CHAPTER 10: STEM CELLS AND THE CARDIOVASCULAR SYSTEM

Stem cell therapy has attracted considerable interest in the treatment of cardiovascular disorders. This is driven by the observation that the heart’s regenerative capacity is insufficient to recover from ischemia or other cardiotoxic insults and the progressive nature of tissue ischemia in patients with coronary artery disease or peripheral arterial disease (PAD). Directed homing of bone marrow–derived stem cells after myocardial injury produced functional improvements in animal studies,^1,2 leading to the hypothesis that enhancing mobilization of bone marrow stem cells or their direct delivery can result in clinical improvement in acute or chronic cardiac injury. Similarly, evidence supporting the incorporation of circulating and bone marrow–derived progenitor cells into newly formed vasculature in limb ischemia models supported the premise that enhanced progenitor cell delivery can reverse tissue ischemia.^3,4

Improvements in our understanding of stem cell biology, cardiac differentiation, and response to cardiovascular injury have advanced the field of cardiac cell therapy.^5,6 These findings have led to numerous studies investigating the efficacy of multiple bone marrow stem cell lineages and other stem cell sources on cardiovascular recovery. To date, no stem cell therapy is approved by the Food and Drug Administration for a cardiovascular indication, but the growing body of literature provides a promising signal to encourage multiple Phase III clinical trials. This chapter will examine the clinical experience and potential applications of stem cell therapy by reviewing each stem cell lineage (Table 10–1).

There are multiple mechanisms by which stem cells may exert a beneficial effect in the cardiovascular system (Fig. 10–1), but some common themes are important to highlight. First, the mechanism by which any stem cell lineage exerts a potential benefit in humans is not yet fully explained. Second, the inability to track stem cells or perform histopathological studies in human subjects impairs the ability to determine cell fate and mechanism of action. Third, most of the mechanisms hypothesized to play a role in the salutatory effect of stem cells are derived from animal models. Lastly, it is unknown (and unlikely) that all stem cell lineages exert their effect on the human cardiovascular system via the same mechanism.

Differentiation is the concept that stem cells form new cardiomyocytes to replace the ones that are lost in injury. This mechanism is now largely accepted to contribute minimally, if at all, to any improvement associated with cardiac functional recovery. Although pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have a robust potential to differentiate into cardiomyocytes, the differentiation capacity of other stem cell lines is more limited.⁷ For example, bone marrow–derived stem cells demonstrate lineage commitment that predisposes them to divide into hematopoietic cells. But to divide into cardiomyocytes, they must transdifferentiate, which involves reversing their lineage commitment to a new somatic cell type. This process may occur in animals (and possibly in humans) endogenously throughout the course of life,^7,8 but it is less likely to be a significant contributor to myocardial improvement as shown by the limited survival of these cells from studies involving direct intramyocardial injections of bone marrow–derived stem cells.^9,10,11

Cell fusion is a phenomenon in which stem cells fuse with native cardiomyocytes and form a more functional cardiomyocyte that is capable of remodeling or regeneration. This infrequent phenomenon is hypothesized to contribute to a favorable remodeling response and has been validated in animal studies.^12,13 However, it remains unknown to what extent this occurs in the human studies performed.

Angiogenesis is the process of new blood vessel formation and is known to be defective in both ischemic and nonischemic cardiomyopathy patients as well as patients with PAD.¹⁴ Thus, enhancing angiogenesis should improve blood flow in those conditions. However, whether stem cells can directly differentiate into new blood vessels or promote new blood vessel formation by secretory factors is currently controversial.^15,16

Paracrine effects refer to changes to the myocardial or vascular environment driven by stem cells that may promote cardiovascular recovery or regeneration. These changes may be conveyed by changes in cytokines, growth factors or interstitium. This is currently the most accepted mechanism of benefit for all bone marrow–derived cell therapies due to the following reasons: (1) this hypothesis is in line with bone marrow cellular mobilization observed in ischemic and postinfarction patients; (2) cell tracking studies suggest that stem cells persist only for a brief period of time in the myocardium, with typically less than 1% survival at 4 weeks; and (3) animal studies show that cell extracts from stem cell cultures can exert a favorable effect on injured myocardium.¹⁷

Lastly, a novel concept in cardiac repair is modulation of dedifferentiation. Dedifferentiation is the process by which cardiomyocytes take on a gene and protein expression profile that is more similar to fetal cardiomyocytes or pluripotent stem cells.^18,19 This phenotype occurs more frequently in acute and chronic injury of the heart and seems to confer a potential survival advantage of these cardiomyocytes. Furthermore, this early progenitor profile may increase the number of endogenous cardiomyocytes capable of dividing to replace lost cardiomyocytes. Prolonged activation of this phenomenon, however, may be deleterious and contribute to adverse cardiac remodeling. Cardiac dedifferentiation may serve as a future target for cardiac regeneration therapy.

Clinically tested methods of cell delivery include using blood as a carrier of stem cells to myocardium, by directly injecting the cells into the target tissue, and tissue engineering constructs. Blood-based approaches include intracoronary arterial infusion, intracoronary venous infusion, peripheral intravenous infusions, and peripheral arterial infusion. Intramyocardial delivery methods include surgical epicardial injection (transepicardial), catheter-based endocardial injection by accessing the left ventricular cavity (transendocardial), and injection by accessing the coronary venous circulation (transvenous myocardial). Direct injection in the case of PAD entails transcutaneous injection into ischemic tissue (most often lower limb). There are a host of tissue engineering constructs that deliver progenitor cells or cardiomyocytes to the cardiovascular system.²⁰ These methods constitute biological delivery systems of progenitor cells taking advantage of natural or synthetic chemicals to create a scaffold that can support the growth of progenitor cells (Table 10–2).

Intracoronary delivery is usually performed during cardiac catheterization after the coronary artery is engaged. Cells are infused into the artery with or without balloon inflations to interrupt flow and slow down stem cell washout from the myocardium. The advantages of intracoronary delivery are (1) it is technically easier, (2) it directly targets perfused areas of the myocardium where stem cells can survive, and (3) it does not require mapping or identification of viability. The disadvantages of this delivery method include the following: (1) it requires access to patent coronary arteries; (2) cells will be preferentially shunted to well-perfused areas, which means that ischemic or border zones will receive fewer cells; and (3) it has reduced retention compared to intramyocardial delivery.²¹

Transendocardial delivery aims to inject stem cell populations directly into the myocardium via a needle catheter by accessing the left ventricular (LV) cavity through the arterial system. This method is aided by electromechanical or magnetic resonance imaging guidance, which is necessary to identify target areas of injection. The cells are injected in healthy, ischemic or border zones—but not infarct zones. Infarct zones are nonperfused and therefore stem cells are unlikely to survive to exert a therapeutic benefit. Additionally, infarct zones are made up of thin scar tissue that is susceptible to perforation. Advantages of transendocardial delivery include the ability to target stem cells to ischemic or border zones and better cellular retention. Disadvantages of the delivery method include the technical complexity (especially if electromechanical mapping is done), cell leakage at injection site, and a higher risk of cardiac perforation or arrhythmia.²²

Transepicardial delivery involves the surgical injection of stem cells by direct visualization into the myocardium, often in conjunction with other cardiac surgery procedures such as coronary artery bypass graft (CABG) surgery. During the surgical injection, visual assessment of the myocardium is often used to determine the target sites for injection. However, the invasive nature of cardiac surgery limits the use of this method to patients undergoing concurrent cardiac surgery.²³

A recent clinical trial compared intracoronary versus intramyocardial (transendocardial) delivery using radiolabeled CD34⁺ cells in patients with non-ischemic cardiomyopathy.²⁴ The transendocardial group showed a higher rate of cardiac signal retention 18 hours postinjection. At the 6-month follow-up, the investigators observed a greater improvement in left ventricular ejection fraction (LVEF) for the transendocardial group. Additionally, the results suggested that early engraftment as detected by imaging can be used to predict late functional outcome, similar to results that were initially reported in animal models.²⁵ However, it is unknown whether these results can be extrapolated to all stem cell lineages or for other cardiac conditions such as acute myocardial infarction (AMI). Nevertheless, these studies highlight the potentially important role that molecular imaging may play in optimizing some of the delivery approaches for cardiac stem cell therapy.²⁶

Another promising approach to cell therapy is endogenous activation of cardiac progenitor cells in host myocardium. This in situ approach entails delivery of viral or nonviral constructs that carry genetic or chemical signals that stand to activate resident stem cells in the cardiovascular system. This approach has shown positive results in animal models, but further studies are needed to validate and refine this approach for clinical testing.^27,28

Skeletal myoblasts are skeletal muscle precursor cells derived from skeletal muscle satellite cells. Interest in this population was supported by similarity to adult cardiomyocytes, the potential for autologous utilization, ease of expansion in vitro, and resistance to hypoxic environment. This lead to a series of early clinical studies that transplanted skeletal myoblasts into small cohorts of ischemic cardiomyopathy patients.^{29,30,31,32,33,34} Subsequently, multiple randomized double-blind placebo-controlled trials examined the broader safety and efficacy of skeletal myoblasts in ischemic heart disease. The largest such study was the Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial, which randomized patients to high or low dose injection of skeletal myoblasts or placebo during CABG.³⁵ There was a small reduction in LV volumes in the high dose group at 6 months, but otherwise no difference in regional or global LV function. However, there was a higher incidence of ventricular arrhythmias in myoblast-treated patients. Three similar studies utilized a cardiac mapping system for transendocardial injections of skeletal myoblasts in ischemic cardiomyopathy patients.^36,37,38 These studies showed a small improvement or trend toward improvement in described functional status but no significant differences in systolic function, LV dimensions, or 6-minute walk tests.

The increased incidence of arrhythmia seen in the MAGIC trial and a large pilot study³⁹ raised significant concern about the long-term safety of skeletal myoblast therapy. This is further supported by histological analysis of skeletal myoblast–treated heart tissue showing islands of myotubes that are not electrically coupled to host myocardium.⁴⁰ Collectively, the clinical and histopathological findings along with animal studies showing increased arrhythymia⁴¹ have tempered interest in this cell population for the treatment of myocardial disorders.

The bone marrow harbors a heterogeneous population of progenitor cells that give rise to mainly hematopoietic or endothelial cells. This is essentially a mixture of multiple stem cell lineages, and many of these populations were later isolated and investigated separately. Interest in bone marrow–derived mononuclear cells (BMMNCs) for cardiac repair was sparked by observations in animal studies that a subpopulation of bone marrow cells mobilize to the heart after myocardial infarction (MI), express early cardiac markers, and improve cardiac function.^1,2 Some animal studies indicated possible improvements after injecting BMMNCs in postinfarct animals.^1,2 These observations supported a hypothesis that bone marrow–derived cells might participate in myocardial recovery or regeneration. The unsorted population of BMMNCs was particularly attractive during the early cell therapy experience because of their relative accessibility via bone marrow biopsy, minimal sorting and processing, and experience utilizing the cells in bone marrow transplantation. With the realization that these progenitor cells mobilize in peripheral blood, some investigators attempted to harvest the progenitor cells from peripheral blood or unprocessed bone marrow extract, but most studies relied on bone marrow biopsy–derived mononuclear cells. The majority of clinical experience in cardiac stem cell trials to date remains with BMMNCs.

Acute Myocardial Infarction

Some of the earliest studies investigating the efficacy of BMMNCs in improving cardiovascular outcomes targeted patients with AMI. This was an attractive indication because of the acute and dramatic nature of cell loss and damage that occurs in the postinfarct period and the potential attenuation benefits of this response.⁴² The trials largely targeted patients with ST-segment elevation MI and treated them with intracoronary infusion of BMMNCs. The initial clinical experience was largely limited to single arm or nonrandomized trials.^{43,44,45,46,47,48} These studies reported an improvement in LV systolic function and/or infarct size 3 to 6 months after cell therapy infusion and few safety issues.

These relatively positive early results led to a series of randomized controlled studies testing the safety and efficacy of BMMNCs (Table 10–3). Given the known risks associated with the coronary infusion process and bone marrow biopsy, these trials were not blinded, and they compared two arms of cell therapy or utilized a nonplacebo control. In other words, control patients underwent optimal standard of care without undergoing bone marrow biopsy or sham intracoronary infusion procedures. This nonuniform exposure to risk in the cell therapy group sometimes resulted in a higher number of periprocedural complications such as increases in cardiac biomarkers of injury or stent thrombosis. Nevertheless, these events did not translate into a significant difference in overall incidence of major serious adverse events. However, in terms of efficacy, the trials provided mixed results with respect to improvement in LVEF, LV volumes, and infarct or scar size. Even positive results were inconsistent as to the specific parameters that were improved among these studies.^{49,50,51,52,53,54,55,56,57,58,59,60,61,62,63}

The third generation studies included randomized placebo-controlled studies that aimed to strengthen blinding and to add a placebo control arm as an alternative to the optimal medical therapy control arm that was utilized previously. These studies validated the safe nature of the bone marrow harvest and intracoronary infusion procedure but unfortunately have not provided a conclusive answer to the efficacy question.^{64,65,66,67,68,69,70} Several meta-analyses have attempted to combine data to determine efficacy but have likewise yielded mixed results.^41,71,72

Several hypotheses have been generated to explain the conflicting results of the trials: the relatively small number of patients, variation in timing of cell infusion, differences in number of cells infused, and different modalities of measuring LV function and dimensions. Another important criticism of the studies is the appropriateness of the surrogate end point used to test for efficacy, namely LV size or function. For example, although angiotensin-converting enzyme (ACE) inhibitors provide superior survival benefit over hydralazine-nitrate in patients with heart failure and reduced LVEF, hydralazine-nitrates provide a greater improvement in LVEF, highlighting the limitation of using surrogate end points.⁷³ Thus, the more relevant question is whether BMMNC therapy provides a hard outcome improvement in AMI that would justify the associated risks and resources committed, similar to other approved therapies such as percutaneous coronary intervention or use of thiopyridine antagonists. To answer this question conclusively, a 3000-patient multicenter randomized clinical trial, the Effect of Intracoronary Reinfusion of BMMNC on All Cause Mortality in Acute Myocardial Infarction (BAMI), is being conducted by European Union scientists, with an expected completion date in the spring of 2018 (retrieved from http://clinicaltrials.gov/ct2, identification NCT01569178).

Chronic Heart Failure

The use of BMMNCs for the treatment of chronic heart failure began with pilot studies that aimed to determine their safety, similar to studies for AMI indication.^{74,75,76,77,78,79,80,81,82,83,84} The studies were mostly positive, showing a mix of symptomatic, contractile, and perfusion improvements regardless of the method of injection at follow-up intervals of 3 to 12 months. This led to multiple randomized trials to confirm efficacy, but the results thus far have been mixed (Table 10–4).^{85,86,87,88,89,90,91,92,93,94,95}

In contrast to the AMI trials, in which cell delivery was largely intracoronary, the method of delivery in chronic heart failure trials included both intracoronary infusion and intramyocardial injections. A number of trials randomized ischemic cardiomyopathy patients undergoing CABG to receive injections of BMMNCs versus standard surgery. No single delivery method seemed to predict a consistent response or lack of response to delivered BMMNCs. The trials primarily targeted ischemic cardiomyopathy, and those that showed positive results found a small improvement in end points such as LVEF and New York Heart Association (NYHA) class. Interestingly, a randomized controlled study of nonischemic patients showed improvement in imaging parameters and functional status.⁹⁶ The safety profile of these studies collectively was once again reassuring. Meta-analysis of chronic heart failure studies did not yield a definitive answer to the question of efficacy.^97,98 Thus, the main conclusion that can be drawn from these studies is that treatment is largely safe and well tolerated, but larger studies will be needed to answer the question of efficacy of BMMNCs in cardiomyopathy patients.

Peripheral Arterial Disease

BMMNCs are the most tested cell therapy for the treatment of PAD (Table 10-5). The first pilot study to investigate the safety and efficacy of cell therapy in PAD examined the injection of BMMNCs versus peripheral blood mononuclear cells (PBMCs) into the gastrocnemius muscle of patients with chronic limb ischemia (CLI). The limbs injected with BMMNCs showed significant improvement in ankle brachial index (ABI), transcutaneous oxygen pressure (TcPO₂), rest pain, and pain-free walking time.⁹⁹ Subsequently, multiple randomized trials tested the safety and efficacy of BMMNCs in patients with PAD, with conflicting results (Table 10–5).^{100,101,102,103,104,105,106,107,108,109} Collectively, the data appear to point toward a small favorable response of measures such as ABI or TcPO₂, but end points such as amputation-free survival did not differ between groups. Two meta-analyses that used rigorous methodological standards concluded there is insufficient evidence for benefit of BMMNCs for the treatment of PAD.^110,111

The majority of studies utilized intramuscular (IM) injection of cell therapy with a minority using intra-arterial (IA) injection. Klepanec et al randomized PAD patients to IM versus IA injection of BMMNCs and observed comparable improvements in quality-of-life questionnaire, TcPO₂ and pain scale in both groups.¹⁰⁷ Terra et al tested the effect of repeated IA infusion of BMMNCs against placebo in 161 patients with critical limb ischemia. After 6 months there was no difference in the primary end point of amputation-free survival or all-cause mortality.¹⁰⁹ There was no increased risk of adverse events associated with BMMNCs in randomized trials.

Mesenchymal stem cells (MSCs) are a population of stromal progenitor cells that are capable of differentiating into multiple cell types, including osteoblasts, adipocytes, and skeletal myocytes.^112,113,114 Initially thought to reside in the bone marrow, they have later been shown to exist in other organs.¹¹⁵ Furthermore, animal studies have suggested that MSCs can differentiate into cardiac progenitors and improve cardiac function in infarct models,^10,116 sparking an interest in clinical application of MSCs for cardiac regeneration. MSCs are relatively accessible via bone marrow biopsy and can be autologous. MSCs have the added advantage of homing to injured myocardial tissue, which facilitates intravenous administration.¹¹⁷ Lastly, MSCs are reported to be immune privileged, and this raises the possibility of using allogeneic MSC populations. The clinical experience with MSCs is limited compared to BMMNCs, but the results to date are arguably more promising (see Table 10–3; Table 10–6).

Acute Myocardial Infarction

One of the earliest studies testing bone marrow–derived MSCs in a randomized fashion was by Chen et al, who randomized post-MI patients to receive MSCs or saline via intracoronary injection.¹¹⁸ The MSC-treated group showed an improvement in LVEF and a reduction in infarct size. Lee et al saw a similar improvement in LVEF with intracoronary infusion of bone marrow–derived MSCs.¹¹⁹ These results diverged from negative findings by Gao et al, who randomized AMI patients to receive intracoronary infusion of bone marrow–derived MSCs versus standard therapy.¹²⁰ Possible reasons for discordance among these studies may relate to differences in number of cells injected and timing of administration post-MI. Similar to the early studies of BMMNCs, one can conclude that MSC therapy has a reasonable safety profile, but larger trials are needed to test efficacy.

Peripheral Arterial Disease

The experience with MSCs in the treatment of PAD is limited to date but preliminary results appear to be positive (Table 10-5). Two randomized studies evaluated the safety and efficacy of bone marrow–derived MSCs in patients with CLI. At 12 weeks, both studies showed a signal toward improvement in the cell therapy group in terms of ulcer healing rate, and one study showed a decrease in amputation rate.^121,122 Subsequently, in a randomized double-blind placebo-controlled study, Gupta et al saw a similar improvement in ABI but not ulcer healing.¹²³ The comparative effectiveness of bone marrow–derived MSCs against BMMNCs was tested in a randomized study of 41 patients with CLIand the results demonstrated improved outcomes in the bone marrow– derived MSCs group.¹²⁴ Multiple randomized trials are currently underway to further test the potential of this population in the treatment of PAD (retrieved from http://clinicaltrials.gov/ct2, identification NCT01456819, NCT01257776, NCT01351610, NCT02145897, NCT02336646, NCT02304588). Some of the trials target patients with intermittent claudication, which represents an earlier stage of ischemic disease and possibly better potential for tissue salvage.

Allogeneic Mesenchymal Stem Cells

Given the immune-privileged properties of MSCs, this opens the door to allogeneic transplantation. Allogeneic transplantation has the advantage of having the cell therapy population ready in advance for clinical use, so called “off the shelf” delivery. Several efforts have investigated the potential for allogeneic bone marrow–derived MSCs. In a randomized double-blind placebo-controlled trial, Hare et al treated post-MI patients with intravenous injection of allogeneic bone marrow–derived MSCs 7 to 10 days after reperfusion.¹²⁵ The study showed a trend of LVEF improvement that did not meet statistical significance. However, there was no significant increase in antibodies in response to the allogeneic cells. In the Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis (POSEIDON) study, the investigators randomized ischemic cardiomyopathy patients to receive either intramyocardial injection of autologous or allogeneic MSCs.¹²⁶ Interestingly, the autologous MSCs group showed improved 6-minute walk distance and Minnesota Living with Heart Failure Questionnaire score, whereas the allogeneic MSCs group showed a reduction in LV end-diastolic dimension. Both groups showed a reduction in infarct size. Only a minor increase in antibody response was seen in the allogeneic group.

A similar safety profile was seen by Perin et al in a dose escalation study using allogeneic MSCs in patients with ischemic or nonischemic cardiomyopathy.¹²⁷ Lastly, a surgical study utilizing allogeneic mesenchymal precursor cells was performed in advanced heart failure patients undergoing left ventricular assist device (LVAD) implantation.¹²⁸ Although imaging surrogates for cardiac recovery failed to reach statistical significance, the study demonstrated no increase in incidence of human leukocyte antigen antibodies. A larger Phase II study investigating intramyocardial injection of MSCs during LVAD implantation is currently underway (retrieved from http://clinicaltrials.gov/ct2, identification NCT02362646). Thus, the aggregate results of these studies support the safety of allogeneic bone marrow-derived MSCs as a potential cell therapy population for clinical use.

Non-Bone Marrow–Derived Mesenchymal Stem Cells

The safe use of allogeneic stem cells opens the door to utilizing MSCs from other sources that may have superior proliferation or engraftment ability. After seeing no effect with intracoronary infusion of bone marrow–derived MSCs in AMI patients,¹²⁰ Gao et al utilized Wharton’s jelly-derived MSCs for intracoronary infusion in patients with AMI.¹²⁹ Wharton’s jelly is a stromal cell population derived from umbilical cord, which the investigators obtained from healthy donors after full-term birth. At 18 months, the cell therapy population showed a significant improvement in LVEF, LV volumes, and perfusion compared to controls.

Comparative Effectiveness

To compare the relative efficacy of BMMNCs against bone marrow–derived MSCs, the Transendocardial Autologous Cells in Ischemic Heart Failure Trial (TAC-HFT) investigators randomized ischemic cardiomyopathy patients to receive intramyocardial injections with either cell therapy or placebo.¹³⁰ Treatment with MSCs improved infarct size, regional contractility, 6-minute walk distance, and Minnesota Living with Heart Failure score. BMMNC-treated patients saw an improvement in Minnesota Living with Heart Failure score but none of the other parameters.

Future Directions for Mesenchymal Stem Cell Studies

Overall, the bone marrow–derived MSC studies are early in the phase of testing compared to the BMMNC studies but appear promising. The early studies have validated the safety profile of cell delivery procedures by widespread inclusion of placebo control groups. Furthermore, there is a sufficient signal for potential efficacy to warrant further studies. Larger studies will be needed to answer the overall question of efficacy with a focus on the optimal source of MSCs, the optimal delivery method, and appropriate indications for use.

This population of progenitor cells mainly give rise to endothelial cells.⁴ Angiogenic progenitor cells (APCs) garnered considerable interest in regenerative capacity as a result of their proposed ability to promote angiogenesis. Improving the microcirculation in theory would provide considerable benefit to patients with cardiovascular disorders because they are known to have reduced number and impaired migratory ability of APCs.^131,132,133 A number of clinical trials investigated the therapeutic potential of APCs for multiple cardiovascular indications, finding a potential benefit for some cardiovascular indications.

Acute Myocardial Infarction

Two studies have tested APCs in MI in a randomized fashion. Bartunek et al randomized AMI patients to receive CD133⁺ cells via intracoronary infusion versus optimal standard therapy, and reported an increase in LVEF and perfusion.¹³⁴ The study showed an increased incidence of adverse events in the cell therapy group, although it is important to point out that the control group did not undergo placebo infusion procedure. Quyyumi et al randomized AMI patients to receive variable doses of CD34⁺ cells and reported an improvement in perfusion in patients with high dose of the cell therapy but no change in LVEF.¹³⁵ The safety profile was adequate to allow further testing of this product in larger studies.

Aldehyde dehydrogenase bright stem cells are a population of stem cells that are thought to have a significant angiogenic and ischemia protective role.¹³⁶ These cells were tested in a small randomized placebo-controlled study by Perin et al, who found a statistically significant reduction in LV end-systolic volume but not in peak oxygen consumption and perfusion.¹³⁷ The trial did achieve its primary end point, which is to demonstrate the safety of the cell therapy, thus opening the door for larger studies to investigate efficacy.

Angina

The angiogenic potential of CD34⁺ cells was particularly attractive in the chronic angina population and was tested in multiple case pilot studies with an overall positive therapeutic response.^{138,139,140,141} In the largest cell therapy study to treat angina to date, Losordo et al randomized 167 patients with refractory angina to receive intramyocardial injections of autologous CD34⁺ cells versus placebo.¹⁴² However, the cell therapy group also experienced a higher incidence of small elevations in cardiac biomarkers of injury associated with cell mobilization and collection procedure. Similar results were seen by Wang et al using an intracoronary delivery method.¹⁴³ Arguably, the studies for angina have yielded some of the most positive results using stem cell therapy for any indication (Table 10–7), and phase III clinical trials to validate the efficacy of CD34⁺ cells are being planned.

Heart Failure

In contrast to the largely positive results seen in AMI and angina, the experience with APCs in ischemic cardiomyopathy is more mixed (see Table 10–4). Two surgical studies investigated the effects of APCs delivered via intramyocardial injection at the time of CABG using routine surgery as the control. One of the studies utilized CD34⁺ cells and showed an increase in LVEF compared to CABG alone at the 6-month follow-up.¹⁴⁴ However, injection of CD133⁺ cells led to improved myocardial perfusion but resulted in a lower LVEF and no difference in functional capacity.¹⁴⁵ This study contradicted findings from prior nonrandomized studies that investigated CD133⁺ cells.¹⁴⁶

Patients with heart failure, including nonischemic cardiomyopathy, have been shown to have an impairment in circulating APCs,¹⁴⁷ which led to the hypothesis that the restoration or mobilization of this cell population might improve heart failure symptoms. Vrtovec et al conducted one of the largest randomized trial with 110 nonischemic cardiomyopathy patients randomized to receive either CD34⁺ intracoronary infusion or standard therapy.^148,149 At 12 and 60 months, the CD34⁺ treatment group showed a significant improvement in LVEF and 6-minute walk distance. Additionally, patients with higher myocardial homing of injected cells showed the greatest improvement.

Peripheral Arterial Disease

Multiple studies have examined the role of APCs in the treatment of PAD, with most showing an excellent safety profile with marginal to no benefit (see Table 10–5).^{142,150,151,152} However, several factors prevent a conclusive determination of the role of APCs in the treatment of PAD. First, these studies were relatively small and primarily were designed to determine safety rather than efficacy. Second, the studies utilized different populations of APCs and thus grouping the results may not be appropriate. A larger study is currently underway to test the efficacy of aldehyde dehydrogenase bright cells on the treatment of PAD.¹⁵³

Adipose tissue harbors a heterogeneous population of progenitor cells that are primarily mesenchymal in nature and show analogous cardiac benefits to bone marrow–derived progenitor cells.^{154,155,156,157} Adipose is more abundant in mesenchymal cells than bone marrow and can be easily obtained by liposuction techniques.¹⁵⁸ Two pilot studies investigated the safety of this therapy. Houtgraaf et al randomized 14 patients with anterior AMI to receive intracoronary infusion of adipose-derived regenerative cells, finding the treatment to be safe, with a trend toward small reductions in infarct size.¹⁵⁹ In a pilot study, Perin et al randomized 27 ischemic cardiomyopathy patients to receive transendocardial injections of adipose stem cells or placebo.¹⁶⁰ There were no serious adverse events, and the researchers observed a signal toward slower decline of maximal oxygen consumption in the cell therapy group, increased LV mass, and improved regional contractility. Similarly, two published Phase I studies demonstrated the feasibility and safety of adipose-derived MSCs for the treatment of PAD.^161,162 Collectively, the studies support the safety of this cell population, although more testing is needed to investigate the potential efficacy of this treatment for cardiovascular indications and compare it to bone marrow cell populations.

Over a decade ago, researchers reported that a group of progenitor cells residing in the heart were capable of differentiating into cardiomyocytes, endothelial, and smooth muscle cells.^163,164,165 Furthermore, animal models suggested that these cells can promote cardiac regeneration in infarct models.¹⁶⁶ The identification of these cells added to the observations challenging the notion that the heart is a terminally differentiated organ.^167,168 Follow-up studies aimed to enrich this scant cell population in hopes of augmenting myocardial regeneration or recovery. Whether the beneficial effects of cardiac progenitor cells occur because of cardiomyocyte differentiation, angiogenesis, or paracrine effects is not fully understood, and these potential mechanisms are under investigation.¹⁶⁹ Nevertheless, two clinical trials have now tested the safety of subtypes of this cell population.

Cardiac stem cells were isolated and tested in randomized fashion in the Cardiac Stem Cell Infusion in Patients With Ischemic CardiOmyopathy (SCIPIO) trial.¹⁷⁰ Bolli et al isolated cardiac stem cells from harvested cardiac tissue during CABG and later injected them via an intracoronary route. The cardiac stem cell group experienced an improvement in LVEF, regional contractility, and NYHA class. In the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dysfunction (CADUCEUS) trial, Makkar et al tested the safety and efficacy of cardiosphere-derived cells (CDCs), which are cardiac progenitor cells that are isolated from adherent clusters of progenitor cells known as cardiospheres.¹⁷¹ The authors randomized 31 post-MI patients to receive intracoronary infusion of CDCs into the culprit artery. At 1 year, the CDC-treated patients had a reduction in infarct size and regional contractility compared to control patients but no overall improvement in LVEF or functional capacity. Both studies provided sufficient evidence for safety and potential efficacy to permit larger studies for further testing. A study investigating the combination of autologous bone marrow–derived MSCs and c-kit+ cardiac stem cells in ischemic cardiomyopathy patients is currently underway (retrieved from http://clinicaltrials.gov/ct2, identification NCT02501811).

First isolated in 1998, human ESCs are PSCs that exist in the inner cell mass of early stage dividing embryo (blastocyst).¹⁷² The pluripotency of ESCs is defined by the ability of the cells to divide into all three germ layers and their cellular derivatives. The plasticity and differentiation potential of ESCs made them an attractive target for human cardiac regeneration. The ability to direct PSCs into cardiomyocytes has evolved over the past decade. Initial techniques relied on formation of embryoid bodies followed by isolation of beating clusters, but more recently monolayer differentiation methods have enabled the creation of cardiac populations with greater than 90% purity.^173,174

However, there are several barriers to widespread adoption of ESCs. First, these cells are derived from human embryos, the use of which is still fraught with significant ethical concerns. Second, the robust differentiation potential of ESCs also carries a potential risk of uncontrolled differentiation that could result in neoplastic tumors.^175,176 Third, ESC derivatives are considered allogenic transplants, with a potential to provoke an immune response.^177,178 This means that the therapeutic use of ESCs may require use of immunosuppressive drugs, which are associated with significant risks of their own. Nonetheless, there have been some small endeavors in utilizing human ESC derivatives for noncardiac indications (mostly ESC-derived retinal pigment cells), all showing a good safety profile so far.^179,180

The first study utilizing ESC-derived cardiac progenitor cells for patients with severe cardiomyopathy undergoing CABG is underway, with an estimated study completion date in June 2017 (retrieved from http://clinicaltrials.gov/ct2, identification NCT02057900).¹⁸¹ This study employs the surgical implantation of a fibrin patch embedded with ESC-derived cardiac progenitor cells, which is one of many tissue engineering approaches under study to improve cell delivery and electromechanical coupling.^20,182,183

In 2006, a pioneering paper by Shinya Yamanaka described induction of pluripotency from mouse somatic cells by transfecting them with a retrovirus carrying the pluripotency-associated transcription factors Oct3/4, Sox2, c-Myx, and Klf4.¹⁸⁴ This process was replicated with human fibroblasts a year later,¹⁸⁵ commencing the widespread adoption of induced pluripotent stem cells (iPSCs). The mechanism underlying this process involves reprogramming somatic cells, such as skin fibroblasts or PBMCs, to express pluripotency transcription factors that allow them to acquire a pluripotent phenotype that closely resembles ESCs. The reprogramming can be driven by viruses, plasmids, microRNA, or proteins.¹⁸⁶ By inducing the adult cells to express major markers of pluripotency, iPSCs take on a phenotype very close to ESCs and are capable of differentiating into all three germ layers and subsequent stem cell types. As a potential source of cardiomyocytes for regenerative therapy, the cells bypass the ethical concerns associated with ESCs and can be used for autologous transplantation, hence lowering the risk of a significant immune response.¹⁸⁷ However, the most efficient reprogramming methods rely on viral vectors, which bring additional concerns of viral immune response (eg, Sendai virus) and integration into recipient genome (eg, lentivirus) that could result in malignant transformation. The first clinical trial utilizing iPSC-derived retinal pigment epithelial cells for treatment of macular degeneration commenced in Japan in 2014 (retrieved from http://www.who.int/ictrp/en/, WHO ICTRP JPRN-UMIN000011929). However, the study was temporarily halted (now restarted) as a result of single nucleotide variations and three copy-number variants identified in the second patient’s iPSCs that were not detectable in the original fibroblasts.¹⁷⁹ To date, no human clinical trial utilizing iPSCs for cardiac regeneration is yet underway.

Independent of their regenerative potential, iPSCs have emerged as a powerful investigative tool in studying cardiovascular diseases driven by the fact that the cells are patient-specific, allowing them to express the underlying genome of the original somatic cell donor. This suggests that iPSC-derived cardiomyocytes (iPSC-CMs) can be surrogates for in vivo cardiomyocytes of the original somatic donor. This concept has led to multiple studies testing the ability of iPSC-CMs to recapitulate disease phenotype in a dish, and results thus far have shown a good correlation with clinical phenotype of long QT syndrome,^188,189,190 catecholaminergic polymorphic ventricular tachycardia,¹⁹¹ arrhythmogenic right ventricular cardiomyopathy,¹⁹² dilated cardiomyopathy,^193,194,195 hypertrophic cardiomyopathy,¹⁹⁶ left ventricular non-compaction,¹⁹⁷ and doxorubicin induced cardiotoxicity.¹⁹⁸ This technology provided the first reliable source of human-like cardiomyocytes that can be used for basic science studies, which have long relied on animal models for such investigations. In addition, iPSC-CMs are emerging as a superior platform for drug cardiac safety testing compared to heterologous expression models,^199,200 and they are being considered as part of the Comprehensive in Vitro Proarrhythmia Assay (CiPA) Initiative.¹⁹⁹ Furthermore, by providing a precision medicine platform for patients with hereditary cardiac disorders, this technology is expected to provide predictive power for improving screening, diagnosis, risk stratification or drug responses (Fig. 10–2).^202,203,204

The past decade witnessed a spurt in studies using stem cell therapy approaches for cardiovascular regeneration. More than 100 studies have been published using various subtypes of stem cells, delivery methods, and study populations. BMMNCs show mixed results in AMI, chronic heart failure, and PAD clinical trials, but they are currently under testing in a large phase III study for AMI in Europe. CD34⁺ cells appear to show an efficacy signal in treating chronic angina symptoms and will be tested in a phase III study. MSCs and cardiac progenitor cells show encouraging preliminary results that promoted larger studies to test efficacy for cardiovascular indications. The first human ESC-derived cardiac progenitor cell trial is now underway. Fortunately, the most consistent finding of all these studies collectively is the relative safety of cell therapy and associated procedures. Moving forward, major efforts are needed to consolidate individual endeavors into larger, more rigorous clinical trials that aim to answer the question of efficacy through robust morbidity and mortality outcomes and not surrogate end points. Implementing mechanistic approaches to identify optimal stem cell type, delivery method, and indication will also be important in guiding the design of future clinical trials. The growing burden of cardiovascular disease for health-care systems and societies worldwide speaks to the urgent need for new therapies to overcome the significant limitations of available medical and surgical therapies. The current science and clinical data support an optimistic view that stem cells will play a role in the treatment for cardiovascular disorders.

We thank Blake Wu and June Wha Rhee for critical reading of the manuscript. We are grateful for funding support from American Heart Association 13EIA14420025 (JCW) and 16FTF30870002 (KS), Burroughs Wellcome Foundation, California Institute for Regenerative Medicine RT3-07709 and DR2A-05394 (JCW), National Institutes of Health (NIH) R01 HL126527, NIH R01 HL128170, and NIH R01 HL123968 (JCW). As a result of space constraints, we could not include and detail all the relevant citations on the subject matter, for which we apologize.