Cardiovascular Surgery

Effect of Repetitive Intra-Arterial Infusion

of Bone Marrow Mononuclear Cells in Patients
With No-Option Limb Ischemia
The Randomized, Double-Blind, Placebo-Controlled Rejuvenating
Endothelial Progenitor Cells via Transcutaneous Intra-arterial
Supplementation (JUVENTAS) Trial
Martin Teraa, MD, PhD*; Ralf W. Sprengers, MD, PhD*; Roger E.G. Schutgens, MD, PhD;
Ineke C.M. Slaper-Cortenbach, PhD; Yolanda van der Graaf, MD, PhD; Ale Algra, MD, PhD;
Ingeborg van der Tweel, PhD; Pieter A. Doevendans, MD, PhD;
Willem P.Th.M. Mali, MD, PhD; Frans L. Moll, MD, PhD; Marianne C. Verhaar, MD, PhD

Background—Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering
clinical trials suggest that bone marrow–derived cell therapy enhances neovascularization, improves tissue perfusion, and
prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of bone marrow
mononuclear cells (BMMNCs) in patients with severe, nonrevascularizable limb ischemia can prevent major amputation.
Methods and Results—The Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation
(JUVENTAS) trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe,
nonrevascularizable limb ischemia. Patients were randomly assigned to repetitive (3 times; 3-week interval) intra-
arterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, ie, major
amputation at 6 months, with major amputation rates of 19% in the BMMNC versus 13% in the placebo group (relative
risk, 1.46; 95% confidence interval, 0.62–3.42). The safety outcome (all-cause mortality, occurrence of malignancy, or
hospitalization due to infection) was not significantly different between the groups (relative risk, 1.46; 95% confidence
Downloaded from http://ahajournals.org by on December 27, 2018

interval, 0.63–3.38), neither was all-cause mortality at 6 months with 5% versus 6% (relative risk, 0.78; 95% confidence
interval, 0.22–2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen
pressure improved during follow-up, but there were no significant differences between the groups.
Conclusions—Repetitive intra-arterial infusion of autologous BMMNCs into the common femoral artery did not reduce
major amputation rates in patients with severe, nonrevascularizable limb ischemia in comparison with placebo. The
general improvement in secondary outcomes during follow-up in both the BMMNC and the placebo group, as well,
underlines the essential role for placebo-controlled design of future trials.
Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00371371.  (Circulation. 2015;
131:851-860. DOI: 10.1161/CIRCULATIONAHA.114.012913.)
Key Words: atherosclerosis ◼ cells ◼ peripheral vascular diseases

S evere limb ischemia, due to advanced peripheral artery dis-

ease (PAD), is associated with a high risk of cardiovascu-
lar events and all-cause mortality1–4 and a poor prognosis with
40%.3,4 Severe limb ischemia is associated with poor quality of
life (QoL)7 and high treatment costs,8 especially when amputa-
tion is inevitable.8,9 With an estimated annual incidence of 500
respect to limb preservation.5,6 In nonrevascularizable, so-called to 1000 new cases per million individuals in Western society,3
no-option patients with severe limb ischemia, 6-month major
amputation rates have been reported to range from 10% to
Clinical Perspective on p 860

Received August 22, 2014; accepted January 5, 2015.

From Department of Nephrology & Hypertension (M.T., M.C.V.), Department of Vascular Surgery (M.T., F.L.M.), Department of Radiology (R.W.S.,
W.P.Th.M.M.), Van Creveldkliniek/Department of Hematology (R.E.G.S.), Cell Therapy Facility/ Department of Clinical Pharmacy (I.C.M.S.-C.), Julius
Center for Health Sciences and Primary Care (Y.v.d.G., A.A., I.v.d.T.), Brain Center Rudolf Magnus, Department of Neurology and Neurosurgery (A.A.),
and Department of Cardiology (P.A.D.), University Medical Center Utrecht, The Netherlands.
*Drs Teraa and Sprengers contributed equally.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.114.
012913/-/DC1.
Correspondence to Marianne C. Verhaar, MD, PhD, University Medical Center Utrecht, Heidelberglaan 100, HP F.03.227, 3584 CX Utrecht, The
Netherlands. E-mail m.c.verhaar@umcutrecht.nl
© 2015 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.114.012913

851
 852  Circulation  March 10, 2015
which is ever increasing in concert with the increase in cardio-
vascular risk factors,10–12 severe limb ischemia poses a substan-
tial burden on patients, healthcare providers, and resources;
hence, new treatment modalities are urgently needed.
Cell-based regenerative therapies aiming at enhanced
neovascularization and improved limb perfusion have been
proposed as novel treatment strategies. In 2002, a small first-
in-man clinical trial reported safety and promising effects
of autologous bone marrow (BM)–derived cell therapy in
patients with critical limb ischemia.13 Since then, several stud-
ies have suggested benefit of BM-derived cell therapy for
advanced PAD. However, studies were small, lacked appro-
priate controls, often did not consider clinically relevant out-
comes, and thus did not provide definite proof on clinical
effectiveness.14,15 Our recent meta-analysis on 12 randomized
clinical trials (RCTs) that studied BM-derived cell therapy in
a total of 510 patients with critical limb ischemia underlined
the promising potential of this therapy but also showed diver-
gent results between placebo-controlled and non–placebo-
controlled RCTs, stressing the need for a large, well-designed,
placebo-controlled RCT with clinically relevant outcomes.15
We designed the Rejuvenating Endothelial Progenitor
Cells via Transcutaneous Intra-arterial Supplementation
(JUVENTAS) trial, a double-blind, placebo-controlled RCT
(NCT00371371) to investigate whether repetitive intra-arte-
rial infusion of BM mononuclear cells (BMMNCs) reduces
amputation rates in a large cohort of patients with severe, non-
revascularizable limb ischemia.16
JUVENTAS was developed and initiated in 2006, and its
Downloaded from http://ahajournals.org by on December 27, 2018
design was based on the available literature at that time. The
rationale has been published elsewhere.16 In short, we chose
the intra-arterial administration route based on preclinical data
suggesting that this route allows the migration to different
zones of ischemic tissue, which is particularly important in
multilevel disease, and facilitates homing to ischemic tissue
with preserved nutrient blood supply, leading to improved
local cell survival,17,18 and to the extrapolation of clinical data,
as well, suggesting the benefit of intracoronary injection of
BMMNC in myocardial infarction.19,20 We chose to admin-
ister BMMNC obtained from 100 mL of BM, which can be
obtained under local anesthesia in an outpatient setting with
a low risk of complications. We adopted a repetitive infusion
scheme assuming that repeated administration would enhance
cell retainment based on observations that only limited num-
bers of cells are retained in injured tissue after injection.
Methods
Detailed methods can be found in the online-only Data Supplement.
Trial Design and Study Population
In this single-center, double-blind, placebo-controlled RCT, we inves-
tigated the clinical effects of repetitive infusion of BMMNC into the
common femoral artery in 160 patients with severe, nonrevasculariz-
able PAD included from September 2006 through June 2012 (clini-
caltrials.gov NCT00371371; Figure 1). Study design and detailed
inclusion and exclusion criteria are described in the Methods in the
online-only Data Supplement and have been reported previously.16
The institutional review board of the University Medical Center
Utrecht approved the study protocol, the study was conducted accord-
ing to the Declaration of Helsinki, and all patients provided written
informed consent before the study interventions.
Patients were randomly assigned by means of computerized block
randomization with variable block sizes to receive either 3 repetitive
intra-arterial infusions of BMMNC or placebo (3-week intervals) into
the common femoral artery of the affected limb. BM aspirates (100
mL) were obtained from the right iliac crest in all patients. BMMNCs
were isolated by density gradient centrifugation (Lymphoprep, Axis-
Shield Inc, Oslo, Norway). For the placebo group, a placebo was
Figure 1. Patient recruitment and trial flow showing
the patient flow during the screening and trial
phase. No patients were lost to follow-up. ABI
indicates ankle-brachial index; and BMMNC, bone
marrow mononuclear cell.
 Teraa et al   Bone Marrow Mononuclear Cells in No-Option Limb Ischemia   853

Table 1.  Demographic and Clinical Characteristics

Characteristic BMMNC (n=81) Placebo (n=79)
Age, y 69 (57–76) 65 (55–74)
Male sex, n (%) 57 (70) 51 (65)
History of cardiovascular disease, n (%)
 Myocardial infarction or chest pain 33 (41) 33 (42)
 TIA or stroke 10 (12) 13 (17)
 Dialysis dependent renal disease 2 (3) 3 (4)
 Angioplasty contralateral leg 18 (22) 19 (24)
 Bypass contralateral leg 11 (14) 16 (20)
 Major amputation contralateral leg 5 (6) 6 (8)
Body mass index, kg/m2 26.2±4.1 26.6±5.1
Smoking, n (%)
 Currently 18 (23) 24 (31)
 Past 47 (59) 46 (59)
Diabetes mellitus, n (%) 29 (36) 31 (39)
 Nonfasting glucose, mmol/L 5.9 (5.2–7.1) 5.8 (5.1–6.9)
Hypertension, n (%)* 70 (86) 72 (91)
 Systolic blood pressure, mm  Hg 130±19 132±21
 Diastolic blood pressure, mm  Hg 73±10 72±10
Hyperlipidemia, n (%)† 74 (91) 71 (90)
 Total cholesterol, mmol/L 4.3±1.1 4.2±1.1
 LDL-cholesterol, mmol/L 2.4±0.9 2.3±1.0
 HDL-cholesterol, mmol/L 1.17 (0.88–1.47) 1.11 (0.84–1.50)
 Triglycerides, mmol/L 1.4 (1.0–2.0) 1.5 (0.9–1.9)
Renal insufficiency, n (%)‡ 20 (25) 16 (20)
 Creatinine, μmol/L 92 (74–122) 86 (74–110)
Cardioprotective drug use, n (%)
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 Antiplatelet therapy 55 (68) 57 (72)

 Coumadines 32 (39) 29 (37)
 Statins 67 (83) 66 (84)
 ACE inhibitors 31 (38) 31 (39)
Disease characteristics treated leg
 Right/ left limb treated, n (%) 47/34 (58/42) 45/34 (57/43)
 Previous bypass ipsilateral, n (%) 37 (46) 43 (54)
 Previous angioplasty ipsilateral, n (%) 48 (59) 47 (60)
 Aorta to popliteal artery patent, n (%) 34 (42) 32 (41)
 <2 patent crural vessels 67 (83) 70 (89)
 Rutherford stage, n (%)
  3 4 (5) 4 (5)
  4 26 (32) 25 (32)
  5 46 (57) 46 (58)
  6 5 (6) 4 (5)
 Ulcer area, cm2 2.0 (1.0–4.5) 1.7 (1.0–4.3)
 Pain-free walking distance, m 50 (20–105) 50 (20–130)
 Unreliable ankle-brachial index, n (%)§ 28 (35) 28 (35)
 Baseline ankle-brachial index║ 0.50 (0.36–0.66) 0.50 (0.39–0.77)
 Baseline tco2, mm Hg 35±22 36±22
Use of cardioprotective drugs was defined at time of inclusion. ACE indicates angiotensin-converting enzyme; BMMNC,
bone marrow mononuclear cell; HDL, high-density lipoprotein; LDL, low-density lipoprotein; and TIA, transient ischemic attack.
*Hypertension was defined as having systolic blood pressure ≥140 mm Hg, or diastolic blood pressure ≥90 mm Hg, or
antihypertensive drug use.
†Hyperlipidemia was defined as having blood levels of total cholesterol ≥6.50 mmol/L, triglycerides ≥2.3 mmol/L, or HDL-
cholesterol ≤1.0 mmol/L, or lipid-lowering drug use.
‡Renal insufficiency was defined as having creatinine blood levels >120 μmol/L.
§Unreliable ankle-brachial index was defined as incompressible arteries or an ankle-brachial index >0.6 not in proportion to
the Fontaine classification.
║Only patients with a reliable ankle-brachial index were included in the analysis (n=53 in the BMMNC and n=51 in the
placebo group).
 854  Circulation  March 10, 2015
prepared by using autologous peripheral blood erythrocytes to match
the color of the BMMNC product. Of both products, one-third was
prepared for direct infusion, and two-thirds were cryopreserved and
stored for subsequent infusions. For cryopreservation of the BMMNC
product, 10% dimethyl sulfoxide was added. Because dimethyl sulf-
oxide releases a specific odor at the time of infusion, the same amount
of dimethyl sulfoxide was added to the cryopreserved placebo prod-
uct to guarantee blinding of the trial staff. Syringes without informa-
tion about the product were provided to the clinical staff at the time
of infusion. At infusion, the product was slowly administered into the
common femoral artery of the affected limb by hand injection.
Numbers of white blood cells, CD34+ hematopoietic progenitor
cells, colony-forming unit granulocytes and monocytes, and burst-
forming unit-erythroid capacity of the BM product were assessed.
Surveillance Protocol and Outcome Assessment
Primary outcome was major amputation, defined as amputation
through or above the ankle joint, within 6 months after randomiza-
tion. The combined safety outcome was all-cause mortality, occur-
rence of malignancy, or hospitalization due to infection. Secondary
outcomes were the combined occurrence of major amputation or
death, minor amputations, changes in clinical status, ulcer size, rest
pain, pain-free walking distance, ankle-brachial index (ABI), trans-
cutaneous oxygen pressure measurements (tco2; Radiometer Medical
ApS, Copenhagen, Denmark), and health-related QoL (Short Form 36
[SF-36] and EuroQoL 5D [EQ-5D]). SF-36 scores were converted to
a norm-based score and the EQ-5D was converted to the Preference-
Based EuroQoL Tariff as reported previously.7,21,22 To allow compari-
sons with recently published clinical trials, we included previously
published composite outcomes for success and failure of cell-based
therapy.23,24 Clinical evaluation by the same investigator, treadmill
exercise testing, and tco2 measurement were performed at baseline
and at 2 and 6 months follow-up.
The assessment of renal and liver function–related laboratory mea-
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surements was performed at inclusion and before and after each intra-
arterial infusion. During conduct of the trial, all adverse events (AEs)
and serious adverse events (SAEs) were recorded and processed
according to national guidelines.
Interim Analyses and Data Monitoring
An independent Data and Safety Monitoring Board evaluated the
results of sequential interim analyses. Every 6 months, or after every
fourth major amputation, or occurrence of a safety outcome, unblinded
analyses were performed on the cumulative database. Based on the
results of these analyses, the Data and Safety Monitoring Board
informed the trial’s executive committee on whether the data provided
sufficient evidence for either efficiency or harm of the intervention
with respect to the primary outcome and on safety and advised the
executive committee whether to (dis)continue the trial (see Methods
in the online-only Data Supplement). During the conduct of the trial,
no recommendations were made to discontinue the trial.
Sample-Size Calculation and Statistical Analyses
Estimation of the sample size of the JUVENTAS trial was based on
the best available evidence at trial initiation in 2006, which reported
a 6-month risk of major amputation in patients with nonrevasculariz-
able chronic critical limb ischemia of 42%25 and a 50% risk reduc-
tion of major amputation by BMMNC infusion.26,27 Based on these
assumptions, it was estimated that with a 2-sided α of 0.05 and a
power of 80%, ≈110 to 160 patients should be included in the trial. For
a sequential design, no fixed sample size estimate can be provided.28
Continuous variables are expressed as means±standard deviation or as
medians and interquartile ranges for nonnormal data. Depending on data
characteristics, group differences for continuous variables were tested
with the independent t test or the Mann-Whitney U test. Dichotomous
variables were analyzed with the Fisher exact test. Analyses were per-
formed in accordance with the intention-to-treat principle. Sequential
analyses28 of the primary outcome, ie, major amputation, and of the
safety outcome were performed by using the PEST program (version
4.4),29 with adjustment of the point and interval estimates for cumula-
tive testing. Additionally, the risks for major amputation and combined
major amputation and death were analyzed by the Kaplan-Meier method
with the use of the log-rank test. Primary analyses were performed at 6
months of follow-up. Major amputation, mortality, and combined major
amputation and death were also analyzed beyond 6 months. Data for
patients were censored at the date of death, last visit, or last known to
be alive. Because of the lack of power, no subgroup analyses were per-
formed. With the exception of the sequential analysis, no adjustments
for multiple statistical testing were made.30
Furthermore, the results with respect to major amputation were
added to our recently published meta-analysis, with an updated lit-
erature review, and reanalyzed in Review Manager (The Cochrane
Collaboration, version 5.3) with the use of a random-effects model
according to the methods published previously.15 Other statistical
analyses were performed by using SPSS for Windows version 20.0
(SPSS Inc., Chicago, IL).
Role of Funding Sources
The sponsors of the study had no role in the study design, data collec-
tion, analysis, interpretation, or writing of the report. All authors had
full access to all data in the study and had final responsibility for the
decision to submit for publication.
Results
Enrollment and Baseline Patient Characteristics
One hundred sixty patients with severe, nonrevasculariz-
able PAD were randomly assigned to repetitive intra-arterial
BMMNC (n=81) or placebo (n=79) infusions. A detailed
overview of the trial flow is depicted in Figure 1. The 2 trial
arms were well comparable with respect to baseline charac-
teristics and concomitant pharmacological therapy during
the study (Table 1). The majority of patients had tissue loss
(Rutherford stage 5 or 6; 63% in both groups). Until primary
trial follow-up at 6 months, no patients were lost to follow-up.
Major amputation, mortality, and combined major amputation
and death were also analyzed beyond the prespecified primary
trial follow-up at 6 months; because this was not a prespecified
outcome, follow-up beyond 6 months was not available for all
patients (n=127; 79% of the total study population, median
follow-up of 9 [6–20] months, not different between groups).
Intervention
Beside a slightly higher number of CD34+ hematopoietic pro-
genitor cells in the BMMNC group, the characteristics of the
BM aspirate did not differ between the 2 groups (Tables 2 and 3).
Table 2.  Characteristics of the Bone Marrow Aspirate and
Cell Product
Characteristics BM Aspirate BMMNC (n=81) Placebo (n=79)
Volume of BM, mL 100 (100–106) 100 (100–106)
Total amount of BMMNC, ×10 6
657 (422–965) 574 (407–801)
Total amount of CD34+ HPC, ×106 11.4 (7.4–15.8) 9.2 (4.8–13.9)
CFU-GM, per ×105 cells plated 264 (176–396) 245 (174–354)
BFU-e, per ×105 cells plated 156 (110–213) 148 (106–205)
Erythrocytes, ×10 /mL*
9
0.08 (0.02–0.14) NA
BFU-e indicates burst-forming unit-erythroid; BM, bone marrow; BMMNC,
bone marrow mononuclear cell; CFU-GM, colony forming unit granulocytes and
monocytes; HPC, hematopoietic progenitor cell; and NA, not applicable for the
placebo group.
*Number of erythrocytes per milliliter after density centrifugation.
 Teraa et al   Bone Marrow Mononuclear Cells in No-Option Limb Ischemia   855

Table 3.  Characteristics of the Cell Product per Infusion

Total Number of
First Infusion Second Infusion Third Infusion Infused Cells
Cell number per infusion
 BMMNC, ×106 199 (133–295) 144 (93–214)* 144 (87–217)* 500 (313–717)
 CD34 HPC, ×10
+ 6
3.6 (2.3–4.9) 2.5 (1.4–4.0)* 2.4 (1.5–3.6)* 8.4 (5.8–11.9)
Colony forming capacity per infusion*
 CFU-GM, per ×105 cells plated 264 (176–396) 151 (95–239)* 147 (92–217)*
 BFU-e, per ×10 cells plated
5
156 (110–213) 123 (92–170)* 129 (92–170)*
Data are only applicable to patients randomly assigned to the BMMNC group. BFU-e indicates burst-forming unit-
erythroid; BMMNC, bone marrow mononuclear cell; CFU-GM, colony forming unit granulocytes and monocytes; and HPC,
hematopoietic progenitor cell.
*P<0.001 compared with first infusion.

Additionally, the colony-forming capacity of patients’ BM was Hemodynamic Measurements and Ulcer Healing
compared with that of healthy controls (n=32; median age, 32 Baseline measurements were comparable for ABI, tco2, and ulcer
[21–36]), which showed no significant differences for both the area (Table 1). For the analyses of ABI, only patients with a reli-
colony-forming unit granulocytes and monocytes and burst- able index at baseline were included (n=53 BMMNC group; n=51
forming unit-erythroid (colony-forming unit granulocytes and placebo group). ABI increased in both groups during follow-up
monocytes 250 [174–355] versus 282 [197–394], P=0.23;
burst-forming unit-erythroid 149 [108–204] versus 174 [139–
Table 4.  Major Amputation, Death, and Composite Outcomes
242], P=0.07, for patients and healthy controls, respectively).
at 2 and 6 Months and Beyond
A total of 15 patients, 6 in the BMMNC and 9 in the pla-
cebo group, did not complete the scheduled 3 infusions Outcome BMMNC Control RR (95% CI) P Value
owing to the occurrence of a major amputation or mortality Major amputation, n (%)
before the completion of the scheduled infusions (Figure 1).  At 2 mo 6/81 (7) 6/79 (8) 0.98 (0.33–2.90) 1.0
In patients who completed 3 BMMNC infusions, total num-  At 6 mo 15/81 (19) 10/79 (13) 1.46 (0.62–3.42) 0.31
bers of 500×106 (313–717) BMMNC and 8.4×106 (5.8–11.9)  Overall* 21/81 (26) 19/79 (24) 1.08 (0.58–2.02) 0.81
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CD34+ hematopoietic progenitor cells were infused.

Death, n (%)

Major Amputation, All-Cause Mortality,  At 2 mo 1/81 (1) 2/79 (3) 0.49 (0.05–5.27) 0.62
and Composite Outcomes  At 6 mo 4/81 (5) 5/79 (6) 0.78 (0.22–2.80) 0.74
The primary outcome was not different between the 2 groups  Overall* 9/81 (11) 11/79 (14) 0.79 (0.53–3.04) 0.60
with a major amputation rate of 19% and 13% at 6 months in Major amputation
the BMMNC and control group (adjusted relative risk BMMNC or death, n (%)†
versus placebo, 1.46; 95% confidence interval [CI], 0.62–3.42;  At 6 mo 19/81 (23) 13/79 (16) 1.43 (0.76–2.69) 0.27
Table 4 and Figure 2A). The combined safety outcome was
 Overall* 28/81 (35) 26/79 (33) 1.07 (0.63–1.84) 0.80
not different between the groups (15% and 10%, respectively;
Other (at 6 mo), n (%)
relative risk, 1.46; 95% CI, 0.63–3.38). All-cause mortality at
6 months was also not different between the groups with 5%  Minor amputation 9/81 (11) 10/79 (13) 0.88 (0.38–2.05) 0.95
versus 6% (relative risk, 0.78; 95% CI, 0.22–2.80), nor was the  Composite therapy 59/81 (73) 63/78 (81) 0.90 (0.76–1.07) 0.26
combined risk for major amputation and death, with a 6-month success Powell et al23‡
risk of 23% in the BMMNC and 16% in the placebo group (rel-  Composite therapy 35/81 (43) 34/77 (44) 0.98 (0.69–1.39) 1.0
ative risk, 1.43; 95% CI, 0.76–2.69; Table 4 and Figure 2B). In success Iafrati et al30a§
the BMMNC group, the number of infused cells did not relate BMMNC indicates bone marrow mononuclear cell; CI, confidence interval;
with any of the outcome measures (data not shown). When fol- and RR, relative risk.
low-up beyond 6 months was considered, no differences with *Overall denotes follow-up also including time beyond 6 months if available;
median follow-up was 9 months, with an interquartile range of 6 to 20 months
respect to major amputation, mortality, and combined major
(the overall results indicate hazard ratios calculated by the Kaplan–Meier
amputation and death were observed between groups (Table 4). method).
†Amputation-free survival defined as being alive without major amputation
Meta-Analysis may be directly derived as (100% – percentage of major amputation or death).
When adding the results of the JUVENTAS trial and newly ‡Composite end point as defined by Powell et al.23 Success is defined as
published RCTs31–34 to our recently published meta-analysis,15 being alive, without major amputation of the index leg, and no doubling in
a subtle, but significant difference was observed for major wound size or de novo gangrene.
§Composite end point as defined by Iafrati et al.30a Success is defined
amputation (Figure 3A) in favor of the cell therapy–treated as the subject A, is alive; B, did not undergo major amputation of the index
group. However, when only the blinded placebo-controlled limb; C, did not worsen in Rutherford classification or visual analogue scale
trials were included, no significant difference between the >30 mm; and D, improved in either Rutherford classification or visual
cell- and placebo-treated groups was observed (Figure 3B). analogue scale >30 mm.
 856  Circulation  March 10, 2015
Figure 2. A, Kaplan–Meier plot of cumulative probability of major amputation according to trial arm. The dashed and continuous lines
represent the BMMNC and placebo group, respectively. No significant differences existed between the groups (P=0.34). B, Kaplan–Meier
plot of cumulative probability of major amputation and death according to trial arm. The dashed and continuous lines represent the
BMMNC and placebo group, respectively. No significant differences existed between the groups (P=0.31). BMMNC indicates bone
marrow mononuclear cell.
(0.11; 95% CI, 0.06–0.15 and 0.08; 95% CI, 0.02–0.13 for the
BMMNC and placebo group; Table 5), without difference in
ΔABI between the groups (0.03; 95% CI, –0.04 to 0.10). tco2 also
improved in the BMMNC (10.4 mm Hg; 95% CI, 4.2–16.6) and
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placebo group (6.7 mm Hg; 95% CI, 1.3–12.1), without differ-
ence between the groups (3.7; 95% CI, –4.4 to 11.9). In patients
with tissue loss, the ulcer area tended to decrease in both groups
without a difference between the groups (difference in Δulcer
area at 6 months –0.1; 95% CI, –6.1 to 5.9). Complete ulcer heal-
ing at 6 months was observed in 37% of the BMMNC in com-
parison with 28% of the placebo group (Table I in the online-only
Data Supplement). Improvement of Rutherford stage, defined as
at least a decrease of 1 Rutherford stage, was observed in 36% of
the BMMNC and 33% in the placebo group.
Quality of Life and Rest Pain
Throughout the study, the proportion of QoL forms completed
to such an extent that total scores could be obtained ranged
from 89% and 97% at inclusion to 80% and 85% at 6 months
follow-up (of those alive without amputation) for the SF-36
and EQ-5D groups, respectively, with no difference between
the groups at any time point. Baseline QoL scores were low on
the physical components of the SF-36, ie, physical function-
ing, role physical, bodily pain, and general health, reflected
by the low Physical Composite Score of 29.5±7.1 in the
BMMNC and 30.7±7.3 in the placebo group. During follow-
up, QoL scores improved over a wide range of SF-36 com-
ponents, the physical components in particular, resulting in a
significant increase of the Physical Composite Score in both
the BMMNC, and in the placebo group, as well. No differ-
ence was observed in ΔPhysical Composite Score at 6 months
between the 2 groups (–0.1; 95% CI, –3.3 to 3.1; Table 5).
The EQ-5D scores 6 months after the first infusion improved
significantly in the BMMNC and placebo groups (0.21; 95%
CI, 0.12–0.31 and 0.19; 95% CI, 0.08–0.31), without differ-
ence between the 2 groups (Table II in the online-only Data
Supplement). The visual analog scale pain score decreased in both
groups at 6 months, with the most pronounced change in visual
analog scale score at 6 months in the placebo group (–2.4; 95%
CI, –3.2 to –1.5; Table II in the online-only Data Supplement).
Safety
During follow-up, no effects on liver or kidney function were
observed in either group. The total number of SAEs and AEs
observed during the study was 213, with 108 (S)AEs and
105 (S)AEs in the BMMNC and placebo groups (difference
of mean number of [S]AEs, 0.0; 95% CI, –0.31 to 0.31). The
number of patients experiencing a SAE in the BMMNC and
placebo groups was 38 and 34 (difference of mean number of
SAE, 0.0; 95% CI, –0.30 to 0.32). All SAEs were identified
as not related to the study interventions, with the exception of
one, an inguinal hematoma as a result of intra-arterial infusion.
Discussion
The JUVENTAS trial is the largest double-blind, placebo-
controlled RCT to study the effects of BM-derived cell admin-
istration in patients with severe PAD to date. The study shows
that repetitive intra-arterial autologous BMMNC administra-
tion was not effective in reducing the incidence of the primary
outcome, ie, major amputation at 6 months, and even leaves
room for a potential negative effect of the intervention. During
the 6-month follow-up period, improvement of secondary
outcomes, including QoL, ABI, tco2, and ulcer healing, was
observed in both treatment groups without any significant dif-
ferences between the groups.
Strategies for BM-derived cell therapy in patients with severe
limb ischemia differ between studies in many aspects, includ-
ing administration route, cell source and cell-subtype, cell dose,
 Teraa et al   Bone Marrow Mononuclear Cells in No-Option Limb Ischemia   857
Downloaded from http://ahajournals.org by on December 27, 2018

Figure 3. A, Meta-analysis of the effect of cell therapy on major amputation in RCTs in CLI. The meta-analysis shows a subtle significant
(P=0.02) beneficial effect on major amputation of cell therapy in comparison with placebo (RR, 0.66; 95% CI, 0.47–0.93). Within Review
Manager, it is not possible to manually adapt the width of the CI to correct for (sequential) interim analyses. This has led to slightly
narrower 95% CI for the RR for major amputation of our study in the meta-analysis in comparison with the results as described in
the text. B, Meta-analysis of the effect of cell therapy on major amputation in placebo-controlled RCTs in CLI. Only blinded placebo-
controlled RCTs with a separate control group were analyzed (ie, trials that use standard of care or the contralateral limb as a control or
lack any form of blinding were excluded). No effect of cell therapy in comparison with the placebo arm is observed on major amputation
(RR, 0.95; 95% CI 0.64–1.39; P=0.78). Within Review Manager, it is not possible to manually adapt the width of the CI to correct for
(sequential) interim analyses. This has led to slightly narrower 95% CI for the RR for major amputation of our study in the meta-analysis in
comparison with the results as described in the text. CI indicates confidence interval; CLI, critical limb ischemia; M-H, Mantel-Haenszel
method; RCT, randomized clinical trial; and RR, relative risk.

and single or repetitive treatments. These variables may influ-
ence study results. For the JUVENTAS trial, initiated in 2006,
we chose a strategy of infusing BMMNCs obtained from a fixed
amount of 100 mL of BM into the common femoral artery on
3 separate occasions. It is unlikely that the lack of benefit in
our study is due to differences in cell administration or dose.
Previously, potential beneficial effects have been reported by
studies that used intra-arterial administration and by studies
using intramuscular administration, as well. Studies that directly
compared the intra-arterial versus intramuscular route did not
show superiority of either route, but lacked the proper design
to draw definite conclusions.35,36 Furthermore, beneficial results
have been reported after the administration of highly variable
doses of BMMNC with volumes of aspirated BM ranging from
50 to 1000 mL, from which varying amounts of BMMNC and
CD34+ cells were retrieved.15 Although some studies have sug-
gested a dose-response relation between numbers of adminis-
tered CD34+ cells or BMMNCs, this has not been a consistent
finding.31,37,38 In the JUVENTAS trial, no relation was found
between cell numbers or cell characteristics of the administered
BM product and clinical improvement. The results of the
Intraarterial Progenitor Cell Transplantation of Bone Marrow
Mononuclear Cells for Induction of Neovascularization
in Patients With Peripheral Arterial Occlusive Disease
(PROVASA) trial suggested benefit of repeated intra-arterial
BMMNC administration.38 This could not be confirmed in our
study. It has also been suggested that assessing potential clini-
cal benefits of cell therapy in patients with severe limb ischemia
requires longer follow-up.38 However, also with longer follow-
up (median, 9 months), we observed no differences between
groups in any of the outcome measures.
Cell-manufacturing procedures have been reported to
influence BMMNC potency.39–41 We used Lymphoprep for
BMMNC isolation and included heparin in our isolation proto-
col, which is similar to previous studies that showed potential
clinical effects.13,38,42 Heparin has been reported to negatively
affect cellular homing by impairment of the SDF-1α/CXCR4
axis.40 We cannot exclude that the use of heparin in our isola-
tion procedure could have reduced the potency of our product.
Contamination of erythrocytes in the BMMNC product may
 858  Circulation  March 10, 2015

Table 5.  Hemodynamic Measurements, Ulcer Healing, and Quality of Life

Difference Between
Outcome BMMNC Control N the Groups
ABI*
 Δ ABI 2 mo 0.05 (0.00 to 0.10) 0.05 (0.00 to 0.10) 40/37    0.00 (–0.07 to 0.06)
 Δ ABI 6 mo 0.11 (0.06 to 0.15) 0.08 (0.02 to 0.13) 40/37    0.03 (–0.04 to 0.10)
tco2, mm Hg*
 Δ tco2 2 mo 6.8 (1.6 to 12.0) 2.4 (–2.0 to 6.8) 62/62    4.4 (–2.4 to 11.2)
 Δ tco2 6 mo 10.4 (4.2 to 16.6) 6.7 (1.3 to 12.1) 57/56    3.7 (–4.4 to 11.9)
Ulcer area, cm2 *
 Δ ulcer area 2 mo –0.8 (–1.1 to 2.8) –0.7 (–1.7 to 0.3) 41/39 –0.1 (–1.1 to 0.8)
 Δ ulcer area 6 mo –0.5 (–4.1 to 5.1) –0.4 (–3.8 to 4.6) 34/37 –0.1 (–6.1 to 5.9)
QoL, SF-36*
 Δ PCS 2 mo 4.0 (2.1 to 6.0) 4.6 (2.8 to 6.5) 53/48 –0.6 (–3.3 to 2.1)
 Δ MCS 2 mo –0.5 (–3.8 to 2.9) 4.8 (1.8 to 7.9) 53/48 –5.3 (–9.8 to 0.8)
 Δ PCS 6 mo 6.3 (4.1 to 8.4) 6.4 (3.9 to 8.8) 46/49 –0.1 (–3.3 to 3.1)
 Δ MCS 6 mo 3.9 (0.0 to 7.8) 1.7 (–2.6 to 5.9) 46/49   2.3 (–3.4 to 7.9)
Values are means and 95% CI. ABI indicates ankle-brachial index; BMMNC, bone marrow mononuclear
cell; CI, confidence interval; MCS, Mental Health Composite Score; PCS, Physical Composite Score; and QoL,
quality of life.
*Patients who underwent major amputation, were deceased, or had missing values at a specific follow-up
occasion were not included in the analysis at that specific time point. With respect to ABI patients with unreliable
ABI at inclusion (incompressible or not in proportion to the clinical status) were not included in the analyses.

also impair its effectiveness.39 Assmus et al showed substantial
impairment of the BMMNC product when >0.2×109 erythro-
cytes/mL are present. Erythrocytes in our product were far
below this threshold (0.08×109/mL±0.06×109/mL) and there-
Downloaded from http://ahajournals.org by on December 27, 2018
in both BMMNC- and placebo-treated patients was observed
emphasizes the essential role of accurate blinding, which,
together with the large population size, is a major strength of
our study. Similar observations have previously been reported

fore unlikely to have influenced our product.
The JUVENTAS trial included mainly elderly (median age,
67 years) white patients with a high systemic atherosclerotic
burden and prevalence of cardiovascular disease, a typical
Western population with advanced PAD. In contrast, several
initial positive studies were conducted in Asian populations,
often including relatively young patients with thromboangi-
itis obliterans.14 Aging and comorbidities have been shown to
induce functional impairment of BMMNCs which may limit
the therapeutic potential of autologous BM-derived cell therapy.
The lack of benefit in our patients could be partly due to BM
cell dysfunction in patients with high atherosclerotic burden.43
The major amputation rate in our study was lower than
expected based on the available literature at the time of the ini-
tiation of the trial.25 More recent data suggest that amputation
rates in no-option patients with advanced PAD have decreased
over the past decades, in line with the observation in our study.44
The low number of major amputations decreased the statistical
power of the study with respect to major amputation, but the
general lack of benefit on secondary outcomes in comparison
with placebo, support a lack of benefit of autologous BMMNCs
as applied in our protocol. Moreover, our observations are in
line with our recently published meta-analysis on cell therapy in
critical limb ischemia.15 The inclusion of the JUVENTAS trial
(and other new trial) results in the meta-analysis of all RCTs
shows a minor significant beneficial effect on major amputa-
tion, whereas analysis of only the blinded placebo-controlled
RCTs shows a clear lack of benefit.
The fact that, in our trial, where rigorous blinding of par-
ticipants and investigators was applied, a general improvement
for cell therapy trials for heart disease. Several recent well-
designed and larger RCTs that studied BMMNC administration
in both acute myocardial infarction, and in chronic heart failure,
as well, failed to prove the beneficial effects of BMMNC admin-
istration on cardiac function in comparison with placebo inter-
vention.45,46 A recent meta-analysis showed that the observed
improvement in left ventricular ejection fraction observed after
BMMNC therapy in myocardial infarction disappeared when
only sham-controlled studies were analyzed.47
In conclusion, this double-blind, placebo-controlled RCT
of autologous BM-derived cell therapy in patients with severe,
nonrevascularizable PAD shows no effect of repetitive intra-
arterial infusion of autologous BMMNC on prespecified out-
comes. The lack of benefit of autologous BMMNCs in our
study does not exclude the potential benefit of other cell
sources or subpopulations, different administration locations
and routes, or in patients with milder disease. Further study is
warranted to investigate whether cell therapy strategies with
selected cell populations, enhanced BM cell function, or dif-
ferent modes of administration can provide therapeutic ben-
efit in patients with advanced PAD. The general improvement
observed in clinical outcomes in both groups during follow-
up stresses the need for future RCTs to implement a rigorous
double-blinded, placebo-controlled design.
Acknowledgments
We thank Dr G.J. de Borst, Dr R.J. Toorop, Dr C.E.V.M. Hazenberg,
Dr J.A. van Herwaarden, Dr P. Berger, Dr E. Waasdorp, and Dr A.Kh.
Jahrome for patient recruitment, screening, and general trial sup-
port. Dr L. te Boome and Dr P.E. Westerweel are acknowledged for
 Teraa et al   Bone Marrow Mononuclear Cells in No-Option Limb Ischemia   859
performing part of the bone marrow aspiration procedures. The Cell
Therapy Facility of the University Medical Center Utrecht, in particular
K. Westinga, is thanked for processing of the trial products. The nurs-
ing staff of the vascular surgery outpatient clinic and surgical wards of
the University Medical Center Utrecht are thanked for their medical-
site support. Furthermore, all referring vascular surgeons, in particu-
lar, from the Sint Antonius Hospital Nieuwegein (Dr J.P.P.M de Vries,
Dr H.D.W.M van de Pavoordt, and Dr R.H.W. van de Mortel) and the
Meander Medical Center Amersfoort (Dr C.H.P. Arts, Dr M.C. Loubert,
Dr A.J.C. Mackaay, and Dr R. Voorhoeve) are kindly acknowledged for
their support. Authors’ contributions: Drs Sprengers, Verhaar, van der
Graaf, and Moll conceived and designed the study, with advise from
Drs Schutgens, Slaper-Cortenbach, Doevendans, and Mali. Dr Teraa
and Sprengers enrolled the patients. Drs Teraa, Sprengers, Algra, van
der Tweel, van der Graaf, Schutgens, Slaper-Cortenbach, Moll, and
Verhaar were involved in data acquisition, analysis, and interpretation.
Drs Teraa and Verhaar wrote the first draft of the report. All authors
edited and approved the report for final submission.
Sources of Funding
The reported work was supported by the “Stichting Vrienden UMC
Utrecht” on behalf of the Dirkzwager-Assink foundation (The
Netherlands, grant CS 06.007), the Dutch Heart Foundation (grant
2008B094), The Netherlands Organization for Scientific Research
(ZonMw-TAS grant 116001026), and foundation “Stichting De Drie
Lichten” (The Netherlands, grant 10/06). Dr Verhaar is supported by
the Netherlands Organization for Scientific Research (NWO; The
Netherlands, Vidi grant 016.096.359).
Disclosures
None.
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Clinical Perspective
The results of our double-blind, placebo-controlled, randomized Rejuvenating Endothelial Progenitor Cells via
Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial show no benefit of repetitive intra-arterial infusion of
autologous bone marrow–derived mononuclear cells over placebo on prespecified clinical outcomes (eg, major amputation,
quality of life, wound healing) in patients with severe, nonrevascularizable peripheral artery disease. The lack of benefit of
autologous bone marrow–derived mononuclear cells in our study does not exclude the potential benefit of alternative cell
sources or subpopulations, different administration locations and routes, or that in patients with milder disease. Further
study is warranted to investigate whether cell therapy strategies with selected cell populations, enhanced bone marrow cell
function, or different modes of administration can provide therapeutic benefit in patients with advanced peripheral artery dis-
ease. The general improvement in clinical outcomes observed in both the placebo- and bone marrow–derived mononuclear
cell–treated groups during follow-up stresses the need for future randomized clinical trials to implement a rigorous double-
blinded, placebo-controlled design.