnaire were among the physical performance measures
examined in our study, and the 100M-group in particular produced
a meaningful outcome in all measures. The 6MWT was originally
designed to evaluate cardiac and pulmonary disorders; recently, its
application has expanded to assess an individual’s exercise capac-
ity at various levels of intensity and their ability to walk safely in a
community setting (36, 37). Similarly, the SPPB is a physical measure
utilized to identify an individual’s future risk of disability, institu-
tionalization, and mortality in the elderly adults (38). CHAMPS was
created to improve physical activity in the elderly (39). The survey
utilizes a series of questions to measure physical activities which are
then employed to estimate caloric expenditure per week (39, 40).
Together with measures of pulmonary function, these factors are of
great importance in an individuals’ ability to remain mobile and active
in a community setting. Other quality of life measures included sexual
function. Although males did not experience any improvement in
erectile dysfunction, women significantly improved their scores on the
SQOL in the 100M-group. This is a particularly meaningful marker
of improved quality of life, as loss of libido in postmenopausal women
Figure 2. Physical markers of frailty. (A) Six-minute walk test (6MWT) increased in mean meters walked in the 100M-group from baseline to 6 months (p = .011)
but not the 200M-group (p = .263) or placebo (p = .112). (B) Short physical performance battery (SPPB) was significant for an overall improvement in the median
total score in the 100M-group from baseline to 6 months (p = .031) but not in the 200M-group (p = .812) or placebo (p = .875). (C) Community Healthy Activities
Model Program for Seniors (CHAMPS) questionnaire was significant for a reduced median total caloric expenditure per week at moderate intensity from
baseline to 6 months in the 200M-group (p = .008) and placebo (p = .039), but not in the 100M-group (p = .641). (D) Forced expiratory volume after 1 second
(FEV1) improved in mean liters from baseline to 6 months in the 100M-group (p = .025) without changes noted in the 200M-group (p = .259) or placebo (p = .883).
* indicates p ≤ .05.
Table 3. Calculated Panel Reactive Antibodies (cPRA)
cPRA Treatment Group
% Increase in donor
specific cPRA (Baseline
to 6 mo)
Allo-100M
(N = 10)
Allo-200M
(N = 10)
Placebo
(N = 10)
Negative (0–10%) 9 8 10
Mild (11–20%) 1 0 0
Moderate (21–79%) 0 2 0
High (≥80%) 0 0 0
Note: cPRAs are from baseline to 6 months, and showed that nine out of ten
patients in the 100M-group had no reaction and 1 had a mild cPRA of 19%
that was donor specific for class I. Eight out of 10 patients in the 200M-group
had no reaction and 2 had a moderate reaction (one patient developed a 29%
cPRA which was donor specific for 1 class II, and another patient developed
36% cPRA which was not donor specific and all were class I). There were
no panel reactive antibodies in the 10 patients in the placebo. Values are the
number of patients in each cPRA category (negative, mild, moderate, and high).
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Figure 3. Immune biomarkers in frailty. All time points are from baseline to 6 months except for TNF-α which begins on Day 1 (infusion) through 6 months. (A)
Early T-cell activation (CD3, CD69) were reduced as a percent change from baseline to 6 months in the 200M-group (p = .004), but not the 100M-group (p = .269) or
placebo (p = .0797). (B) Late T-cell activation (CD3, CD25) was reduced as a percent change from baseline to 6 months in the 100M and 200M-groups (p = .007 and
p = .048 respectively), but not in the placebo (p = .119). (C) % CD8 T-cells decreased from baseline to 6 months in the 200M-group (p = .022) and no changes were
noted in the 100M-group (p = .978) or placebo (p = .0797). (D) CD4/CD8 ratio increased from baseline to 6 months in the 200M-group (p = .014) and no changes
were found in the 100M-group (p = .609) or placebo (p = .104). (E) Serum TNF-α decreased in pg/mL from baseline to 6 months in the 100M-group (p = .031)
without a change in the 200M-group (p = .129) or placebo (p = .094). (F) %B cells expressing intracellular TNF-α decreased from baseline to 6 months in the 100M
(p < .0001) and 200M-groups (p = .002) without a significant change in placebo (p = .869). * indicates p ≤ .05.
is intrinsically linked to hypoactive sexual desire disorder (HSDD), a
disorder marked by clinically significant personal distress (41).
Frailty in advancing age is associated with a heightened state
of inflammation termed “inflammaging” (42). Markers of chronic
inflammation, such as TNF-α and leukocytosis, are all associated
with aging and age-related diseases (43). TNF-α in particular has
been correlated with increased mortality in the elderly adults (44).
MSCs harbor immunomodulatory properties and have been shown
to decrease inflammatory markers in several studies (45,46).
Therefore, it is not surprising that both the 100M- and 200M-cell
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doses significantly modulated the immune systems of the treated par-
ticipants. Serum TNF-α was significantly reduced in the 100M-group,
while B cell intracellular TNF-α was reduced in both the 100M- and
200M-groups, and as compared to placebo. Furthermore, there
was a suppression of the late/chronically activated T-cells (CD25)
at 6 months postinfusion. The 200M-group produced clinically sig-
nificant decreases in markers of early and late/chronic T-cell acti-
vation. Most interestingly, allo-hMSCs significantly reduced the
percentage of CD8 T-cells. The risk for infection is increased in aging
and is marked by a CD4/CD8 ratio less than one (29). Six months
post-allo-hMSC treatment, there was a significant improvement
in the immune risk phenotype of the 200M-group. These immune
responses due to MSCs are likely to contribute salutary effects and
could enhance health span in individuals with aging frailty.
Aging is characterized by a diminished reserve in all organ sys-
tems, with impaired stem cell production and/or function being
implicated as contributing to the body’s inability to repair itself (47).
Chronic inflammation in particular is not only associated with frailty,
but also creates a detrimental environment for stem cells and their
ability to oppose disease processes (48). Currently, most research on
frailty has focused on improvements on physiologic reserve, with
a focus on the dysregulation of inflammation (49). Utilizing young
healthy individuals as donors for hMSCs, this study addresses both
physiologic and inflammatory aspects of aging frailty.
This study is limited by a small sample size. The lack of differ-
ences between groups, with the exception of intracellular TNF-α,
is due to the study’s small size which limits statistical power. Of
note, the point estimate between the 100M-group and placebo in
the physical performance metric 6MWD would require 30 patients
per group for appropriate statistical power to detect a difference
between groups. A future larger study is planned to address this.
In summary, the present study indicates that intravenous allo-
hMSC delivery is safe in individuals with aging frailty. Given this
excellent safety profile coupled with promising indications of effi-
cacy in the 100M cell group, pivotal clinical trials are warranted
to further establish the efficacy of allo-hMSCs in this multisystem
disorder, to define optimal dosing of MSCs in this population, and
to validate the use of inflammatory biomarkers as a useful surrogate
of clinical outcome.
Supplementary Material
Supplementary data is available at The Journals of Gerontology,
Series A: Biological Sciences and Medical Sciences online.
Funding
This work was supported by The Soffer Family Foundation and The Starr
Foundation.
Acknowledgments
The Data Safety and Monitoring Board members are: Wilson Colucci (Boston
University School of Medicine), Douglas Cowart (Therapeutic Development
Consultants LLC), Michael Thorn (Statistical Resources, Inc.) and Diane
Schneider (University of California, San Diego).
Conflict of Interest
J.M.H. has a patent for cardiac cell-based therapy; he holds equity in Vestion Inc.;
maintains a professional relationship with Vestion as a consultant and member
of the Board of Directors and Scientific Advisory Board; and is a shareholder
in Longeveron LLC. E.A., J.E.-K., and A.M. are employees of the EMMES
Corporation. D.L.D., A.K., and A.M.L. maintain a professional relationship
with Longeveron, LLC as consultants. A.A.O., G.G., and A.M. are employees of
Longeveron, LLC. The other authors declare that they have no competing interests.
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Journals of Gerontology: Medical Sciences
cite as: J Gerontol A Biol Sci Med Sci, 2017, Vol. 72, No. 11, 1513–1521
doi:10.1093/gerona/glx137
Advance Access publication July 17, 2017This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Translational Article
Stem Cell Transplantation for Frailty
Research Article
Allogeneic Mesenchymal Stem Cells Ameliorate Aging
Frailty: A Phase II Randomized, Double-Blind, Placebo-
Controlled Clinical Trial
Bryon A. Tompkins, MD,
1,2
Darcy L. DiFede, RN, BSN,
1,5
Aisha Khan, Msc, MBA,
1
Ana Marie Landin, PhD,
1
Ivonne Hernandez Schulman MD,
1,3
Marietsy V. Pujol, MBA,
1
Alan W. Heldman, MD,
1
Roberto Miki, MD,
3
Pascal J. Goldschmidt-Clermont, MD,
3
Bradley J. Goldstein, MD,
1,2
Muzammil Mushtaq, MD,
3
Silvina Levis-Dusseau, MD,
3
John J. Byrnes, MD,
3
Maureen Lowery MD,
3
Makoto Natsumeda, MD,
1
Cindy Delgado, MA, CCRC,
1
Russell Saltzman, BS.Ed,
1
Mayra Vidro-Casiano, MPH,
1
Moisaniel Da Fonseca, AA,
1
Samuel Golpanian, MD,
2
Courtney Premer, PhD,
1
Audrey Medina, BSc,
5
Krystalenia Valasaki, MSc,
1
Victoria Florea, MD,
1
Erica Anderson, MA,
4
Jill El-Khorazaty, MS,
4
Adam Mendizabal, PhD,
4
Geoff Green, BA, MBA,
5
Anthony A. Oliva, PhD,
5
and Joshua M. Hare, MD
1,3
1
The Interdisciplinary Stem Cell Institute,
2
Department of Surgery, and
3
Department of Medicine, University of Miami Miller School of
Medicine, Florida.
4
EMMES Corporation, Rockville, Maryland.
5
Longeveron LLC, Miami, Florida.
Address correspondence to: Joshua M. Hare, MD, Louis Lemberg Professor of Medicine, Director, Interdisciplinary Stem Cell Institute, University
of Miami Miller School of Medicine, Biomedical Research Building, 1501 N.W. 10
th
Ave., Room 824, P.O. Box 016960 (R125), Miami, FL 33101. E-mail:
jhare@med.miami.edu
Received: April 14, 2017; Editorial Decision Date: June 15, 2017
Decision Editor: Anne Newman, MD, MPH
Abstract
Background: Aging frailty, characterized by decreased physical and immunological functioning, is associated with stem cell depletion. Human
allogeneic mesenchymal stem cells (allo-hMSCs) exert immunomodulatory effects and promote tissue repair.
Methods: This is a randomized, double-blinded, dose-finding study of intravenous allo-hMSCs (100 or 200-million [M]) vs placebo delivered
to patients (n = 30, mean age 75.5 ± 7.3) with frailty. The primary endpoint was incidence of treatment-emergent serious adverse events (TE-
SAEs) at 1-month postinfusion. Secondary endpoints included physical performance, patient-reported outcomes, and immune markers of
frailty measured at 6 months postinfusion.
Results: No therapy-related TE-SAEs occurred at 1 month. Physical performance improved preferentially in the 100M-group; immunologic
improvement occurred in both the 100M- and 200M-groups. The 6-minute walk test, short physical performance exam, and forced expiratory
volume in 1 second improved in the 100M-group (p = .01), not in the 200M- or placebo groups. The female sexual quality of life questionnaire
improved in the 100M-group (p = .03). Serum TNF-α levels decreased in the 100M-group (p = .03). B cell intracellular TNF-α improved in
both the 100M- (p < .0001) and 200M-groups (p = .002) as well as between groups compared to placebo (p = .003 and p = .039, respectively).
Early and late activated T-cells were also reduced by MSC therapy.
Translational
Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/72/11/1513/3977809 by guest on 28 March 2019
Conclusion: Intravenous allo-hMSCs were safe in individuals with aging frailty. Treated groups had remarkable improvements in physical
performance measures and inflammatory biomarkers, both of which characterize the frailty syndrome. Given the excellent safety and efficacy
profiles demonstrated in this study, larger clinical trials are warranted to establish the efficacy of hMSCs in this multisystem disorder.
Clinical Trial Registration: www.clinicaltrials.gov: CRATUS (#NCT02065245).
Keywords: Immunomodulation, Tumor necrosis factor-α, Regenerative medicine
There is increasing recognition of the health burden of frailty, a syn-
drome that increases in incidence with aging. Frailty confers an increased
vulnerability to adverse health outcomes and mortality in response to
stressors (1 ,2). Of note, the frailty syndrome is driven mostly by bio-
logical aging processes that include inflammation and stem cell dysfunc-
tion, as opposed to chronological aging (2–5). Early intervention may
improve quality of life, reduce hospitalizations, and nursing home costs
(6,7). Therefore, it is increasingly important to recognize the clinical
onset of frailty, and to develop effective therapeutic strategies.
There are two main models used to define frailty (7): The defi-
cit and the physical phenotype model. The deficit model accounts
for a person’s geriatric syndromes, diseases, psychosocial, physical,
and cognitive impairments, and combines them to create a “Frailty
Index” (8). The physical phenotype model consists of the identifi-
cation of at least three factors: weight loss, exhaustion, weakness,
slowness, and decreased physical activity, which together comprise
an underlying state of multisystem dysregulation (9,10). Despite the
use of different criteria for evaluating frailty, both models show evi-
dence that the prevalence of the syndrome increases with age and is
higher among women (9.6%) than men (5.2%) (11). In a study of
over 44,000 community-dwelling elderly adults, the overall preva-
lence of frailty was found to be 10.7% (11).
Currently, several multimodal interventions are employed to man-
age frailty, namely resistance/aerobic exercise, caloric support, vita-
min D, and optimization of polypharmacy (7 ). However, there are
no specific medical or biologic treatments that ameliorate or reverse
frailty (12, 13). Stem cell depletion is a key mechanism postulated to
contribute to frailty (14–16). In this regard, we recently conducted a
phase I open label study of human allogeneic mesenchymal stem cells
(allo-hMSCs) intravenously infused for frailty, which showed that the
cells could be safely administered, improved measures of functional
capacity, and reduced inflammation (17). Therefore, we conducted the
current phase II double-blinded and placebo-controlled study in order
to test the hypothesis that exogenous allo-hMSCs could reverse signs
and symptoms of frailty in older individuals. Similar approaches have
been shown to exert beneficial effects on the cardiovascular system,
with functional improvements on various types of heart disease (18–
20), endothelial function (21), and systemic inflammation (22). Given
their pleiotropic mechanisms of action, which include antifibrotic,
anti-inflammatory, proangiogenic properties (23), and their ability to
stimulate endogenous progenitor cells (21, 24), we hypothesize that
their use may offer a novel treatment strategy in frail patients.
Methods
The AllogeneiC Human Mesenchymal Stem Cells in Patients
with Aging FRAilTy via IntravenoUS Delivery (CRATUS) study
(#NCT02065245) is a phase II, randomized, double-blinded, pla-
cebo-controlled study of allo-hMSCs delivered intravenously (IV)
in frail individuals to test the safety and efficacy of allo-hMSCs in
reducing markers of inflammation and improving markers of physi-
cal and mental functioning and quality of life (15,25).
Study Design, Stem Cell Procurement and
Randomization
The study design and phase I of the CRATUS study have been
recently published (17,26). Screening and patient randomization
are outlined in Figure 1 (26), and available in the Supplementary
Material.
Patient Inclusion Criteria and Timeline
The inclusion criteria were as follows: (i) Patients were pro-
vided written informed consent, (ii) patients were aged ≥60 and
≤95 years at the time of signing the Informed Consent Form, and
(iii) they showed the signs of frailty based on physician assessment,
apart from a concomitant condition, by a score between 4 and 7
as denoted by the Canadian Study on Health Aging (25, 27,28).
Major exclusion criteria and a detailed timeline have been pub-
lished (26) .
Study Endpoints
The primary endpoint was the safety of allo-hMSCs at 1 month,
assessed by treatment emergent-serious adverse events (TE-SAE).
TE-SAEs were defined by the following: death, nonfatal pulmonary
embolism, stroke, hospitalization for worsening dyspnea, and clini-
cally significant laboratory abnormalities.
The secondary endpoints assessed the efficacy of the therapy.
Efficacy was demonstrated by differences in the rate of change of
frailty markers as defined by: reduced activity (Community Healthy
Activities Model Program for Seniors (CHAMPS) questionnaire),
slowing of mobility (6-minute walk test (6MWT), 4-m gait speed test
(4MGST), and the short physical performance battery (SPPB) score,
comprised of balance tests, gait speed tests, and chair stand tests),
weight loss, diminished hand grip strength (dynamometry), exhaus-
tion-multidimensional fatigue inventory (MFI), quality of life assess-
ments (Sexual Quality of Life-Female (SQOL-F) and International
Index of Erectile Dysfunction (IIEF) Questionnaires), dobutamine-
induced ejection fraction (EF) via echocardiography, C-reactive pro-
tein (CRP), IL-6, D-dimer, complete blood cell count (CBC) with
differential, and TNF-α.
Immune Monitoring
Immune biomarkers were measured at baseline and 6 months as
described previously (17) and in the Supplementary Material.
Statistical Analysis
No formal statistical justification was performed to determine sam-
ple size for this study. Sample size was determined to be appro-
priate for an early phase study to assess safety in this population.
Due to the early phase nature of this study, no adjustments were
made for multiple analyses (26). Statistical analysis was completed
by statisticians at the Emmes Corporation and is available in the
Supplementary Material.
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Figure 1. Study flow chart. Patient screening, follow-up, and randomization in a 1:1:1 fashion to either the 100M-group, 200M-group, or placebo. M = Million.
Results
Patient Population
Table 1 shows the baseline characteristics of the enrolled patients.
Sixty percent of the patients were White males and the mean age was
75.5 ± 7.3 years.
Safety
No TE-SAEs occurred in any of the three groups in the first 30 days.
Similarly, there were no cumulative treatment-related SAEs in either
group throughout the duration of the study (Table 2). None of the
patients showed any signs of adverse cardiopulmonary reaction
following the intravenous infusion. There were no clinically signifi-
cant changes in basic hematologic and chemistry laboratory tests
throughout the duration of the study.
Long-term Adverse Events
One patient in the 200M-group died of an unrelated event prior
to the 12-month follow-up. Additionally, one patient in the placebo
had an unrelated stroke 307 days postinfusion. The proportion of
patients with adverse events at 12 months did not differ between
groups at the 6- and 12-month time points (p = .300 and p = .141,
respectively).
Hospitalization
There were four patients who required hospitalization within the
12-month follow-up. Two of the hospitalizations were reported in
one patient in the 100M-group, both of which were moderate in
severity; however, none of the hospitalizations were secondary to the
procedure. No patients in the 200M-group were hospitalized. The
remaining three patients belonged to the placebo group; one patient
had two moderate hospitalizations and one severe, another had a
hospitalization that was moderate in severity, and another had one
severe hospitalization. None of the hospitalizations were related to
the procedure.
Functional Status, Quality of Life, and Pulmonary
Function
Quality of life and functional status were monitored throughout the
study. These outcomes preferentially improved in patients randomized
to receive 100M allo-hMSCs. The 6MWT increased in the 100M-group
from baseline to 6 months (345.9 ± 103.4 to 410.7 ± 155.4 m, p = .011;
Figure 2A). There was no significant change at 6 months (p = .263) in
either the 200M-group or placebo (p = .112). The 4MGST showed no
significant differences among groups (p = .659) at 6 months. Consistent
with the improvement in 6MWT, the SPPB total score was significantly
improved in the 100M-group from baseline to 6 months (median
10.5, IQR 9.0, 12.0 to 12.0, IQR 11.0, 12.0; p = .031; Figure 2B).
However, there were no significant differences in the 200M-group
(p = .812) or placebo (p = .875). The CHAMPS-questionnaire showed
a reduced total caloric expenditure per week at moderate intensity in
the 200M-group from baseline to 6 months (median 5,118.8, IQR
1,470.0, 1,4542 to 1,509.4, IQR 472.5, 6,090.0; p = .008) and pla-
cebo (median 3,386.3, IQR 1,286.3, 4,042.0 to 2,021.3, IQR 682.5,
3,150.0; p = .039; Figure 2C). Conversely, there was no significant
reduction in the 100M-group at 6 months (p = .641). There were no
differences between groups in weight loss (p = .7599), MFI, which
assessed mental fatigue (p = .548), and handgrip strength as assessed
via the average of dominant hand scores (p = .676). Ejection fraction,
assessed by dobutamine stress echo, remained stable throughout the
study in all patients. FEV1 improved in the 100M-group from base-
line to 6 months (2.5 ± 0.66 to 2.6 ± 0.77 L/min, p = .025) without
significant changes in the 200M-group (p = .259) or placebo (p = .883;
Figure 2D).
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Immune Biomarkers
Immunotolerability was assessed using a calculated PRA (cPRA)
measured at baseline and 6 months postinfusion on each patient.
Three patients had a mild/moderate increase in donor specific anti-
bodies (one mild in the 100M- and two moderate in the 200M-group).
There were two other patients in the 200M-group that had a mild/
moderate increase in cPRA but were not donor-specific reactions
(Table 3). There were no clinically significant immune reactions
reported. Both the 100M and 200M doses were effective in modu-
lating immune parameters whereas placebo was not. Reduction
in the early-activation CD69 cells was noted in the 200M-group
(27.0 ± 4.30 to 16.4 ± 7.25%, p = .004; Figure 3A) at 6 months.
There were no reductions in the 100M-group (p = .269) or placebo
(p = .0797; Figure 3A). There was a reduction in the late-activation
CD25 cells in both the 100M-group (12.6 ± 6.87 to 6.9 ± 3.30%,
p = .007) and 200M-groups (12.0 ± 6.68 to 8.0 ± 4.64%, p = .048)
from baseline to 6 months. No significant reduction was noted in the
placebo (p = .119; Figure 3B). The CD8 T-cell marker decreased signif-
icantly in the 200M-group from baseline to 6 months (28.7 ± 15.04
to 19.9 ± 10.03%, p = .022; Figure 3C). This is a crucial finding
as aging is marked by an expansion of CD8 cells (29). There were
no significant changes in the 100M-group (p = .978) or placebo at
6 months (p = .0797; Figure 3C). There were no significant changes
in CD4 cells in the 200M-group (p = .052), 100M-group (p = .135),
or placebo (p = .540). The CD4/CD8 ratio appropriately increased
in the 200M-group at 6 months (1.2 ± 1.05 to 1.9 ± 1.17, p = .014),
however there were no changes in the 100M-group (p = .609) or
placebo (p = .104; Figure 3D).
Serum TNF-α decreased in the 100M-group at 6 months (median
3.2, IQR 2.8, 3.8 to 1.2, IQR 1.0, 2.8, p = .031), whereas it did
not significantly change in the 200M-group (p = .129) or placebo
(p = .094; Figure 3E). Similarly, B cell intracellular TNF-α signifi-
cantly decreased in both the 100M- and 200M-groups (17.3 ± 1.8 to
7.0 ± 1.0, p < .0001, and 17.1 ± 2.0 to 8.4 ± 1.1, p = .001, respectively;
Figure 3F) with no improvement in placebo at 6 months (p = .69).
The reductions in both the 100M- and 200M-groups were significant
compared to placebo (p < .00001 and p = .00002; Figure 3F). Finally,
there were no significant changes noted in IL-6, CRP, D-dimer, CBC,
or fibrinogen at 6 months in any group (data not shown).
Sexual Quality of Life
Among female patients, the SQOL-F exhibited a remarkable increase
in the 100M-group at 6 months (59.8 ± 15.3 to 76.0 ± 12.9, p = .035),
but no changes were observed in the 200M-group (p = .882) or pla-
cebo (p = .941; Figure 4). Conversely, there were no differences among
male participants in the IIEF from baseline to 6 months (p = .666).
Discussion
The CRATUS trial is a randomized, double-blind, placebo-con-
trolled evaluation of allo-hMCSs to treat the signs and symptoms of
Table 1. Baseline Characteristics
Treatment Group
Total (N = 30) N (%)Characteristics Allo-100M (N = 10) N (%) Allo-200M (N = 10) N (%) Placebo (N = 10) N (%)
Gender
Male 6 (60%) 6 (60%) 6 (60%) 18 (60%)
Female 4 (40%) 4 (40%) 4 (40%) 12 (40%)
Ethnicity
Hispanic or Latino 1 (10%) 1 (10%) 2 (20%) 4 (13%)
Not Hispanic or Latino 9 (90%) 9 (90%) 8 (80%) 26 (87%)
Race
American Indian/Alaskan Native0 (0%) 1 (10%) 0 (0%) 1 (3%)
White American 10 (100%) 9 (90%) 10 (100%) 29 (97%)
Age at infusion (years) 75.0 ± 7.4 76.3 ± 8.4 75.3 ± 6.8 75.5 ± 7.3
Infusion status
Yes 10 (100%) 10 (100%) 10 (100%) 30 (100%)
No 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Unknown 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Canadian Clinical Frailty Score
4 5 (50%) 7 (70%) 5 (50%) 17 (57%)
5 3 (30%) 1 (10%) 5 (50%) 9 (30%)
6 2 (20%) 2 (20%) 0 (0%) 4 (13%)
7 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Mini-mental state examination 29.3 ± 0.8 28.5 ± 1.1 29.5 ± 1.0 29.1 ± 1.0
Hemoglobin level (g/dL) 14.1 ± 1.2 13.5 ± 1.3 14.3 ± 1.2 14.0 ± 1.3
WBC count (cells/mm
3
) 7,160 ± 2,438 6,600 ± 1,304 7,070 ± 2,215 6,943 ± 1,989
Platelet count (cells/mm
3
) 207,000 ± 64,389 194,500 ± 37,936 194,500 ± 57,880 198,667 ± 52,999
AST (U/L) 24.5 ± 7.6 20.7 ± 3.6 29.3 ± 11.1 24.8 ± 8.5
ALT (U/L) 23.0 ± 16.2 16.5 ± 6.0 31.9 ± 15.6 23.8 ± 14.5
Six-min walk test (m) 345.9 ± 103.4 390.6 ± 148.9 385.8 ± 83.1 374.1 ± 112.9
FEV1 (L) 2.5 ± 0.7 2.3 ± 0.9 2.3 ± 0.5 2.4 ± 0.7
FEV1 (percent predicted) 90.6 ± 10.4 86.9 ± 25.4 87.9 ± 15.2 88.5 ± 17.6
Tumor necrosis factor-α (pg/mL) 3.2 (2.8, 3.8) 3.2 (2.6, 3.4) 2.4 (1.1, 3.1) 3.1 (2.1, 3.4)
Note: Values are mean ± SD, N (%), or median (interquartile range [IQR]). FEV1 (Liters) = Forced Expiratory Volume in one second. Hemoglobin (grams/
deciliter). WBC (cells/millimeters = White blood cells. AST (U/L) = Aspartate Aminotransferase (units/liter). ALT = Alanine Aminotransferase. Six-min walk test
distance (m, meters). Tumor necrosis factor-α (pg/mL, picogram/milliliter).
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Table 2. Safety Summary
System Organ Class
MedDRA Preferred
Term
Treatment Group
Allo-100M (N = 10) Allo-200M (N = 10) Placebo (N = 10) Total (N = 30)
Events PatientsEvents PatientsEvents PatientsEvents Patients
N (%) N (%) N (%) N (%) N (%) N (%) N (%) N (%)
General disorders and administration site conditions
Death 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Hepatobiliary disordersCholecystitis 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Infections and infestations
Gastroenteritis 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Musculoskeletal and connective tissue disorders
Flank pain 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Spinal column stenosis 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Spondylolisthesis 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Neoplasms benign,
malignant and
unspecified (incl cysts
and polyps)
Glioblastoma 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Renal and urinary
disorders
Ureteric stenosis 1 (50%) 1 (10%) 1 (10%) 1 (3%)
Vascular disorders Aneurysm 1 (16%) 1 (10%) 1 (10%) 1 (3%)
Hypotension 1 (17%) 1 (10%) 1 (10%) 1 (3%)
Total 2 (100%)1 (10%)2 (100%)2 (20%)6 (100%)4 (40%)10 (100%)7 (23%)
Note: There were no TE-SAEs at 1 month or SAEs in either group. One patient died in the 200M-group unrelated to the treatment. There were seven hospitaliza-
tions, two in the 100M-group, and five in the placebo. None were related to the procedure. Details of the hospitalizations are located in the supplementary material.
SAE = Serious adverse events; TE-SAE = Treatment-emergent serious adverse events.
frailty. The results support the safety and feasibility of administering
allo-hMSCs in this population. With regard to efficacy, there was a
preferential effect towards improvement of functional capacity and
patient reported outcome measures in patients receiving lower dose
MSCs, although immunologic bioactivity was evident with both
doses. Together, these findings suggest that allo-hMSCs may be an
effective biological modifier of aging frailty, and support ongoing
investigation of allo-hMSCs alone or as an adjunct to current physi-
cal training strategies for aging frailty.
These findings are in agreement with a recently completed
dose-finding phase I safety study (17). In that study, two impor -
tant sets of observations were made. First, a constellation of physi-
cal performance measures improved with cell therapy, and second,
100M cells represented the peak responsiveness dose, with a pla-
teau and/or reduction in efficacy being noted with 200M cells.
Accordingly, this study was based, in part, on phase I. Importantly,
randomizing patients in a double-blind fashion to 100M cells, 200M
cells, or placebo was performed to validate the results of the earlier
study and to confirm both the constellation of physical performance
findings and the dose–response.
The findings here replicate in large part the results of the earlier
open label study, support the concept that MSCs have bioactivity
against aging frailty, and confirm the fact that 100M represents a
superior dose level compared to 200M. The reasons underlying the
inverse dose relationship noted here remain incompletely understood.
The 100M dose group produced significant improvements in both
physiologic and immunologic markers of frailty, while the high dose
group solely demonstrated positive immunomodulatory effects. It is
important to note that there is a precedent for this in earlier studies,
and a number of stem cell-based clinical trials exhibit greater effects
with lower doses (19, 30). However, the available preclinical and clin-
ical evidence regarding dose relationship in stem cell therapy is con-
flicting (31), with some studies reporting that lower cell dosage and/
or infusion cell concentration may provide the most benefit (19, 32),
while others finding either a direct or nonlinear relationship (33).
There are several factors that could contribute to nonlinear dose
response curves with cell-based therapy. These include variation in
functional activity of the cells rather than the absolute number of cells
infused. In this regard, higher cell concentrations could impair cell
activity through physical effects such as concentration-dependent cell
aggregation, or damage of cells due to excessive shear forces on cells
during infusion that could influence the relationship between cell dose
and clinical benefit (31). Therefore, studies have recently been focus-
ing on cell activity and or genetic modification to enhance their activ-
ity, rather than quantity (34, 35). However, as with all progenitor cell
types in various disease processes, whether modified or not, exact dos-
ing has yet to be established, and thus is a weakness of this particular
study. Given the novel use of MSCs in frailty, a patient population for
whom a successful therapy has yet to be developed, dosing was based
on safety as established by previous studies (18, 19) and phase I (17),
and was further investigated in the current study. Importantly, safety
was ultimately established in both cell-dose groups. The optimal effec-
tive dosing will be investigated in future larger randomized trials.
In the current study, we employed allo-hMSCs, which can
target two pathways implicated in the pathogenesis of aging
frailty—inflammation and stem cell depletion. The current
findings support the idea that biological modification of aging
frailty is not only feasible but has the potential to meaningfully
impact the physical performance of older individuals with mild
to moderate. It is noteworthy that the effects on inflammatory
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cytokines may have practical value by providing a clinical use-
ful biomarker. Importantly, both circulating TNF-α levels as well
as B-cell intracellular TNF-α appear to be candidate biomakers
that could be used to index the efficacy of hMSCs in this patient
population.
The 6MWT, SPPB-questionnaire, FEV1, and the CHAMPS-
questionnaire were among the physical performance measures
examined in our study, and the 100M-group in particular produced
a meaningful outcome in all measures. The 6MWT was originally
designed to evaluate cardiac and pulmonary disorders; recently, its
application has expanded to assess an individual’s exercise capac-
ity at various levels of intensity and their ability to walk safely in a
community setting (36, 37). Similarly, the SPPB is a physical measure
utilized to identify an individual’s future risk of disability, institu-
tionalization, and mortality in the elderly adults (38). CHAMPS was
created to improve physical activity in the elderly (39). The survey
utilizes a series of questions to measure physical activities which are
then employed to estimate caloric expenditure per week (39, 40).
Together with measures of pulmonary function, these factors are of
great importance in an individuals’ ability to remain mobile and active
in a community setting. Other quality of life measures included sexual
function. Although males did not experience any improvement in
erectile dysfunction, women significantly improved their scores on the
SQOL in the 100M-group. This is a particularly meaningful marker
of improved quality of life, as loss of libido in postmenopausal women
Figure 2. Physical markers of frailty. (A) Six-minute walk test (6MWT) increased in mean meters walked in the 100M-group from baseline to 6 months (p = .011)
but not the 200M-group (p = .263) or placebo (p = .112). (B) Short physical performance battery (SPPB) was significant for an overall improvement in the median
total score in the 100M-group from baseline to 6 months (p = .031) but not in the 200M-group (p = .812) or placebo (p = .875). (C) Community Healthy Activities
Model Program for Seniors (CHAMPS) questionnaire was significant for a reduced median total caloric expenditure per week at moderate intensity from
baseline to 6 months in the 200M-group (p = .008) and placebo (p = .039), but not in the 100M-group (p = .641). (D) Forced expiratory volume after 1 second
(FEV1) improved in mean liters from baseline to 6 months in the 100M-group (p = .025) without changes noted in the 200M-group (p = .259) or placebo (p = .883).
* indicates p ≤ .05.
Table 3. Calculated Panel Reactive Antibodies (cPRA)
cPRA Treatment Group
% Increase in donor
specific cPRA (Baseline
to 6 mo)
Allo-100M
(N = 10)
Allo-200M
(N = 10)
Placebo
(N = 10)
Negative (0–10%) 9 8 10
Mild (11–20%) 1 0 0
Moderate (21–79%) 0 2 0
High (≥80%) 0 0 0
Note: cPRAs are from baseline to 6 months, and showed that nine out of ten
patients in the 100M-group had no reaction and 1 had a mild cPRA of 19%
that was donor specific for class I. Eight out of 10 patients in the 200M-group
had no reaction and 2 had a moderate reaction (one patient developed a 29%
cPRA which was donor specific for 1 class II, and another patient developed
36% cPRA which was not donor specific and all were class I). There were
no panel reactive antibodies in the 10 patients in the placebo. Values are the
number of patients in each cPRA category (negative, mild, moderate, and high).
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Figure 3. Immune biomarkers in frailty. All time points are from baseline to 6 months except for TNF-α which begins on Day 1 (infusion) through 6 months. (A)
Early T-cell activation (CD3, CD69) were reduced as a percent change from baseline to 6 months in the 200M-group (p = .004), but not the 100M-group (p = .269) or
placebo (p = .0797). (B) Late T-cell activation (CD3, CD25) was reduced as a percent change from baseline to 6 months in the 100M and 200M-groups (p = .007 and
p = .048 respectively), but not in the placebo (p = .119). (C) % CD8 T-cells decreased from baseline to 6 months in the 200M-group (p = .022) and no changes were
noted in the 100M-group (p = .978) or placebo (p = .0797). (D) CD4/CD8 ratio increased from baseline to 6 months in the 200M-group (p = .014) and no changes
were found in the 100M-group (p = .609) or placebo (p = .104). (E) Serum TNF-α decreased in pg/mL from baseline to 6 months in the 100M-group (p = .031)
without a change in the 200M-group (p = .129) or placebo (p = .094). (F) %B cells expressing intracellular TNF-α decreased from baseline to 6 months in the 100M
(p < .0001) and 200M-groups (p = .002) without a significant change in placebo (p = .869). * indicates p ≤ .05.
is intrinsically linked to hypoactive sexual desire disorder (HSDD), a
disorder marked by clinically significant personal distress (41).
Frailty in advancing age is associated with a heightened state
of inflammation termed “inflammaging” (42). Markers of chronic
inflammation, such as TNF-α and leukocytosis, are all associated
with aging and age-related diseases (43). TNF-α in particular has
been correlated with increased mortality in the elderly adults (44).
MSCs harbor immunomodulatory properties and have been shown
to decrease inflammatory markers in several studies (45,46).
Therefore, it is not surprising that both the 100M- and 200M-cell
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doses significantly modulated the immune systems of the treated par-
ticipants. Serum TNF-α was significantly reduced in the 100M-group,
while B cell intracellular TNF-α was reduced in both the 100M- and
200M-groups, and as compared to placebo. Furthermore, there
was a suppression of the late/chronically activated T-cells (CD25)
at 6 months postinfusion. The 200M-group produced clinically sig-
nificant decreases in markers of early and late/chronic T-cell acti-
vation. Most interestingly, allo-hMSCs significantly reduced the
percentage of CD8 T-cells. The risk for infection is increased in aging
and is marked by a CD4/CD8 ratio less than one (29). Six months
post-allo-hMSC treatment, there was a significant improvement
in the immune risk phenotype of the 200M-group. These immune
responses due to MSCs are likely to contribute salutary effects and
could enhance health span in individuals with aging frailty.
Aging is characterized by a diminished reserve in all organ sys-
tems, with impaired stem cell production and/or function being
implicated as contributing to the body’s inability to repair itself (47).
Chronic inflammation in particular is not only associated with frailty,
but also creates a detrimental environment for stem cells and their
ability to oppose disease processes (48). Currently, most research on
frailty has focused on improvements on physiologic reserve, with
a focus on the dysregulation of inflammation (49). Utilizing young
healthy individuals as donors for hMSCs, this study addresses both
physiologic and inflammatory aspects of aging frailty.
This study is limited by a small sample size. The lack of differ-
ences between groups, with the exception of intracellular TNF-α,
is due to the study’s small size which limits statistical power. Of
note, the point estimate between the 100M-group and placebo in
the physical performance metric 6MWD would require 30 patients
per group for appropriate statistical power to detect a difference
between groups. A future larger study is planned to address this.
In summary, the present study indicates that intravenous allo-
hMSC delivery is safe in individuals with aging frailty. Given this
excellent safety profile coupled with promising indications of effi-
cacy in the 100M cell group, pivotal clinical trials are warranted
to further establish the efficacy of allo-hMSCs in this multisystem
disorder, to define optimal dosing of MSCs in this population, and
to validate the use of inflammatory biomarkers as a useful surrogate
of clinical outcome.
Supplementary Material
Supplementary data is available at The Journals of Gerontology,
Series A: Biological Sciences and Medical Sciences online.
Funding
This work was supported by The Soffer Family Foundation and The Starr
Foundation.
Acknowledgments
The Data Safety and Monitoring Board members are: Wilson Colucci (Boston
University School of Medicine), Douglas Cowart (Therapeutic Development
Consultants LLC), Michael Thorn (Statistical Resources, Inc.) and Diane
Schneider (University of California, San Diego).
Conflict of Interest
J.M.H. has a patent for cardiac cell-based therapy; he holds equity in Vestion Inc.;
maintains a professional relationship with Vestion as a consultant and member
of the Board of Directors and Scientific Advisory Board; and is a shareholder
in Longeveron LLC. E.A., J.E.-K., and A.M. are employees of the EMMES
Corporation. D.L.D., A.K., and A.M.L. maintain a professional relationship
with Longeveron, LLC as consultants. A.A.O., G.G., and A.M. are employees of
Longeveron, LLC. The other authors declare that they have no competing interests.
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