Stem cell therapy for chronic ischaemic heart disease and congestive heart failure (Review)

Stem cell therapy for chronic ischaemic heart disease and congestive heart failure (Review) Fisher SA, Brunskill SJ, Doree C, Mathur A, Taggart DP, Martin-Rendon E This is a reprint of a Cochrane review, prepared and maintained by The Cochrane Collaboration and published in The Cochrane Library 2014, Issue 4 http://www.thecochranelibrary.com

T A B L E O F C O N T E N T S HEADER....................................... 1 ABSTRACT...................................... 1 PLAIN LANGUAGE SUMMARY.............................. 2 SUMMARY OF FINDINGS FOR THE MAIN COMPARISON................... 3 BACKGROUND.................................... 6 OBJECTIVES..................................... 7 METHODS...................................... 7 RESULTS....................................... 10 Figure 1...................................... 11 Figure 2...................................... 14 Figure 3...................................... 16 DISCUSSION..................................... 21 AUTHORS CONCLUSIONS............................... 24 ACKNOWLEDGEMENTS................................ 24 REFERENCES..................................... 24 CHARACTERISTICS OF STUDIES............................. 34 DATA AND ANALYSES.................................. 107 Analysis 1.1. Comparison 1 Stem cells versus no stem cells, Outcome 1 Mortality (any)........... 112 Analysis 1.2. Comparison 1 Stem cells versus no stem cells, Outcome 2 LVEF (%): Short-term follow-up (< 12 months). 114 Analysis 1.3. Comparison 1 Stem cells versus no stem cells, Outcome 3 LVEF (%): Long-term follow-up ( 12 months). 116 Analysis 1.4. Comparison 1 Stem cells versus no stem cells, Outcome 4 Adverse effects........... 117 Analysis 1.5. Comparison 1 Stem cells versus no stem cells, Outcome 5 Infarction............. 118 Analysis 1.6. Comparison 1 Stem cells versus no stem cells, Outcome 6 Rehospitalisation due to heart failure.... 120 Analysis 1.7. Comparison 1 Stem cells versus no stem cells, Outcome 7 Angina episodes per week: short term follow-up (<12 months).................................. 121 Analysis 1.8. Comparison 1 Stem cells versus no stem cells, Outcome 8 NYHA Classification: short-term follow-up (< 12 months).................................... 122 Analysis 1.9. Comparison 1 Stem cells versus no stem cells, Outcome 9 NYHA Classification: long-term follow-up ( 12 months).................................... 123 Analysis 1.10. Comparison 1 Stem cells versus no stem cells, Outcome 10 CCS class: short-term follow-up (< 12 months).................................... 124 Analysis 1.11. Comparison 1 Stem cells versus no stem cells, Outcome 11 Exercise capacity: short-term follow-up (< 12 months).................................... 125 Analysis 1.12. Comparison 1 Stem cells versus no stem cells, Outcome 12 Exercise capacity: long-term follow-up ( 12 months).................................... 126 Analysis 1.13. Comparison 1 Stem cells versus no stem cells, Outcome 13 LVESV (ml): short-term follow-up (< 12 months).................................... 127 Analysis 1.14. Comparison 1 Stem cells versus no stem cells, Outcome 14 LVESV (ml): long-term follow-up ( 12 months).................................... 129 Analysis 1.15. Comparison 1 Stem cells versus no stem cells, Outcome 15 LVEDV (ml): short-term follow-up (< 12 months).................................... 130 Analysis 1.16. Comparison 1 Stem cells versus no stem cells, Outcome 16 LVEDV (ml): long-term follow-up ( 12 months).................................... 132 Analysis 1.17. Comparison 1 Stem cells versus no stem cells, Outcome 17 Stroke volume index: short term follow-up (<12 months).................................... 133 Analysis 1.18. Comparison 1 Stem cells versus no stem cells, Outcome 18 Stroke volume index: long term follow-up ( 12 months).................................... 134 Analysis 2.1. Comparison 2 Cell dose: subgroup analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months). 135 Analysis 2.2. Comparison 2 Cell dose: subgroup analysis, Outcome 2 NYHA Classification: short-term follow-up (< 12 months).................................... 137 i

Analysis 3.1. Comparison 3 Baseline cardiac function: subgroup analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months)................................... 138 Analysis 3.2. Comparison 3 Baseline cardiac function: subgroup analysis, Outcome 2 NYHA Classification: short-term follow-up (< 12 months)............................... 139 Analysis 4.1. Comparison 4 Route of cell administration: subgroup analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months).................................. 141 Analysis 4.2. Comparison 4 Route of cell administration: subgroup analysis, Outcome 2 NYHA Classification: short-term follow-up (< 12 months)............................... 142 Analysis 5.1. Comparison 5 Cell type: subgroup analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months). 144 Analysis 5.2. Comparison 5 Cell type: subgroup analysis, Outcome 2 NYHA Classification: short-term follow-up (< 12 months).................................... 146 Analysis 6.1. Comparison 6 Participant diagnosis: subgroup analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months).................................... 147 Analysis 6.2. Comparison 6 Participant diagnosis: subgroup analysis, Outcome 2 NYHA Classification: short-term followup (< 12 months)................................. 149 Analysis 7.1. Comparison 7 Method of measurement: sensitivity analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months)................................... 150 Analysis 8.1. Comparison 8 Risk of bias: sensitivity analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months). 152 Analysis 9.1. Comparison 9 Co-intervention/comparator sensitivity analysis, Outcome 1 LVEF (%): short-term follow-up (< 12 months).................................. 154 ADDITIONAL TABLES.................................. 155 APPENDICES..................................... 162 CONTRIBUTIONS OF AUTHORS............................. 166 DECLARATIONS OF INTEREST.............................. 166 SOURCES OF SUPPORT................................. 166 DIFFERENCES BETWEEN PROTOCOL AND REVIEW..................... 167 ii

[Intervention Review] Stem cell therapy for chronic ischaemic heart disease and congestive heart failure Sheila A Fisher 1, Susan J Brunskill 1, Carolyn Doree 1, Anthony Mathur 2, David P Taggart 3, Enca Martin-Rendon 4 1 Systematic Review Initiative, NHS Blood and Transplant, Oxford, UK. 2 Department of Clinical Pharmacology, William Harvey Research Institute, London, UK. 3 Oxford Heart Centre, John Radcliffe Hospital, Oxford, UK. 4 Stem Cell Research Department, NHS Blood and Transplant, Oxford, UK Contact address: Enca Martin-Rendon, Stem Cell Research Department, NHS Blood and Transplant, John Radcliffe Hospital, Headington, Oxford, OX3 9BQ, UK. Enca.Rendon@ndcls.ox.ac.uk. Editorial group: Cochrane Heart Group. Publication status and date: New, published in Issue 4, 2014. Review content assessed as up-to-date: 22 May 2013. Citation: Fisher SA, Brunskill SJ, Doree C, Mathur A, Taggart DP, Martin-Rendon E. Stem cell therapy for chronic ischaemic heart disease and congestive heart failure. Cochrane Database of Systematic Reviews 2014, Issue 4. Art. No.: CD007888. DOI: 10.1002/14651858.CD007888.pub2. Background A B S T R A C T A promising approach to the treatment of chronic ischaemic heart disease (IHD) and heart failure is the use of stem cells. The last decade has seen a plethora of randomised controlled trials (RCTs) developed worldwide which have generated conflicting results. Objectives The critical evaluation of clinical evidence on the safety and efficacy of autologous adult bone marrow-derived stem cells (BMSC) as a treatment for chronic ischaemic heart disease (IHD) and heart failure. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library, 2013, Issue 3), MEDLINE (from 1950), EMBASE (from 1974), CINAHL (from 1982) and the Transfusion Evidence Library (from 1980), together with ongoing trial databases, for relevant trials up to 31st March 2013. Selection criteria Eligible studies included RCTs comparing autologous adult stem/progenitor cells with no autologous stem/progenitor cells in participants with chronic IHD and heart failure. Co-interventions such as primary angioplasty, surgery or administration of stem cell mobilising agents, were included where administered to treatment and control arms equally. Data collection and analysis Two review authors independently screened all references for eligibility, assessed trial quality and extracted data. We undertook a quantitative evaluation of data using fixed-effect meta-analyses. We evaluated heterogeneity using the I² statistic; we explored considerable heterogeneity (I² > 75%) using a random-effects model and subgroup analyses. 1

Main results We include 23 RCTs involving 1255 participants in this review. Risk of bias was generally low, with the majority of studies reporting appropriate methods of randomisation and blinding, Autologous bone marrow stem cell treatment reduced the incidence of mortality (risk ratio (RR) 0.28, 95% confidence interval (CI) 0.14 to 0.53, P = 0.0001, 8 studies, 494 participants, low quality evidence) and rehospitalisation due to heart failure (RR 0.26, 95% CI 0.07 to 0.94, P = 0.04, 2 studies, 198 participants, low quality evidence) in the long term ( 12 months). The treatment had no clear effect on mortality (RR 0.68, 95% CI 0.32 to 1.41, P = 0.30, 21 studies, 1138 participants, low quality evidence) or rehospitalisation due to heart failure (RR 0.36, 95% CI 0.12 to 1.06, P = 0.06, 4 studies, 236 participants, low quality evidence) in the short term (< 12 months), which is compatible with benefit, no difference or harm. The treatment was also associated with a reduction in left ventricular end systolic volume (LVESV) (mean difference (MD) -14.64 ml, 95% CI -20.88 ml to -8.39 ml, P < 0.00001, 3 studies, 153 participants, moderate quality evidence) and stroke volume index (MD 6.52, 95% CI 1.51 to 11.54, P = 0.01, 2 studies, 62 participants, moderate quality evidence), and an improvement in left ventricular ejection fraction (LVEF) (MD 2.62%, 95% CI 0.50% to 4.73%, P = 0.02, 6 studies, 254 participants, moderate quality evidence), all at longterm follow-up. Overall, we observed a reduction in functional class (New York Heart Association (NYHA) class) in favour of BMSC treatment during short-term follow-up (MD -0.63, 95% CI -1.08 to -0.19, P = 0.005, 11 studies, 486 participants, moderate quality evidence) and long-term follow-up (MD -0.91, 95% CI -1.38 to -0.44, P = 0.0002, 4 studies, 196 participants, moderate quality evidence), as well as a difference in Canadian Cardiovascular Society score in favour of BMSC (MD -0.81, 95% CI -1.55 to -0.07, P = 0.03, 8 studies, 379 participants, moderate quality evidence). Of 19 trials in which adverse events were reported, adverse events relating to the BMSC treatment or procedure occurred in only four individuals. No long-term adverse events were reported. Subgroup analyses conducted for outcomes such as LVEF and NYHA class revealed that (i) route of administration, (ii) baseline LVEF, (iii) cell type, and (iv) clinical condition are important factors that may influence treatment effect. Authors conclusions This systematic review and meta-analysis found moderate quality evidence that BMSC treatment improves LVEF. Unlike in trials where BMSC were administered following acute myocardial infarction (AMI), we found some evidence for a potential beneficial clinical effect in terms of mortality and performance status in the long term (after at least one year) in people who suffer from chronic IHD and heart failure, although the quality of evidence was low. P L A I N L A N G U A G E S U M M A R Y Stem cell treatment for chronic ischaemic heart disease and congestive heart failure Those suffering from heart disease and heart failure are currently treated with drugs and, when possible, the blood supply is restored in the heart (revascularisation) either by opening the arteries with a tiny balloon in a procedure called primary angioplasty (or percutaneous coronary intervention (PCI)) or by heart surgery (or coronary artery bypass graft (CABG)). Revascularisation has reduced the death rate associated with these conditions. In some people heart disease and heart failure symptoms persist even after revascularisation. Those people may not have other treatments available to them. Recently, bone marrow stem/progenitor cells have been investigated as a new treatment for people with heart disease and heart failure, whether they are also treated for revascularisation or not. Results from 23 randomised controlled trials, covering more than 1200 participants, to 2013 indicates that this new treatment leads to a reduction in deaths and readmission to hospital and improvements over standard treatment as measured by tests of heart function. At present, these results provide some evidence that stem cell treatment may be of benefit in people both with chronic ischaemic heart disease and with heart failure. Adverse events are rare, with no long-term adverse events reported. However, the quality of the evidence is relatively low because there were few deaths and hospital readmissions in the studies, and individual study results varied. Further research involving a large number of participants is required to confirm these results. 2

S U M M A R Y O F F I N D I N G S F O R T H E M A I N C O M P A R I S O N [Explanation] Bone marrow stem cells(bmsc)(intervention) compared with control(no intervention) for chronic ischaemic heart disease and congestive heart failure Patient or population: People with chronic ischaemic heart disease and congestive heart failure Settings:[setting] Intervention: Bone marrow stem cells Comparison: Control(no cells) Outcomes Illustrative comparative risks*(95% CI) Relative effect (95% CI) Mortality - Short-term follow-up(<12 months) Mortality - Long-term follow-up( 12 months) Assumed risk Control 25per1000 148per1000 Corresponding risk BMSC 14per1000 (9to35) 27per1000 (8to78) No of Participants (studies) RR 0.68(0.32 to 1.41) 1138 participants (21 studies) RR 0.28(0.14 to 0.53) 494 participants (8 studies) Quality of the evidence (GRADE) low low Comments A combination of low events and discordant results from one study leads to low confidence in the estimate of the effect. This is likely to change with further research As above. Rehospitalisation due to heart failure- Short-term follow-up(<12 months) 95per1000 33per1000 (5to101) RR 0.36(0.12 to 1.06) 236 participants (4 studies) low As above. Rehospitalisation due to heart failure- Long-term follow-up( 12 months) 92per1000 23per1000 (3to86) RR 0.26(0.07 to 0.94) 198 participants (2 studies) low As above. 3

LVEF(%):meanchange from baseline to end of study(<12 months) LVEF(%):meanchange from baseline to end of study( 12 months) NYHA classification: mean value at end ofstudy- Class I to IV (< 12 months) See comments See comments MD 4.22%(3.47 to 4.97) 746 participants (18 studies) See comments See comments MD 2.62%(0.50 to 4.73) 254 participants (6 studies) See comments See comments MD-0.63(-1.08 to-0.19) 486 participants (11 studies) moderate moderate moderate LVEF measurements are given in percentage. There is lack of appropriate blinding in a number of studies, which increases the risk of bias and moderate statistical heterogeneity LVEF measurements are given in percentage. There is lack of appropriate blinding in a number of studies, which increases the risk of bias and moderate statistical heterogeneity NYHAClassI(1),II(2),III (3)andIV(4). There is lack of appropriateblindinginanumberof studies which increases the risk of bias, and high statistical heterogeneity NYHA classification: mean value at end ofstudy- Class I to IV ( 12 months) See comments See comments MD-0.91(-1.38 to-0.44) 196 participants (4 studies) moderate NYHAClassI(1),II(2),III (3)andIV(4). There is lack of appropriateblindinginanumberof studies which increases the risk of bias, and high statistical heterogeneity *The assumed risk is provided as the median control group risk across studies. The corresponding risk(and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention(and its 95% CI). CI: Confidence interval; RR: Risk Ratio; MD: Mean Difference. 4

GRADE Working Group grades of evidence High quality: Further research is very unlikely to change our confidence in the estimate of effect. Moderate quality: Further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate. Lowquality:Furtherresearchisverylikelytohaveanimportantimpactonourconfidenceintheestimateofeffectandislikelytochangetheestimate. Very low quality: We are very uncertain about the estimate. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 5

B A C K G R O U N D Description of the condition Ischaemic heart disease (IHD) is a major cause of mortality and morbidity and the main cause of congestive heart failure (CHF) in Western societies (BHF 2008). Despite current therapies having increased the short-term survival of people suffering from myocardial infarction (MI), the number of people with CHF is rapidly increasing. In IHD, although some of the myocardium has been replaced by scar tissue, the heart may prevent the death of cardiomyocytes by reducing the energy demands of contraction. This results in non-contracting or hibernating, but viable, myocardium. This is a physiological response to chronic hypoxic stress, which is identifiable by electromechanical dissociation, and potentially reversible by revascularisation of the hibernating myocardium in order to restore cardiac function. As with acute MI (AMI), pharmacological therapy, angioplasty and bypass surgery are the standard treatments offered to those suffering from CHF. In the acute setting, angioplasty restores the normal flow in infarct-related arteries in more than 90% of those who have suffered MI (Grines 1999; Stone 1998). This early revascularisation of the occluded artery after AMI has improved the prognosis, although a significant number of people still develop CHF. Preventing the progression of IHD and the development of CHF therefore remains a challenge. In some cases, people who have already received angioplasty or bypass surgery are treated with maximal medical therapy, but they still present symptoms of chronic myocardial ischaemia, sometimes with refractory angina. Alternative therapies for CHF, such as stem/progenitor cell transplantation, are being investigated to complement current pharmacological therapies, primary angioplasty and cardiac surgery. This approach to the treatment of CHF developed from the observation in animal models that mononuclear cells from bone marrow or mobilised peripheral blood were effective in cardiac repair (Deb 2003; Orlic 2001a; Orlic 2001b; Yoon 2005). Later, it was demonstrated in the first non-randomised trials that cardiac function was improved when bone marrow stem/progenitor cells were transplanted into the infarcted myocardium (Assmus 2002; Strauer 2002). Although the stem cell type contributing to the repair of damaged tissue was not well defined, a study by Stamm 2003 indicated that the delivery of CD133 progenitor cells from haematopoietic tissues (e.g. bone marrow and blood) into the ischaemic cardiac muscle could improve revascularisation. This has resulted in a number of larger randomised controlled trials (RCTs) worldwide, for AMI (Janssens 2006; Lunde 2006; Meyer 2006; Schächinger 2006) and chronic heart failure (Assmuss 2006; Erbs 2005; Hendrikx 2006; Patel 2006; Stamm 2007). The procedure is currently as follows: either the bone marrow is harvested from the recipient or stem/progenitor cells are mobilised into circulation by a growth factor stimulant (most commonly granulocyte colony-stimulating factor (G-CSF)). In the first procedure, cells are usually collected (sometimes under general anaesthesia) from the pelvic bone, using large suction needles. Thereafter, the cells are separated from other bone marrow cells in sterile conditions. The bone marrow harvest and separation of stem cells may take several hours. In the G-CSF mobilisation procedure, stem/progenitor cells are collected as a blood sample and then separated from other blood cells in sterile conditions. In both procedures, the stem/progenitor cells are infused directly into the recipient s coronary arteries or heart. The first procedure delivers the cells to the coronary arteries via a special balloon-catheter during angioplasty (e.g. percutaneous coronary intervention (PCI)) using a stop-flow technique. The second procedure administers the cells into the heart muscle during an angioplasty-like procedure using electromechanical mapping and direct intramyocardial injection (e.g. NOGA system) or during cardiac surgery (e.g. coronary artery bypass grafting (CABG)). The interval between the collection of the stem cells and their reinfusion varies, but as the stem cells are used fresh the time cannot be too long unless they undergo some form of culture and expansion ex vivo. The collection of the stem cells is most probably undertaken by a haematologist. A specialised technician or scientist undertakes the separation of the stem/progenitor cells from the other bone marrow cells and the cardiologist or cardiac surgeon undertakes the infusion or intramyocardial injection of the cells. There are no adverse effects associated with the administration of stem cells as a treatment for people with chronic IHD or heart failure. In those trials where G-CSF has been administered prior to the stem cell harvest, transient complications arising from the G-CSF treatment have been described. However, no long-term adverse effects have been reported. This treatment is currently only available in research-associated facilities, but it is conceivable that, if long-term effectiveness is confirmed, this procedure may be available to some or all people with chronic heart disease, since bone marrow harvest is a standard procedure used in bone marrow transplantation. The most effective results so far have been found in recipients with low left ventricular ejection fraction (LVEF) and heart failure symptoms (Pokushalov 2010). The costs may be high, depending on the procedures used, and currently relate to the costs of the stem/progenitor cell procedure (stem cell harvest) and to the costs of the collection of the stem/ progenitor cells (approximately a tenth of the overall cost of the trial). The potential for a large, funded clinical trial is limited, as there are no intellectual property rights associated with this procedure in its current form, rendering it unattractive to private company funding. Description of the intervention 6

How the intervention might work The mechanisms of the beneficial effects of bone marrow-derived stem cells (BMSC) remain unclear, and clinical trials in which BMSC have been administered to participants with acute myocardial infarction (AMI) and chronic myocardial ischaemia have produced divergent results. The type of stem cell contributing to the repair of the damaged tissue or the amelioration of tissue damage is still not well defined and the mechanism of action is not yet fully understood. Bone marrow-, cord blood- or peripheral bloodderived stem cells may exert their effects on cardiac function by increasing vascularity via endothelial progenitor cell incorporation into the ischaemic tissue, by generating cardiomyocytes, by modulating cardiac remodelling and/or, in a paracrine fashion, by producing cytokines or other factors that may help to promote cardiac repair and limit fibrosis in the affected area (Beltrami 2003; Carr 2008; Martin-Rendon 2008a; Mathur 2004; Stuckey 2006; Yoon 2005). Why it is important to do this review Stem cell therapy represents an exciting new form of treatment for many diseases. Heart disease is one of the clinical settings in which to address this new form of therapy, although the exact clinical role for stem cell therapy remains to be defined. A recent systematic review (Martin-Rendon 2008b; Martin-Rendon 2008c) of stem cell treatment for AMI found that stem cell treatment may lead to some improvements over conventional therapy as measured by surrogate tests of heart function, although further trials are required to confirm that these changes translate into improvements in long-term survival and are not accompanied by side effects. A number of RCTs have been undertaken and published exploring a clinical role for stem cell treatment in people with chronic IHD and heart failure. This is a clinical group with defined treatment options and problems sufficiently different from those who have suffered an AMI to indicate the need for a new systematic review. In addition, several RCTs have generated contradictory results. Since the use of stem/progenitor cells for cardiac tissue repair is such a recent intervention in clinical practice, it is important that a systematic review is undertaken at an early stage to assess the safety and efficacy of this intervention. We define safety as the absence of adverse events (e.g. increased mortality and morbidity, increased risk of infarction, restenosis and arrhythmias), and efficacy as a significant improvement in cardiac function, clinical outcomes and quality of life. O B J E C T I V E S The critical evaluation of clinical evidence on the safety and efficacy of autologous adult bone marrow-derived stem cells (BMSC) as a treatment for chronic ischaemic heart disease (IHD) and heart failure. M E T H O D S Criteria for considering studies for this review Types of studies Randomised controlled trials (RCTs). Types of participants Anyone with a clinical diagnosis of IHD or congestive heart failure (CHF), excluding people with acute myocardial infarction (AMI). Types of interventions Studies involving the administration of autologous adult stem cells on their own or in combination with co-interventions, such as cardiac surgery, as treatment for IHD or CHF. Participants in the comparator treatment arm of the trial received either no intervention or a placebo (e.g. the medium in which the stem cells were suspended or plasma). Trials where co-interventions (e.g. coronary artery bypass graft (CABG), percutaneous coronary intervention (PCI), granulocyte colony-stimulating factor (G-CSF), extracorporal shockwave therapy) were additionally administered were eligible as long as the co-interventions were equal in both arms and administered to an equivalent proportion of participants. In summary: 1. Any autologous human adult stem cells 2. Any single dose 3. Any method of stem cell isolation 4. Any route of administration 5. Any co-intervention 6. Repeated intervention or multiple doses. Types of outcome measures We divided beneficial outcomes into clinically-based and surrogate endpoints. At the protocol stage of this review, we had intended to consider clinical and surrogate endpoint data at 30 days, six months and 12 months after baseline; however, this was not possible due to the variation in follow-up periods reported in individual studies. We therefore stratified outcome data into short-term (up to 12 months) and long-term (12 months or longer) follow-up. 7

Primary outcomes Clinical Mortality. Surrogate endpoints Left ventricular ejection fraction (LVEF). Adverse events Searching other resources We searched the following trial registers for ongoing trials on 31 March 2013: Current Controlled Trials (ISRCTN), ClinicalTrials.gov, WHO International Clinical Trials Registry Platform (IC- TRP), UMIN-CTR (Japanese Clinical Trials Registry) and the Hong Kong Clinical Trials Register. We checked the reference lists of all identified eligible papers and relevant narrative reviews. We applied no language or date restrictions to any of the searches. Data collection and analysis Secondary outcomes Clinical 1. Morbidity (infarction, heart failure, arrhythmias); 2. Composite outcome of morbidity and infarction; 3. Health-related quality of life; 4. Performance status; Surrogate endpoints 1. Engraftment and survival of the infused stem cells; 2. End-systolic volume; 3. End-diastolic volume; 4. Wall motion score; 5. Stroke volume index. Search methods for identification of studies Electronic searches We identified relevant studies from searches of the Cochrane Central Register of Controlled trials (CENTRAL) on The Cochrane Library 2013, Issue 3, and the Cochrane Heart Group s Trials Register, MEDLINE (1948 to 31 March 2013), PubMed (epublications only, 31 March 2013), EMBASE (1974 to 31 March 2013) and CINAHL (1982 to 31 March 2013). We combined the MEDLINE search with RCT search filters based on the validated Cochrane MEDLINE filter according to the current version of the Cochrane Handbook for Systematic Reviews of Interventions, section 6.4.11.1 (Higgins 2011). We also searched the databases LILACS, KoreaMed, IndMed, PakMediNet and the Transfusion Evidence Library on 31 March 2013, using a selection of keywords. See Appendix 1 for details of the search strategies. Selection of studies The information specialist (CD) conducted the electronic search for potentially relevant papers and removed references that were duplicates, clearly irrelevant and/or included in previous search results. Two review authors (SAF, EMR) screened all titles and abstracts identified by the review search strategy for relevance to the review question. We excluded only studies that were clearly irrelevant at this stage, and assessed all other studies as full-text articles for inclusion or exclusion using the criteria indicated above (type of studies, participants, interventions and outcome measures). At this point, two review authors (SAF, EMR) independently assessed eligibility using ad hoc eligibility forms, and resolving disagreements between them by discussion. Data extraction and management We extracted data onto tailored data extraction forms which were created and piloted specifically for this review. Two review authors (EMR, SAF) undertook data extraction for all eligible studies independently. Aside from details relating to the quality of included studies, we extracted the following two groups of data: (1) Study characteristics: place of publication, date of publication, population characteristics, setting, detailed nature of intervention, detailed nature of comparator, detailed nature of outcomes. A key purpose of these data was to explain clinical heterogeneity between included studies independently from analysis of the results; (2) Results of included studies for each of the main outcomes indicated in the review question. For dichotomous outcomes we recorded the numbers of outcomes in treatment and control groups. For continuous outcomes, we recorded the mean and standard deviation. We resolved data extraction disagreements by consensus between the review authors. When disagreements regarding any of the above were unclear, we attempted to contact authors of the original trials to provide further details. One review author (SAF) then transcribed the data into the systematic review computer software Review Manager 5 (Review Manager 2012). 8

Assessment of risk of bias in included studies The two review authors (SAF, EMR) undertaking the data extraction independently assessed the risk of bias for each trial using the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), and resolving any disagreements by discussion. We assessed the design, conduct and analysis of the trial using a three-point scale: low, high or unclear risk of bias. To assess risks of bias, the authors included the following questions in the Risk of bias table for each included study: 1. Was the allocation sequence adequately generated? 2. Was allocation adequately concealed? 3. Was knowledge of the allocated intervention adequately prevented (i.e. blinded) throughout the study? 4. Were incomplete outcome data adequately addressed for every outcome? 5. Were reports of the study free of selective outcome reporting? 6. Was the study apparently free of other problems that could put it at risk of bias? With reference to (1) to (6) above, the review authors assessed the likely magnitude and direction of the bias and whether they considered it likely to impact on the findings. Measures of treatment effect We expressed dichotomous data for each arm in a particular study as a proportion or risk and the treatment effect as a risk ratio (RR) with 95% confidence intervals (CIs). We expressed continuous data for each arm in a particular study as a mean and standard deviation, and the treatment effect as the mean difference (MD) if outcomes were measured in the same way across trials. For outcomes measured using different methods, we combined the treatment effect data and analysed them using the standardised mean difference (SMD). We used fixed-effect models in the first instance, but random-effects models in the presence of considerable heterogeneity (I² greater than 75%) or for outcomes which were measured using different methods across studies. Although we intended to analyse continuous outcomes as mean change from baseline, several studies only reported baseline and endpoint data. Where possible, we calculated the standard deviation of the mean change from baseline based on reported confidence intervals or P values, and we used these values in the analysis. However, for several studies, insufficient information was reported to calculate the standard deviation. Since the mean difference based on the change from baseline can be assumed to address the same underlying intervention effects as an analysis based on final measures (i.e. the differences in mean final values will on average be the same as the differences in mean change scores), we combined studies reporting mean change from baseline values with those reporting endpoint values, but presented mean change and endpoint values separately as well as in combined analyses for clarity. We did not conduct this pooling of studies by method of reporting of continuous measures for analyses of exercise capacity, since the assumption of consistent underlying effects does not hold for standardised mean differences. Several studies reported surrogate endpoints (left ventricular end systolic volume (LVESV), left ventricular end diastolic volume (LVEDV), stroke volume, left ventricular ejection fraction (LVEF)), using different measures (magnetic resonance imaging (MRI), echocardiography, single-photon emission computed tomography (SPECT), left ventricular angiography) and in some cases, results from several different methods were reported within a single study. In this case, we used MRI data as the preferred measure followed by left ventricular angiography, SPECT and echocardiography. Unit of analysis issues In studies in which there were multiple interventions in the same trial, we combined the intervention trial arms for a single comparison with the comparator (control) arm to avoid double counting of participants and potential correlation of results. However, for subgroup and sensitivity analyses, where the two intervention arms were classified into different categories (for example, type of cell, cell dose, route of administration of cells), we included results for each treatment arm in the corresponding group, with the control group included in both groups. In order to avoid unit of analysis issues, we treated cross-over trials as parallel trials and included them in the review up to the point of cross-over, i.e. first phase data only. Dealing with missing data We attempted to contact the authors of 15 studies by email for clarification of methods (randomisation, allocation concealment and blinding), potential overlapping of studies and/or requests for additional data. In five cases we failed to make contact with the authors by email. Of the remaining 10 studies, we received replies from three study authors who provided additional data. In one study (Assmus 2006), results were reported for a pooled randomised cohort and a non-randomised pilot study cohort; the authors of this study provided full clinical and surrogate endpoint data for the randomised cohort alone, as well as details of the method of randomisation. The authors of a second study (Hendrikx 2006) provided LVESV and LVEDV data (as only LVESV/LVEDV index values were reported). For a third study (Hu 2011), authors provided mean change from baseline data for surrogate endpoint measures. Assessment of reporting biases Although we believe that we made every effort to identify unpublished studies, we assessed publication bias using a funnel plot for the primary outcome of mortality. We accept that asymmetry, of 9

which publication bias may be one cause, is difficult to detect with the small numbers of studies (i.e. fewer than 10) often encountered in systematic reviews. Data synthesis We undertook meta-analyses using the Review Manager 5 software (Review Manager 2012), where there were sufficient data of suitable type. We used a fixed-effect model to combine data in the first instance. Where we detected considerable heterogeneity (I² greater than 75%) using a fixed-effect model, we repeated the analysis using a random-effects model. Although quantitative synthesis was the main method of analysis, we incorporated insights from a qualitative evaluation of studies for an overall interpretation of the data. We based conclusions on patterns of results identified across clearly-tabulated results of included studies as well as summary measures, taking both direction and magnitude of any effect into account. Subgroup analysis and investigation of heterogeneity We examined statistical heterogeneity using the I² statistic ( Higgins 2003) and by visual inspection of forest plots. We rated values of I² greater than 75% as indicating a considerable level of heterogeneity at which summary estimates should be explored and results interpreted with caution. We explored potential reasons for observed heterogeneity in comparisons with at least 10 studies and statistical significance in the observed effect (P < 0.05). We placed particular emphasis on study population, treatment, outcome measurements and study quality differences between the included studies. We assessed clinical heterogeneity based on the data extracted on the characteristics of the included studies. We planned subgroup analysis for mortality and LVEF (primary clinical and surrogate endpoint outcomes) as well as for outcomes which met the above conditions for further exploration of heterogeneity. Subgroup analysis considered the following factors: 1. Dose of stem cells administered; 2. Route of cell administration; 3. Baseline cardiac function; 4. Type of cell administered (mononuclear cells; circulating progenitor cells; haematopoietic progenitor cells; and mesenchymal stem cells); 5. Participant diagnosis (chronic IHD; heart failure (secondary to IHD); intractable/refractory angina); 6. Eligibility for revascularisation. We regard the latter three subgroup comparisons listed above as hypothesis-generating. Sensitivity analysis We assessed the robustness of the overall results for the primary outcome of LVEF for sensitivity to the following factors: 1. Risk of bias (selection bias; performance bias; detection bias; attrition bias); 2. Co-intervention or comparator; 3. Method of measurement (MRI, left ventricular angiography, SPECT, echocardiography). Differences in methods of reporting for continuous outcomes across trials led us to combine mean change from baseline and endpoint data for several outcomes (LVESV, LVEDV, stroke volume index, LVEF) (see Measures of treatment effect above). We present results separately as well as in combination to assess the sensitivity of the results to the method of reporting. R E S U L T S Description of studies Results of the search Given that a wide variety of products and terms have been used in the comparator arms of the included studies, for ease of reference we use the term control throughout this review to refer to the comparator treatment arm. We identified 7704 references from electronic database searches. De-duplication and removal of all clearly irrelevant references by the Information Specialist (CD) excluded 5370 references. Initial screening of the remaining 2334 citations against inclusion criteria excluded a further 2225 references. Of the remaining 109 citations, we subsequently excluded 25 references (describing 21 independent studies), as they did not fully meet the inclusion criteria (see Excluded studies). Eight further references described six independent study protocols (see Ongoing studies). Nine studies (14 references) were published in abstract form only and although they appeared to meet the inclusion criteria, did not contain sufficient data for inclusion; these have been identified as Studies awaiting classification. The remaining 62 citations describe a total of 23 independent RCTs (see Included studies). A summary of study classification is displayed in a PRISMA flow diagram (Figure 1). 10

Figure 1. PRISMA flow diagram. 11

Searching of ongoing trial databases identified 837 trial records. De-duplication and removal of clearly irrelevant trials by the Information Specialist (CD) excluded 651 records. Of the remaining 186 records, 25 ongoing trials met the eligibility criteria and are shown in Ongoing studies. Included studies Twenty-three studies met the inclusion criteria for this review and included a total of 1137 participants (659 bone marrow-derived stem cells (BMSC) and 478 control) who were assessed for the primary outcomes of the study. The mean age of participants ranged from 53.4 years to 69.8 years and the proportion of men ranged from 50% to 100%. All trials were presented as full journal articles with the exception of one trial (Assmus 2012) which was published in the form of a conference abstract. Five studies (Losordo 2007; Losordo 2011; Perin 2011; Perin 2012a; Tse 2007) were multicentre trials. Studies were based worldwide, including China (Chen 2006; Hu 2011; Wang 2009; Wang 2010; Yao 2008; Zhao 2008), Germany (Assmus 2006; Assmus 2012; Erbs 2005; Honold 2012; Turan 2011), the United States (Losordo 2007; Losordo 2011; Perin 2011; Perin 2012a; Perin 2012b), United Kingdom (Ang 2008), Belgium (Hendrikx 2006), The Netherlands (Van Ramshorst 2009), Russia (Pokushalov 2010), Hong Kong/Australia (Tse 2007), Korea (Kang 2006) and Argentina (Patel 2005). Two studies included publications in Chinese (Hu 2011; Wang 2009); these studies was translated into English for this review. Ten studies included participants with chronic ischaemic heart disease (IHD) (Ang 2008; Assmus 2006; Assmus 2012; Chen 2006; Erbs 2005; Hendrikx 2006; Honold 2012; Kang 2006; Turan 2011; Yao 2008), normally defined as multivessel disease with persistent ischaemia and at least 30 days from the last myocardial infarction (MI), with the exception of one study that defines old MI as only 14 days post-infarction (Kang 2006). Seven studies included people with congestive heart failure (CHF), defined as severe ischaemic heart failure and post-infarction heart failure (secondary to IHD) (Hu 2011; Patel 2005; Perin 2011; Perin 2012a; Perin 2012b; Pokushalov 2010; Zhao 2008) and six studies were of people with intractable or refractory angina (Losordo 2007; Losordo 2011; Tse 2007; Van Ramshorst 2009; Wang 2009; Wang 2010). All trials maintained the participants with a standard set of drugs including aspirin, clopidogrel, heparin, blockers, statins, angiotensin converting enzyme (ACE) inhibitors, nitrates and/or diuretics. Duration of follow-up ranged from three months (Assmus 2006), four months (Assmus 2012; Hendrikx 2006), six months (Ang 2008; Hu 2011; Kang 2006; Losordo 2007; Patel 2005; Perin 2011; Perin 2012a; Perin 2012b; Tse 2007; Van Ramshorst 2009; Wang 2009; Wang 2010; Yao 2008; Zhao 2008), 12 months ( Chen 2006; Losordo 2011; Pokushalov 2010; Turan 2011), 15 months (Erbs 2005) and five years (Honold 2012). Eighteen trials isolated the stem cells by bone marrow aspiration and further separation of the mononuclear cells using ficoll gradient centrifugation (Ang 2008; Assmus 2006; Assmus 2012; Chen 2006; Hendrikx 2006; Hu 2011; Patel 2005; Perin 2011; Perin 2012a; Perin 2012b; Pokushalov 2010; Tse 2007; Turan 2011; Van Ramshorst 2009; Wang 2009; Wang 2010; Yao 2008; Zhao 2008). Three of these trials enriched the stem cell fraction in CD34-positive haematopoietic progenitors by magnetic separation (Patel 2005; Wang 2009; Wang 2010), whilst one trial enriched the stem cell fraction in aldehyde dehydrogenase (ALDH)- positive haematopoietic progenitors (Perin 2012b), and one trial cultured the mononuclear cell population from bone marrow ex vivo to enrich in mesenchymal progenitors (Chen 2006). In one three-arm trial (Assmus 2006), bone marrow mononuclear cells were compared with circulating progenitor cells (CPCs), and with mononuclear cells isolated from venous peripheral blood. In the CPC arm, cells were isolated from peripheral blood by leukapheresis. In the remaining five trials, bone marrow stem cells were mobilised into circulation with granulocyte colony-stimulating factor (G-CSF) and subsequently isolated from blood via leukapheresis (Erbs 2005; Honold 2012; Kang 2006; Losordo 2007; Losordo 2011). Whilst previous trials reported severe but transient complications associated with G-CSF treatment (Kang 2006), the most recent pilot study by Honold 2012 demonstrated that G-CSF can be safely administered to people suffering from IHD since none of the participants included in this trial developed the type of adverse events previously associated with G-CSF treatment. Two of these trials further enriched the stem cell population in CD34-positive progenitors by magnetic separation (Losordo 2007; Losordo 2011). The dose of bone marrow mononuclear cells administered varied between 2 x 10 cells (Perin 2011) and 2 x 10 cells (Assmus 2006), whilst the dose of CD34-positive cells varied between 1 x 10 cells (Wang 2009) to 5.6 x 10 cells (Losordo 2011). The doses of ALDH-positive cells (Perin 2012b) and mesenchymal progenitors (Chen 2006) administered averaged 2.96 x 10 cells and 5 x 10 cells respectively. In the trial where bone marrow mononuclear cells were compared to CPCs, the dose of CPCs administered was 2.2 x 10 cells (Assmus 2006). Eleven trials administered the treatment via a coronary artery (intracoronarily (IC)) (Assmus 2006; Assmus 2012; Chen 2006; Erbs 2005; Honold 2012; Hu 2011; Kang 2006; Turan 2011; Wang 2009; Wang 2010; Yao 2008), whilst 11 trials delivered the treatment intramyocardially (IM) (Hendrikx 2006; Losordo 2007; Losordo 2011; Patel 2005; Perin 2011; Perin 2012a; Perin 2012b; Pokushalov 2010; Tse 2007; Van Ramshorst 2009; Zhao 12

2008). Nine out of these 11 trials aided their delivery into the heart muscle using electromechanical mapping of the heart. The other two (Hendrikx 2006; Zhao 2008) did not report whether the IM delivery of stem cells was aided in any other way. Only one trial had three arms comparing IC and IM delivery of stem cells with control (Ang 2008). Thirteen studies (Assmus 2012; Erbs 2005; Hendrikx 2006; Hu 2011; Losordo 2007; Losordo 2011; Perin 2012a; Perin 2012b; Tse 2007; Van Ramshorst 2009; Wang 2010; Yao 2008; Zhao 2008) compared stem cell therapy with administration of a placebo that consisted of a cell-free solution, either a heparin saline solution or a saline solution containing the participant s own serum; one further study (Perin 2011) used a simulated mock injection procedure for participants in the control arm, but without administering a placebo solution. The remaining nine trials compared treatment to no treatment (Ang 2008; Assmus 2006; Chen 2006; Honold 2012; Kang 2006; Patel 2005; Pokushalov 2010; Turan 2011; Wang 2009). Three studies included a three-way comparison involving two interventions, including intracoronary versus intramyocardial cell administration (Ang 2008), mononuclear cells versus circulating progenitor cells (Assmus 2006) and high versus low cell dose (Losordo 2011). Data for both intervention arms were combined for the main analyses, although we used individual intervention trial arms for subgroup analyses where applicable. A fourth study (Assmus 2012) which involved a co-intervention of shockwave therapy also included an additional trial arm involving BMSC treatment, but since the co-intervention was not administered in this treatment arm, we include only the first two treatment arms in this review. One three-arm trial was also a cross-over study (Assmus 2006); we include only data up to the point of cross-over (three months) in this review. One study described aortic cross-clamping during surgery with clamp times exceeding 25-30 minutes (Hendrikx 2006). Aortic cross-clamping isolates the systemic circulation during surgery but causes ischaemia. Although increasing times of aortic crossclamping has been identified as a predictor of mortality, the effect of cross-clamping in this study was not as strong as might be expected. This may be due to the fact that the cause of cardiac damage is multifactorial, including coronary lesions. The majority of included studies reported the primary outcomes of this review, i.e. mortality, LVEF and adverse events. One study which was published only in abstract form (Assmus 2012) did not report mortality, and all but four studies (Losordo 2007; Losordo 2011; Wang 2009; Wang 2010) reported LVEF. For a summary details of the included studies, see the Characteristics of included studies tables. Excluded studies We excluded 21 studies (described by 25 references) from the review following full-text assessment against the eligibility criteria (see Characteristics of excluded studies tables). In summary, the reasons for exclusion were as follows: 14 studies were not RCTs, four studies included participants with AMI, one trial included participants with idiopathic dilated cardiomyopathy, one trial provided a review of imaging techniques for cardiac stem cell therapy, and one trial described outcomes not included in the protocol of this review. Risk of bias in included studies See the Characteristics of included studies tables for details of our assessment of risk of bias for each study; a summary of risk of bias is shown in Figure 2. 13

Figure 2. Risk of bias summary: review authors judgements about each risk of bias item for each included study. 14