Cell Therapy for Critical Limb Ischemia: A Meta-Analysis of Randomized Controlled Trials by Vaclav Prochazka

Original Article Angiology 1-12 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003319715595172 ang.sagepub.com

Cell Therapy for Critical Limb Ischemia: A Meta-Analysis of Randomized Controlled Trials Aaron Liew, MB, BCh, BAO, MRCPI, MRCPSG, PhD1, Vish Bhattacharya, MB, BS, FRCS, FRCS2, James Shaw, BSc, MB ChB, PhD, FRCP1, and Gerard Stansby, MA, MB, MChir, FRCS3

Abstract Early-phase trials showed the feasibility and potential efficacy of cell therapy in patients with critical limb ischemia (CLI). For systematic review, randomized controlled trials (RCTs) of cell therapy versus no cell therapy in CLI were searched from PubMed and the Cochrane library databases. Outcome measures included major amputation, complete ulcer healing, ankle– brachial index (ABI), and all-cause mortality. Data were pooled using 16 RCTs, involving 774 patients. Compared with no cell therapy, cell therapy significantly reduced major amputation (odds ratio [OR]: 0.54; 95% CI: 0.34-0.87: P ¼ .01) and improved ulcer healing (OR: 2.90; 95% confidence interval [CI]: 1.44-5.82; P < .01) and ABI (OR: 5.91; 95% CI: 1.85-18.86: P < .01). Peripheral blood-derived mononuclear cells (PB-MNCs; OR: 0.29; 95% CI: 0.12-0.72; P < .01) and bone marrow concentrate (OR: 0.44; 95% CI: 0.21-0.93; P ¼ .03) significantly lowered the risk of major amputation. The PB-MNCs also significantly increased ulcer healing (OR: 5.77; 95% CI: 1.77-18.87; P < .01). All-cause mortality was similar in both groups (OR: 0.78; 95% CI: 0.44-1.40; P ¼ .41). However, all estimates were nonsignificant following reanalysis using placebocontrolled RCTs only. Cell therapy remains a potential therapeutic option in CLI, but further larger placebo-controlled RCTs are needed. Keywords meta-analysis, critical limb ischemia, cell therapy

Introduction Critical limb ischemia (CLI) is associated with high mortality and disability and represents a significant social and economic burden. Its prevalence is rising in parallel with an aging society and increasing prevalence of diabetes mellitus. The current treatments for CLI aim at improving distal perfusion by surgical, endovascular, or a combination of both approaches.1 However, the presence of existing comorbidities or predominantly distal vessel disease renders many patients unsuitable for these procedures, often resulting in amputation. There remains a significant unmet medical need, and novel approaches, including cell therapy toward augmented vascular regeneration with resultant limb salvage, are urgently needed. Several earlyphase trials have shown great promise using various types of cell therapy, including mononuclear cells derived from peripheral blood or bone marrow, bone marrow concentrate, and mesenchymal stromal cells.2-18 The exact mechanism of action of these various cell therapies is not yet fully understood, and there is a lack of firm evidence on the efficacy and long-term safety of cell therapy for these patients due to the paucity of phase 3 randomized

controlled trials (RCTs). We performed a meta-analysis of available RCTs comparing cell therapy with no cell therapy in patients with CLI. We hypothesized that cell therapy would be associated with a significant reduction in amputation and all-cause mortality, as well as an improvement in ulcer healing and ankle–brachial index (ABI), as compared with no cell therapy in patients with CLI. Our research questions were: In patients with CLI, does cell therapy, compared with no cell therapy, result in: (1) reduction in major amputation; (2) increase in complete ulcer healing; (3) improvement in ABI; and (4) reduction in all-cause mortality?

Institute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom 2 Queen Elizabeth Hospital, Gateshead, United Kingdom 3 Freeman Hospital, High Heaton, Newcastle upon Tyne, United Kingdom Corresponding Author: Aaron Liew, Institute of Cellular Medicine, Newcastle University, 4th Floor William Leech Building, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Emails: liew.aaron@gmail.com

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Methods This meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (http://www.prisma-statement.org/; Appendix A). The RCTs comparing cell therapy as a therapeutic option (vs no cell therapy) in patients with CLI were identified by computerized search of the PubMed and Cochrane library databases (from January 1, 2000, to January 15, 2015, restricted by English language). The following cell types with relevant search terms were included: mononuclear cells derived from either bone marrow or peripheral blood (with or without granulocyte-colony stimulating factor [G-CSF] stimulation), endothelial progenitor cells, hematopoietic stem cells, bone marrow concentrates, and mesenchymal stromal cells. Appendices B and C described the detailed strategy and search terms for each database. The main outcomes of interest were: (1) number of major amputations; (2) number of complete ulcer healing; (3) number of patients with improvement of ABI (>0.1 or >15%); and (4) all-cause mortality. To be included, trials had to meet all of the following criteria: (1) RCTs; (2) inclusion of patients with CLI only in their protocols; (3) compared cell therapy in the intervention arm with no cell therapy in the comparator arm; (4) report at least one of the following outcomes: (a) major amputation; (b) complete ulcer healing; (c) improvement in ABI (>0.1 or >15%); and (d) all-cause mortality; and (5) outcomes were reported in binary form. Studies were excluded if they: (1) involved administration of either similar or alternative cell therapy in the comparator arm; or (2) included patients with intermittent claudication, unless the outcomes on patients with CLI were reported separately. The number of all-cause mortality will be considered as nil by default if it was not specifically mentioned in the full-text article. Two reviewers (AL and VB) independently extracted data from published sources (regarding study design, number of patients, method of randomization, blinding, intervention, duration of follow-up, and outcomes). Agreement was assessed using Cohen unweighted k statistic. Disagreements were resolved by joint review and consensus. Analyses were based on the per protocol principle. The included RCTs were pooled according to the 4 main cell types: (1) peripheral blood-derived mononuclear cells (PB-MNCs); (2) bone marrow-derived mononuclear cells (BM-MNCs); (3) bone marrow-derived mesenchymal stromal cells (BMMSCs); and (4) bone marrow concentrate. Pooled results are presented as odds ratios (ORs) and absolute risk reduction/ increase with their respective 95% confidence intervals (CIs). Heterogeneity assessment was performed using the I2 index, t2 test, and chi square (w2) test. We calculated the pooled OR using a random effects model (DerSimonian and Laird method).19 A random effects model was used in expectation of clinical heterogeneity, irrespective of statistical heterogeneity. The absolute risk reduction is a weighted estimate of the difference in the event rates.19 We also reanalyzed the data by including only placebo-controlled RCTs.

Publication bias was assessed using the Funnel plot. A 2-sided P value of <.05 was considered as statistically significant. Meta-analysis was performed using RevMan version 5.3.

Results We searched PubMed and the Cochrane databases for RCTs comparing cell therapy with no cell therapy in patients with CLI from January 1, 2000, to January 15, 2015. The articles included in the search were restricted to the English language only. Sixteen RCTs were included and the result of the electronic search was shown in Appendix D. Benoit et al6 and Iafrati et al7 reported the same RCT with the same number of patients but different time points (6 and 3 months, respectively). Iafrati et al7 was also included in part of this metaanalysis solely to derive data on improvement in ABI at 3 months, as this outcome was not reported by Benoit et al6, Powell et al20 and Powell et al11 also reported the same RCT, but the number of patient in the former (interim) study was significantly less than that of the latter study and in order to avoid double counting, the former study was not included in our main analysis. Nevertheless, the inclusion of the former study resulted in a similar conclusion for complete ulcer healing outcome (odds ratio [OR] 2.80; 95% confidence interval [CI]: 1.49-5.23; P < .01). The Cohen weighted k was 1.0 (standard deviation ¼ 0) for the assessment of the agreement between reviewers at the level of study selection from full-text articles. The characteristics of the included 16 studies are summarized in Table 1. All the included studies were published between 2005 and 2015. Study sample size ranged from 6 to 160 patients with a follow-up duration ranging from 1 month to 2 years. The type of cell therapy varied, being PB-MNCs, CD34 cells, CD133þ cells, BM-MNCs, bone marrow-derived mesenchymal stromal cell, or bone marrow concentrate. The PB-MNCs were mobilized with G-CSF in all included RCTs2,10,14,16,17 with the exception of Szabo et al.15 Cells were administered via either intramuscular or intra-arterial routes with varying cell concentrations. The comparator arm received standard care with or without placebo (either saline, autologous blood, or both). All included RCTs, except that from Lu et al, compared cell therapy between patients.8 Lu et al compared cell therapy with placebo on ipsilateral and contralateral limbs, respectively, in the same recipient.8 Compared with no cell therapy, cell therapy was associated with a significant reduction in major amputation (OR: 0.54; 95% CI: 0.34-0.87: P < .01; Absolute risk reduction [ARR] ¼ 14%, Number needed to treat [NNT] ¼ 8; Figure 1) and improvement in complete ulcer healing (OR: 2.90; 95% CI: 1.44-5.82; P < .01; ARR ¼ 23%; NNT ¼ 5; Figure 2) and ABI (OR: 5.91; 95% CI: 1.85-18.86; P < .01; ARR ¼ 33%, NNT ¼ 4; Figure 3). In comparison with other cell types, PB-MNCs (OR: 0.29; 95% CI: 0.12-0.72; P < .01; ARR ¼ 28%, NNT ¼ 4) and bone marrow concentrate (OR: 0.44; 95% CI: 0.21-0.93; P ¼ .01; ARR ¼ 18%, NNT ¼ 6) significantly lowered the risk of major amputation (Figure 1). The

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Table 1. Randomized Controlled Trials Comparing Cell Therapy Versus No Cell Therapy in Patients With Critical Limb Ischemia: Summary of Trial Design and Characteristics. References

Trial Design

Huang et al 20052 Nonplacebo controlled Arai et al 20063

Nonplacebo controlled

Barc´ et al 20064

Nonplacebo controlled

Nonplacebo controlled Prochazka et al 20105 a Benoit et al 20116 Double blind (2:1), placebo and and Iafrati et al sham-controlled 20117 Lu et al 20118 Double blind, placebo controlled Walter et al 20119 Losordo et al 201214 Ozturk et al 201210 Powell et al 201211 Gupta et al 201312 Mohammadzadeh et al 201317 Li et al 201313 Szabo et al 201315 Raval et al 201416 Teraa et al 201518

Outcome Follow-Up

Cell Therapy Arm

Comparator Arm

Autologous G-CSF mobilized PB-MNCs via IM administration (n ¼ 14) Autologous BM-MNCs via IM administration (n ¼ 13) Autologous BM-MNCs via IM (n ¼ 10) or IM plus IA administration (n ¼ 4) Autologous BM concentrate via IM administration (n ¼ 42) Autologous BM concentrate via IM administration (n ¼ 34)

Standard care plus IV prostaglandin 3 months E1 (n ¼ 14) Standard care (n ¼ 12) 1 month

Autologous BM-MNCs via IM administration (n ¼ 21 limbs) Autologous cultured BM-MSCs via IM administration (n ¼ 20 limbs) Double blind (1:1), Autologous BM-MNC via IA administration placebo controlled (n ¼ 19) Double blind, placebo Autologous G-CSF mobilized PB-MNCs via and sham-controlled IM administration (low dose, n ¼ 7; high dose, n ¼ 9) Open label, nonplacebo Autologous G-CSF mobilized PB-MNCs via controlled IM administration (n ¼ 20) Double blind (2:1), Autologous BM-MSCs via IM administration placebo-controlled (n ¼ 48) Double blind, Allogeneic cultured BM-MSCs via IM placebo-controlled administration (n ¼ 10) Placebo-controlled Autologous G-CSF mobilized PB-MNCs via IM administration (n ¼ 7) Single blind (1:1), Autologous BM-MNCs via IM administration placebo-controlled (n ¼ 29) Open-label, nonplacebo Autologous expanded PB-MNCs via IM controlled administration (n ¼ 10) Double blind, placebo & Autologous CD133þ cells from G-CSF sham-controlled mobilized PB-MNCs via IM administration (n ¼ 3) Double blind, placebo Autologous BM-MNCs via thrice IA and sham-controlled administrations (n ¼ 79)

Standard care (n ¼ 15)

6 months

Standard care (n ¼ 54)

120 days

Standard care plus blood (n ¼ 14)

6 months

Standard care plus saline (n ¼ 41 contralateral limbs)

24 weeksb

Standard care plus blood (n ¼ 21)

3 months

Standard care plus saline and blood 12 months (n ¼ 12) Standard care (n ¼ 20)

12 weeks

Standard care plus saline (n ¼ 24)

12 months

Standard care plus saline (n ¼ 10)

2 years

Standard care plus G-CSF and saline 3 months (n ¼ 14) Standard care plus saline (n ¼ 29) 6 months Standard care (n ¼ 10)

3 monthsc

Standard care plus saline (n ¼ 3)

12 months

Standard care plus blood (n ¼ 81)

6 months2

Abbreviations: BM-MNCs, bone marrow derived mononuclear cells; BM-MSCs, bone marrow derived mesenchymal stromal cells; G-CSF, granulocyte-colony stimulating factor; IA, intra-arterial; IM, intramuscular; PB-MNCs, peripheral blood derived mononuclear cells. a Benoit et al6 and Iafrati et al7 are from the same RCT with 6 and 3 months duration of follow-up, respectively. b 12 weeks for complete ulcer healing. c Data beyond 6 months for all-cause mortality.

PB-MNCs also significantly increased complete ulcer healing rate (OR: 5.77; 95% CI: 1.77-18.87; P < .01; ARR ¼ 40%; NNT ¼ 3; Figure 2). However, these estimates were not statistically significant when only placebo-controlled RCTs were included (Table 2, Supplementary Figures 1–4). There was no statistically significant reduction in all-cause mortality between both groups (OR: 0.78; 95% CI: 0.44-1.40; P ¼ .41; Figure 4). The overall risk of bias for the included study was low and was summarized in Figure 5 and Supplementary Figure 5. There was no significant statistical evidence of

heterogeneity for all outcomes except for ABI (I2 ¼ 62%; t2 ¼ 0.99; w2 ¼ 10.39; P ¼ .03). There was no evidence of heterogeneity following reanalysis of the outcome for ABI with the exclusion of the study by Teraa et al, which involved a total of 3 repeated cell administrations via intra-arterial route (I2 ¼ 0%; t2 ¼ 0.00; w2 ¼ 0.59; P ¼ .90). All funnel plots with the exception of that for improvement in ABI were symmetrical in shape, suggesting low susceptibility to publication bias. However, the exclusion of study by Teraa et al resulted in a symmetrical funnel plot (supplementary Figures 6-10).

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Figure 1. Odds ratio of major amputation in patients with CLI treated with cell therapy versus no cell therapy (random effects model).

Discussion Early-phase RCTs have demonstrated that cell therapy is feasible for patients with CLI, using intramuscular and intra-arterial (or combination of both) routes, either by single or repeated administration.2-18 Direct comparison of intramuscular and intra-arterial routes of cell delivery in patients with CLI by Gu et al and Klepanec et al has shown similar outcomes.21,22 These RCTs have also demonstrated a reassuring short-term safety profile with promising efficacy signal.2-18 Intriguingly, various cell populations, ranging from a specific cell type (CD133Ăž cells or CD34Ăž cells) to unfractionated bone marrow concentrate, appeared to be beneficial for these indications. However, direct comparison of different cell types showed conflicting results. Tateishi-Yuyama et al showed that treatment with BM-MNCs was associated with significant

improvement in ABI, pain-free walking distance, and transcutaneous oxygen pressure, as compared with PB-MNCs.23 In contrast, Onodera et al showed that treatment with either BM-MNCs or PB-MNCs was associated with no significant difference in long-term prognosis in patients with CLI.24 Lu et al showed that BM-MSCs therapy was associated with better lower limb perfusion and ulcer healing in diabetic patients with CLI than BM-MNCs.8 These trials suggested that the difference in the therapeutic effect of various cell types may be inherently related to the respective cell potency. Assessment of cell potency, underpinning an important element of standardization of a particular cell product, will be crucial to prove consistency and enable meaningful comparison with other approaches. Several meta-analyses assessing the potential therapeutic effect of cell therapy for PAD have been published.25-31 The

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Figure 2. Odds ratio of complete ulcer healing in patients with CLI treated with cell therapy versus no cell therapy (random effects model).

majority have included studies from patients with intermittent claudication.25,26,29-31 Some of these meta-analyses included non-RCTs.26,27 Others25,30 also include an RCT23 that compared 2 different cell types within the same patients. The conclusion of all these meta-analyses remains unchanged, suggesting the beneficial effect of cell therapy in patients with PAD.25-31 In this meta-analysis, we have specifically included patients with either Rutherford categories 4 to 6 or Fontaine categories 3 to 4 to minimize the heterogeneity related to their disease severity. We also included only RCTs that compared cell therapy versus no cell therapy in patients with CLI to determine the potential efficacy of cell therapy, updating previous meta-analyses which included 7 RCTs.27,28 Furthermore, we have updated previous meta-analysis, which reported specifically the results from placebo-controlled RCTs only and showed similar overall finding for major amputation.31 In addition, we have also included various types of cell therapies and stratified them into 4 groups, allowing further analysis of the

potential differential therapeutic effect of these distinct cell types. We have specifically chosen 3 hard objective outcomes (major amputation, complete ulcer healing, and all-cause mortality) and a surrogate objective outcome (ABI). The latter is less susceptible to placebo effects, compared with more subjective outcomes such as alleviation of pain. Our meta-analysis showed that PB-MNCs were associated with a significant reduction in major amputation and improvement in ulcer healing in patients treated with cell therapy as compared with no cell therapy. We also showed that bone marrow concentrate significantly lowered major amputation rate in this cohort. It is important to point out that these results do not imply that cell types other than PB-MNCs, and bone marrow concentrate were not efficacious, since the majority of these RCTs have shown a trend toward positive therapeutic effect on several outcomes. Intriguingly, the nonplacebo-controlled RCTs in general appeared to demonstrate more marked therapeutic effect in all

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Figure 3. Odds ratio of improvement in ABI (>0.1 or >15%) in patients with CLI treated with cell therapy versus no cell therapy (random effects model).

Table 2. The Summary of Odds Ratios: All Included RCTs Versus Placebo-Controlled RCTs Only. Outcomes

All Included RCTs (Placebo-Controlled and Nonplacebo-Controlled)

Major amputation Complete ulcer healing Improvement in ABI All-cause mortality

0.54; 95% 2.90; 95% 5.91; 95% 0.78; 95%

CI: CI: CI: CI:

0.34-0.87: P ¼ .01a 1.44-5.82; P < .01c 1.85-18.86; P < .01e 0.44-1.40; P ¼ .41g

Placebo-Controlled RCTs Only 0.83; 1.92; 4.45; 0.85;

95% CI: 95% CI: 95% CI: 95% CI:

0.51-1.34; P ¼ .44b 0.74-5.03; P ¼ .18d 0.85-23.22; P ¼ .08f 0.43-1.67; P ¼ .63h

Abbreviations: CI, confidence interval; RCT, randomized controlled trial. a Heterogeneity: I2 ¼ 20%; t2 ¼ 0.18; w2 ¼ 19.94 (P ¼ .22). b Heterogeneity: I2 ¼ 0%; t2 ¼ 0.00; w2 ¼ 10.74 (P ¼ .47). c Heterogeneity: I2 ¼ 20%; t2 ¼ 0.27; w2 ¼ 12.57 (P ¼ .25). d Heterogeneity: I2 ¼ 26%; t2 ¼ 0.38; w2 ¼ 6.77 (P ¼ .24). e Heterogeneity: I2 ¼ 62%; t2 ¼ 0.99; w2 ¼ 10.39 (P ¼ .03). f Heterogeneity: I2 ¼ 66%; t2 ¼ 1.40; w2 ¼ 5.89 (P ¼ .05). g Heterogeneity: I2 ¼ 0%; t2 ¼ 0.00; w2 ¼ 4.12 (P ¼ .90). h Heterogeneity: I2 ¼ 0%; t2 ¼ 0.00; w2 ¼ 3.14 (P ¼ .87).

outcomes as compared with the placebo-controlled RCTs. This, in combination with the lower number of RCTs in the secondary analysis, might explain why primary estimates were not statistically significant with the assessment of placebo-controlled RCTs only. Hence, the nonsignificant result following exclusion of nonplacebo-controlled RCTs does not imply that cell therapy has no therapeutic effect in patients with CLI. Rather,

more placebo-controlled RCTs adequately powered through sufficient randomized participants are required for the reassessment of the potential efficacy of cell therapy with the respective different cell types. There are several limitations of our meta-analysis, which were inherent to the included RCTs. Firstly, the cell types, cell concentrations, route of administration, methods of assessment,

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Figure 4. Odds ratio of all-cause mortality in patients with CLI treated with cell therapy versus no cell therapy (random effects model).

time of outcome assessment, and duration of follow-up were inconsistent between RCTs. For instance, the cell type varied from a specific cell type (CD133Ăž cells or CD34Ăž cells) to unfractionated bone marrow concentrate. The number of cells administered could be up to over 300 million cells per subject. The cells were administered either intramuscularly (predominantly) or intra-arterially. The method of assessment varied and the time of assessment and duration of follow-up ranged from 1 month to 2 years. Nevertheless, there was no evidence of significant heterogeneity, apart from the possible direct effect of repeated cell administration via intra-arterial route on ABI outcome. Secondly, the results from this meta-analysis may still remain hypothesis generating from the efficacy standpoint since a definitive efficacy profile can only be determined from phase 3 RCTs. Thirdly, we considered the all-cause mortality as a surrogate marker of efficacy rather than a safety outcome. The latter is more accurately assessed with the inclusion of all

published studies and not limited to RCTs. Nevertheless, the trend toward lower all-cause mortality is reassuring. Fourthly, there remains a lack of clarity in terms of the cell purity in the majority of included RCTs, which may have attenuated their potential efficacy signal. Finally, the cell potency underpinning its therapeutic effect was not evaluated, predominantly due to the paucity of currently available standardized potency assays. This is relevant since Altaner et al have shown that the therapeutic effect of BM-MNCs in patients with CLI is determined by differential expression of cell surface markers, secreted factors, and gene expressions.32 While cumulative data on cell therapy for CLI are promising, more data are awaited from well-designed large, placebo-controlled, double-blind RCTs (BONMOT-CLI,33 NCT01483898, NCT01245335, NCT00434616, and NCT00539266). The phenotype of the cells needs to be much more clearly defined and the exact mode of action of the cell

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Angiology therapy requires further elucidation, in particular, whether its action is exerted in a paracrine manner, since this might lead to meaningful markers of functional potency. Thus, optimally designed future trials must include fixed cell numbers with well-defined phenotype and potency in a large double-blind, sham, and placebo-controlled phase 3 RCTs, in a homogenous patient cohort (stratified based on the presence of ulcer, diabetes, or other cardiovascular risk factors), to allow better comparison between trials.

Conclusion In summary, our meta-analysis suggests that cell therapy for patients with CLI is feasible and safe, with preliminary assessments of efficacy of these cell types, encouraging. However, additional, carefully designed future double-blind, sham, and placebo-controlled RCTs in a large, homogenous patient cohort, with a clearly defined cell type, cell number, and potency are needed to confirm its true potential therapeutic effect. Finally, data on the long-term safety profile and exact mechanism of action of cell therapy will be a prerequisite if cell therapy is to progress toward routine clinical practice.

Figure 5. Risk of bias summary: review authorsâ&#x20AC;&#x2122; judgments about each risk of bias item for each included study.

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Appendix A Table A1. PRISMA Checklist.a Section/topic TITLE Title ABSTRACT Structured summary

INTRODUCTION Rationale Objectives

# Checklist item

Reported on page #

1 Identify the report as a systematic review, meta-analysis, or both.

Title page

Abstract page 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. 3 Describe the rationale for the review in the context of what is already Introduction section known. 4 Provide an explicit statement of questions being addressed with reference to Introduction section participants, interventions, comparisons, outcomes, and study design (PICOS).

METHODS Protocol and registration

5 Indicate if a review protocol exists, if and where it can be accessed (eg, Web address), and, if available, provide registration information including registration number. Eligibility criteria 6 Specify study characteristics (eg, PICOS, length of follow-up) and report characteristics (eg, years considered, language, publication status) used as criteria for eligibility, giving rationale. Information sources 7 Describe all information sources (eg, databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. Study selection 9 State the process for selecting studies (ie, screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). Data collection process 10 Describe method of data extraction from reports (eg, piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. Data items 11 List and define all variables for which data were sought (eg, PICOS, funding sources) and any assumptions and simplifications made. Risk of bias in individual 12 Describe methods used for assessing risk of bias of individual studies, and studies how this information is to be used in any data synthesis. Summary measures 13 State the principal summary measures (eg, risk ratio, difference in means). Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (eg, I2) for each meta-analysis. Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (eg, publication bias, selective reporting within studies). Additional analyses 16 Describe methods of additional analyses (eg, sensitivity or subgroup analyses, meta-regression), if done, indicating which were prespecified. RESULTS Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. Study characteristics 18 For each study, present characteristics for which data were extracted (eg, study size, PICOS, follow-up period) and provide the citations. Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). Results of individual studies

N/A

Method section

Method section, appendices B&C Method section Method section

Method section Method section Method section Method section Method section Method section

Results and Discussion section

Results and Discussion section, Table 2, supplementary Figures 6-10 20 For all outcomes considered (benefits or harms), present, for each study: (a) Results and Discussion section, Table 2, simple summary data for each intervention group (b) effect estimates and Figures 1-4, supplementary confidence intervals, ideally with a forest plot. Figures 6-10 (continued)

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Table A1. (continued) Section/topic

# Checklist item

Reported on page #

Synthesis of results

21 Present results of each meta-analysis done, including confidence intervals and Results and Discussion section, measures of consistency. Table 2 Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15). Results and Discussion section, Table 2 Additional analysis 23 Give results of additional analyses, if done (eg, sensitivity or subgroup Results and Discussion section, analyses, meta-regression [see Item 16]). Table 2 DISCUSSION Summary of evidence 24 Summarize the main findings including the strength of evidence for each main Results and Discussion section outcome; consider their relevance to key groups (eg, health care providers, users, and policy makers). Results and Discussion section Limitations 25 Discuss limitations at study and outcome level (eg, risk of bias), and at review-level (eg, incomplete retrieval of identified research, reporting bias). Conclusions 26 Provide a general interpretation of the results in the context of other Conclusion section evidence, and implications for future research. FUNDING Funding 27 Describe sources of funding for the systematic review and other support (eg, N/A supply of data); role of funders for the systematic review. Abbreviation: N/A, not applicable. a Adapted from Moher D et al.34

Appendix B PubMed Database Search Strategy

Appendix C. (continued)

The following search terms were used in the PubMed database: ‘‘critical limb ischaemia’’ OR ‘‘critical limb ischemia’’ OR ‘‘peripheral artery disease’’ OR ‘‘peripheral arterial disease’’ OR ‘‘peripheral vascular disease’’ OR ‘‘peripheral arteriosclerosis’’ OR ‘‘arteriosclerosis obliterans’’ AND ‘‘stem cell’’ OR ‘‘bone marrow concentrate’’ OR ‘‘bone marrow derived mononuclear cell’’ OR ‘‘peripheral blood derived mononuclear cell’’ OR ‘‘peripheral mononuclear cell’’ OR ‘‘granulocyte colony stimulating factor’’ OR ‘‘GCSF’’ OR ‘‘CD34’’ OR ‘‘CD133’’ OR ‘‘mesenchymal stromal cell’’ OR ‘‘mesenchymal stem cell’’ OR ‘‘adipose tissue derived stem cell’’ OR ‘‘umbilical cord derived mononuclear cell’’ OR ‘‘umbilical cord derived mesenchymal stem cell’’ OR ‘‘umbilical cord derived mesenchymal stromal cell’’ OR ‘‘umbilical cord stem cell’’ OR ‘‘hematopoietic stem cell’’ OR ‘‘haematopoietic stem cell’’ OR ‘‘endothelial progenitor cell’’ OR ‘‘circulating angiogenic cell’’ OR ‘‘outgrowth endothelial cell.’’

Appendix C Cochrane Library Database Search Strategy ID

#1 #2

MeSH descriptor: [Stem Cell Transplantation] explode all trees MeSH descriptor: [Peripheral Blood Stem Cell Transplantation] explode all trees MeSH descriptor: [Hematopoietic Stem Cell Mobilization] explode all trees

#4 #5

MeSH descriptor: [Bone Marrow Cells] explode all trees MeSH descriptor: [Granulocyte Colony-Stimulating Factor] explode all trees MeSH descriptor: [Mesenchymal Stem Cell Transplantation] explode all trees MeSH descriptor: [Mesenchymal Stromal Cells] explode all trees MeSH descriptor: [Leukocytes, Mononuclear] explode all trees MeSH descriptor: [Cord Blood Stem Cell Transplantation] explode all trees MeSH descriptor: [Bone Marrow Transplantation] explode all trees bone marrow cell endothelial progenitor cell circulating angiogenic cell adipose derived stem cell mononuclear cell bone marrow concentrate (BM-MNC* or PB-MNC* or M-PB-MNC* or BM-MSC* or AT-MSC* or ADSC* or UC-MSC* or EPC* or CAC* or OEC*): ti, ab, kw MeSH descriptor: [Arteriosclerosis Obliterans] explode all trees MeSH descriptor: [Arteriosclerosis] explode all trees MeSH descriptor: [Atherosclerosis] explode all trees MeSH descriptor: [Arterial Occlusive Diseases] explode all trees MeSH descriptor: [Peripheral Vascular Diseases] explode all trees critical limb isch* or CLI #1 or #2 or #3 or #4 or #5 or #6 or #7 or #8 or #9 or #10 or #11 or #12 or #13 or #14 or #15 or #16 or #17 #18 or #19 or #20 or #21 or #22 or #23 #24 and #25

#6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17

#18 #19 #20 #21 #22 #23 #24 #25 #26

(continued)

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11 AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Pfizer, Daichii Sankyo, Merck Sharp & Dohme, Novo Nordisk, Novartis, Sanofi-Aventis, and Medtronic over the last ten years. He is an author on a patent. Professor James Shaw perceives no conflict of interest with this manuscript but has previously served as a member of the speakers’ bureau for Lilly Deutschland GmbH. Both Professor Gerard Stansby and Mr. Vish Bhattacharya have declared no conflict of interest.

Appendix D

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental Material The online data supplements are available at http://ang.sagepub.com/ supplemental.

References

Figure D1. Study Flow Diagram.

Author Contribution AL, VB, and GS were involved in data acquisition. All authors: (1) had a substantial contributions to the conception and design of this study; (2) were involved in analysis and interpretation of data; (3) were involved in drafting the article and revising the article critically for important intellectual content; and (4) have given final approval of the version to be published.

Declaration of Conflicting Interests Dr. Aaron Liew perceives no conflict of interest with this manuscript but has previously received educational and research support from

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