Cardiovascular Pathology 24 (2015) 133–140

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Cardiovascular Pathology

Innovations in Cardiovascular Pathology, Pathobiology, Interventions and Technology

The winding road to regenerating the human heart

Kaytlyn A. Gerbin a, Charles E. Murry a,b,c,⁎a Department of Bioengineering, Center for Cardiovascular Biology and the Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USAb Department of Pathology, Center for Cardiovascular Biology and the Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USAc Department of Medicine/Cardiology, Center for Cardiovascular Biology and the Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA

a b s t r a c ta r t i c l e i n f o

⁎ Corresponding author. Center for Cardiovascular BRoom 454, Seattle, WA 98109, USA. Tel.: +1-206-616-86

E-mail address: murry@uw.edu (C.E. Murry).

http://dx.doi.org/10.1016/j.carpath.2015.02.0041054-8807/© 2015 The Authors. Published by Elsevier Inc. T

Article history:Received 19 December 2014Received in revised form 10 February 2015Accepted 10 February 2015

Keywords:Myocardial infarctionHeart regenerationCell transplantationStem cell-derived cardiomyocytes

Regenerating the human heart is a challenge that has engaged researchers and clinicians around the globe fornearly a century. From the repair of thefirst septal defect in 1953, followed by thefirst successful heart transplantin 1967, and later to the first infusion of bone marrow-derived cells to the human myocardium in 2002, signifi-cant progress has been made in heart repair. However, chronic heart failure remains a leading pathological bur-denworldwide.Whyhas regenerating the human heart been such a challenge, and how close arewe to achievingclinically relevant regeneration? Exciting progress has been made to establish cell transplantation techniques inrecent years, and new preclinical studies in large animal models have shed light on the promises and challengesthat lie ahead. In this review, we will discuss the history of cell therapy approaches and provide an overview ofclinical trials using cell transplantation for heart regeneration. Focusing on the delivery of human stem cell-derived cardiomyocytes, current experimental strategies in the field will be discussed as well as their clinicaltranslation potential. Although the human heart has not been regenerated yet, decades of experimental progresshave guided us onto a promising path.Summary: Previouswork in clinical cell therapy for heart repair using bonemarrowmononuclear cells, mesenchy-mal stem cells, and cardiac-derived cells have overall demonstrated safety and modest efficacy. Recent advance-ments using human stem cell-derived cardiomyocytes have established them as a next generation cell type formoving forward, however certain challenges must be overcome for this technique to be successful in the clinics.

© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Amyocardial infarction (MI) transforms healthy and contractile myo-cardium into an akinetic, fibrotic tissue, resulting in a heart that cannotpumpblood at full capacity. As theheart is oneof the least regenerative or-gans in the body, this often leads to the development of chronic heart fail-ure— a diseasewith a 50% survival rate over 5 years [1]. Current treatmentoptions are limited and consist primarily of palliative drugs, organ replace-ment by heart transplant (available to b0.1% of heart failure patients), ormechanical assist devices (with complications related to infection, throm-bosis, and power supply). While these available treatments have greatlyimpacted the trajectory of patient health after an MI, ischemic heart dis-ease remains the number one cause of death and disabilityworldwide [2].

In recent years, the field of heart regeneration has emerged from afar-fetched notion to the forefront of cardiac research. Heart regenera-tion is an interdisciplinary field with the goal of restoring functionalmyocardium after cardiac injury [3]. Approaches to repair the injuredheart have beenwidespread and include cell transplantation, gene ther-apy, stimulating innate repair pathways, direct cellular reprogramming,cardiac tissue engineering, and biomaterial delivery. The most

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his is an open access article under the C

established strategy for heart repair has been the delivery of exogenouscells. Nearly every cell type imaginable has been transplanted into thedamaged myocardium, from skeletal myoblasts to pluripotent stemcells and their derivatives. It is an exciting but challenging time for phy-sicians, scientists, and engineers in the field — we now have over a de-cade of experience in clinical trials contributing to heart regenerationresearch, and there are several promising preclinical strategies emerg-ing as contenders to our current clinical approaches.

In this review, we provide an overview of the clinical trial progres-sion using cell therapy to regenerate the heart after ischemic injuryand discuss strengths and limitations of these trials. We will then dis-cuss current experimental strategies designed to improve upon whatwe have learned in these clinical trials, focusing on the advancementsin stem cell-derived cardiomyocyte transplantation and the clinicaltranslatability of this approach for heart repair.

2. Cell therapy clinical trials for heart repair

Approximately 1 billion cardiomyocytes are lost during anMI [3]. Asthe adult human heart has an extremely limited regenerative capacity,this damaged myocardial tissue is replaced by fibrotic scar. There is in-creasing evidence of the slow cardiomyocyte turnover rate during nor-mal organ growth and development (reviewed in Ref. [4]), andfollowing myocardial injury [5], however, this turnover accounts for a

C BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

134 K.A. Gerbin, C.E. Murry / Cardiovascular Pathology 24 (2015) 133–140

low percentage of cells. Up to 3% of preexisting cardiomyocytes near theinjury region undergo cell division while most DNA replication occurswithout cytokinesis as a hypertrophic response, and there is minimalcontribution from progenitor cells [5]. As a result, the innate generationof de novo cardiomyocytes post-MI falls orders of magnitude short ofmeaningful regeneration.

Exogenous cell transplantation aims to repair damagedmyocardial tis-sue either by delivering cells that act via paracrine-mediated effects or byproviding de novo cardiomyocytes that directly contribute to force pro-duction. Toward this goal, numerous clinical trials have been conductedusing cell types including skeletal myoblasts, bone marrow-derived he-matopoietic cells, mesenchymal stem cells (MSCs, also known as marrowstromal cells), adipose-derived cells, endothelial progenitor cells, andcardiac-derived cells (CDCs) (reviewed in Refs. [6–9]). A schematic over-view of the derivation, deliverymode, and proposedmechanism of actionfor themajor groups of cell therapies is provided in Fig. 1. An ideal cell typefor replacing damagedmyocardial tissuewould have contractile and elec-trophysiological properties, the ability to survive and integrate into an is-chemic area, proliferation potential, and the ability to elicit a paracrineeffect to stimulate endogenous regeneration (e.g., vascularization;discussed in detail in Refs. [9] and [10]). Despite the plethora of celltypes tested in clinical trials to date, nonehasmet all of these expectations.The type of cell used for transplantation inherently places restrictions onimportant variables that may affect the success of cell therapy, making itdifficult todirectly compare results across trials. These include thedeliverymode (intracoronary catheter, transendocardial catheter, or epicardialcatheter delivery compared to epicardial delivery in tissue patches), theavailability of autologous or allogenic cells, and the timing of cell deliverydependent on the need for in vitro cell expansion (i.e., MSCs require ex-tensive in vitro expansion, while unfractionated bone marrow cells maybe delivered the same day of isolation).

The field hasmade tremendous progress in terms of establishing clin-ical trial design, delivery techniques, and demonstrating safety; however,the clinical benefits have been modest at best. This indicates that there isroom for improvement on our cell source. The two major cell sourcesused in the clinics thus far have been bonemarrow-derived cells and car-diac explant-derived cells, which are discussed below.

2.1. Bone marrow-derived cells

Following closely behind the first major wave of clinical trials in thefield using skeletal myoblasts [11], bone marrow-derived cells pavedtheway for intracoronary cell therapy in the heart, transitioning quicklyinto the clinic despite the scarcity of published evidence supportingtheir role in heart regeneration at the time [12,13].

2.1.1. Bone marrow-derived mononuclear cell derivativesMost bone marrow-derived cell transplantation trials in the heart

have used an unfractionated subpopulation called bonemarrowmono-nuclear cells (BMMNCs) (reviewed in Ref. [14]). Referring to BMMNCsas a stem cell preparation is a misnomer because true stem cells com-prise well below 0.1% of the total mononuclear cell population.Unfractionated BMMNCs principally consist of a heterogeneous popula-tion of hematopoietic cells including monocytes, committed myeloidprogenitor cells and lymphocytes, and a small population of hematopoi-etic and mesenchymal stem cells [9,15].

Intracoronary transplantation of BMMNCs into patients with acuteMI was first reported in 2002 [13], and while this trial has beendiscredited for ethics violations, it was followed by a flurry of more rig-orously performed studies.Most of these early BMMNC studies enrolledacute MI patients with ST-segment elevation and a baseline ejectionfraction of 40–50%, and they reported functional improvement aftertreatment. One such studywas the BOOST trial [16] inwhich autologousBMMNCs (characterized as b1% CD34+) were isolated from patientsand delivered by intracoronary infusion to the infarct-related arterythe same day. No serious adverse events were reported in either

group, and cardiacmagnetic resonance imaging (MRI) at 6months indi-cated a significant increase in left ventricular (LV) ejection fraction aftercell treatment (compared to placebo control), providing evidence thatintracoronary infusion of BMMNCs improves systolic function in acuteMI patients. In longer-term follow-up studies, however, the controlgroup showed a “catch-up” period of recovery, such that benefits ofBMMNCs could no longer be demonstrated [17]. Results from theREPAIR-AMI trial [18] further supported efficacy for BMMNCs, reportinga 5.5% increase in LVejection fraction at 4months after intracoronary in-fusion of BMMNCs compared to a 3.0% improvement in controls. Whilethe results of this studywere hindered by the use of quantitative LV an-giography to assess function as opposed to cardiac MRI, the enrollmentof over 200 patients made this the largest BMMNC trial at the time andset the standard for expected systolic improvement, albeit a modest in-crease, after cell therapy. The same group reported that functional im-provement persists up to 5 years posttreatment in a subset of patientswhowere enrolled in the TOP-CARE-AMI trial [19–21], which comparedthe benefits of BMMNCs to those of autologous circulating progenitorcells isolated from venous blood.

Despite these and other studies reporting functional improvementafter BMMNC treatment (reviewed in Ref. [22]), larger trials employinggreater degrees of randomization, placebo controls, and blinding con-ducted in the years following have not replicated these results. The Car-diovascular Cell Therapy Research Network (CCTRN) was designed tofacilitate cell-based therapies in the United States [23] and sponsoredthe FOCUS-CCTRN trial [24], which was one of the first trials to targetpatients with chronic LV dysfunctionwho had not qualified for revascu-larization therapy post-MI. Enrolled patients had a mean baseline ejec-tion fraction of 30–32% andNewYork Heart Association (NYHA) class of2 or 3, andwhile therewas no improvement in the primary endpoints ofLV end systolic volume or maximal oxygen consumption, there was asmall yet statistically significant 1.4% improvement in LV ejection frac-tion over baseline at 6 months. The CCTRN also sponsored the TIMEand LateTIME trials to assess the influence of BMMNC delivery timingon LV function [25–27]. Each of these double-blinded and placebo-controlled trials enrolled successfully reperfused MI patients and deliv-ered 150×106 autologous BMMNCs by intracoronary perfusion either atday 3 or 7 (TIME) or at 2–3weeks (LateTIME) afterMI. Neither studyde-tected any functional benefit by cardiacMRI at 6months after cell treat-ment, regardless of delivery timing. Similar in cell dose and design, theSWISS-AMI study [28] compared BMMNC delivery at days 5–7 to deliv-ery at weeks 3–4 after post-MI reperfusion and again detected no im-provement in LV ejection fraction at 4 months. Collectively, thesestudies challenge the earlier reports of functional improvement, butthey differ in using a double-blinded study design and in targeting pa-tients with significantly worse baseline cardiac function (for example,the median ejection fraction of SWISS-AMI patients was 37%). It seemsunlikely to us that this difference in baseline cardiac function underliesthe difference, however, because the REPAIR-AMI trial found that the pa-tientswith theworst ejection fractions showed the greatest improvementwith treatment. Results are eagerly awaited from the 3000-patient enroll-ment,multicenter Phase 3 trial (the BAMI trial), which is currently under-way in Europe, as itwill help clear up someof the conflicting results in thefield (clinical trial identifier NCT01569178 [29]).

2.1.2. Mesenchymal stem cellsNumerous trials have been conducted using MSCs purified from

bone marrow, which are adult cells characterized for their osteogenic,chondrogenic, and adipogenic differentiation potential [30,31]. Lessthan 0.01% of the cells isolated from bone marrow are consideredMSCs [32,33], therefore obtaining clinically relevant cell numbers re-quires ex vivo expansion.

The first clinical trial investigating the intracoronary injection ofMSCs reported an improvement in LV ejection fraction and increasedmyocardial perfusion 3months after treatment [33], echoing the resultsreported using BMMNCs at the time. A few studies have directly

paracrine signaling electromechanical coupling paracrine factor

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Fig. 1. Cell transplantation techniques and proposedmechanisms of cell therapy for heart regeneration. (A) Cell transplantation afterMI. (1) Cardiac-derived cells (CDCs) are isolated fromeither the atrial appendage or the septal wall, expanded in vitro, and transplanted via intracoronary catheter delivery. (2) Bonemarrowmononuclear cells (BMMNCs) andmesenchymalstem cells (MSCs) are harvested from the bone marrow. BMMNCs may undergo purification steps followed by transplantation via intracoronary catheter delivery or intramyocardial in-jection,whileMSCs require in vitro expansion prior to transplantation,most commonly by intramyocardial injection. (3)Humancardiomyocytes are derived fromhumanpluripotent stemcells (hPSCs) after in vitro expansion and directed cardiac differentiation. The proposed clinical delivery method for hPSC-cardiomyocytes (hPSC-CMs) is via transepicardial ortransendocardial catheter-based injection. (B) Proposed mechanism of action after cell transplantation. Bone marrow-derived cells and CDCs work primarily though paracrine signaling,in which transplanted cells secrete paracrine factors to the surrounding infarcted myocardium. hPSC-cardiomyocytes act primarily though the direct electromechanical integration withneighboring host cardiomyocytes. Paracrine factors may also be secreted by the hPSC-cardiomyocytes.

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compared the safety profile and efficacy of BMMNCs toMSCs, includingthe TAC-HFT trial [34]. In this study, chronic MI patients received atransendocardial injection of BMMNCs (harvested the day of implant)or MSCs (expanded 4–6 weeks in vitro prior to implantation). Whilethere was no difference between groups in the 1-year serious adverseevent rate or LV ejection fraction, patients receiving MSCs showed in-creased regional function by strain analysis and an improvement in exer-cise capacity. These studies used autologous MSCs; however, a limitationof this approach is that autologous MSCs require a significant expansionperiod between the time of bone marrow aspiration and implantation.The POSEIDON trial was designed to address this and to directly comparethe safety andefficacy of autologousMSCs to allogeneicMSCs [35]. Chron-ic MI patients received a dose of 20, 100, or 200 million autologous or al-logeneic MSCs, injected into the myocardium via a transendocardialcatheter, and the study concluded that neither cell source stimulated asignificant adverse immune response. Curiously, there was an inversedose response in terms of improved ejection fraction and reversed LV re-modeling, with more improvement detected in the 20 million cell dosethan the 200 million cell dose.

Up until this point, studies focused their efforts on testing cells intheir native MSC state, but the C-CURE trial took a unique approach bytreating autologous MSCs with a cytokine cocktail prior to transplanta-tion [36]. Guided by NOGA electromechanical mapping, these cytokine-stimulated MSCs were transplanted transendocardially into chronicheart failure patients an average of 1540 days post-MI. Cell-treated pa-tients showed an absolute improvement of 7% in their ejection fractionand enhanced exercise capacity, compared to no improvement in con-trols. This is a surprisingly large treatment effect, given the long dura-tion postinfarction in these patients. As previously reviewed [37],most MSC studies have demonstrated that the cells die off within a

week or two posttransplantation with little direct cardiac differentia-tion.Mechanismsof benefit in this trial could not be determined, but an-imal studies suggest it is likely a paracrine action.

2.1.3. Comments on bone marrow-derived cell therapyThrough the successful completion of numerous Phase I clinical tri-

als, bone marrow-derived cell therapies have established an importantfeasibility and safety baseline for delivering cells into the myocardium[14,38]. While there have been a few reports of significant functionalimprovement, for themost part these therapies have resulted in amod-est reduction in scar size after infarction with little (at best) improve-ment in systolic function. Because the majority of transplanted cellsdie off within a fewweeks [39] and there is no solid evidence of cardio-genic potential, all benefits are believed to be paracrine mediated(Fig. 1B) [3]. Therapies using BMMNCs andMSCs suggest that interven-tion by cell therapy can change the trajectory of wound healing and theinflammatory response after an infarction, but with no long-term im-provement in global heart function or long-term engraftment, thesetherapies are not truly regenerating the heart.

2.2. Cardiac-derived cells

The most recent addition to the clinical trials has been CDCs, whichare derived from myocardial biopsies and grown as explants in cultureto obtain an autologous CDC population. Studies in rodents have sup-ported their potential to be a more effective cell source than BMMNCsand MSCs [40], and CDCs were originally postulated to be progenitorcells capable of forming new cardiomyocytes. However, most investiga-tors now think that these cells, like bone marrow cells, show minimallong-term engraftment or cardiac differentiation and instead work

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principally through paracrine signaling pathways. The three leading tri-als using CDCs to date are described below.

2.2.1. The SCIPIO TrialThe first trial using CDCs focused on cells expressing the surface anti-

gen c-kit, whichwere first isolated and characterized in the rat [41]. Sim-ilar to bone marrow-derived cells, initial animal studies suggested thatc-kit+ cells gave rise to cardiomyocytes; however, lineage tracing studieshave determined that these cells show minimal long-term engraftmentand only extremely low rates of cardiac differentiation in the adultheart [42–44]. The SCIPIO (cardiac stem cell infusion in patients with is-chemic cardiomyopathy) trial enrolled 33 heart failure patients withchronicMI (mean ejection fraction of 27.5% at baseline), who underwenta right atrial appendage biopsy during coronary bypass surgery. This atri-al tissue was used to isolate a putative cardiac progenitor cell thatexpressed the surface antigen c-kit and was negative for hematopoieticand cardiovascular lineagemarkers. After 4months of in vitro expansion,0.5–1 million cells were injected via the coronary arteries perfusing theischemicmyocardium of 20 patients, while 13 patients remained as con-trols. Analysis of heart function by 3D echocardiography or cardiac MRIshowed an 8.2% and a 12.3% improvement in LV ejection fraction at 4and 12 months, respectively, and, somewhat surprisingly, a reductionin infarct size in a subset of patients [45]. Readers should know, however,that results from this study have been flagged with an “expression ofconcern” by the editors of the Lancet relating to an ongoing investigationpertaining to data integrity as of this writing [46].

2.2.2. The CADUCEUS TrialThe next trial of CDCs involved “cardiosphere-derived cells”, a mesen-

chymal cell population obtained by explant culture of endomyocardial bi-opsies, followed by transient growth as cellular spheroids [47].Cardiosphere-derived cells are heterogeneous by surface markers butare primarily CD105+/CD45−. In the Phase I CADUCEUS (cardiosphere-derived autologous stem cells to reverse ventricular dysfunction) trial[48,49], patients with a mean LV ejection fraction of 39% undergoing pri-mary angioplasty 2–4 weeks after MI had a right ventricular biopsy re-moved to expand autologous cells. After a 1- to 3-month expansionperiod, 25 million cardiosphere-derived cells were delivered as anintracoronary infusion into the infarct-related artery. Although the prima-ry endpoint was safety, cardiacMRI at 6 and 12months after cell deliveryrevealed a reduction in infarct size (identified as a reduced region of de-layed gadolinium enhancement) and an increase in viable myocardium.Although there was no significant change in global ejection fraction,cell-treated patients showed improved regional systolic wall thickeningthatwasmaintained from4months to 1 year after treatment. The authorsinterpreted the increase in viable myocardium seen by MRI as regenera-tion, but pathological hypertrophy of preexisting cardiomyocytes cannotbe ruled out as an alternate explanation. Although not statistically signif-icant, cell-treated patients experiencedhigher levels of nonsustained ven-tricular tachycardia and serious adverse events at the 1-year follow-up,which will require closer evaluation in future trials.

2.2.3. The ALCADIA TrialThe ALCADIA (autologous human cardiac-derived stem cell to treat is-

chemic cardiomyopathy) trial is ongoing at the time of this review (clin-ical trial identifier NCT00981006) and takes a combined cell therapy andcontrolled growth factor-release approach that was first established in aporcinemodel of chronicMI [50]. The trial enrolled advanced heart failurepatientswith LVejection fraction of 15–45% andNYHAclass of 3 or 4,withprimary endpoints of 1-year safety and secondary endpoints of assessingfunctional improvement by echocardiography and MRI, NYHA class, andexercise capacity at 6 months. At the time of coronary artery bypassgrafting, patients received a transepicardial injection of autologousCD105+/CD90+ CDCs grown from an endocardial biopsy (0.5 millioncells per kilogram of patient body weight). Injection sites were subse-quently covered with a biodegradable gelatin sheet that was loaded

with basic fibroblast growth factor (bFGF) by incubation with bFGFprior to implantation. Although there were only six patients enrolledand no controls at the time, preliminary reports suggest an increase inejection fraction, a decrease in infarct size, and an increase in patient aer-obic exercise capacity [51]. If successful in larger-enrollment trials, thisdual cell delivery and biomaterialsmethodmay promote a shift in clinicalapproaches in the future toward the combined use of cell and drug deliv-ery, and this is a progressive approach that deservesmore attention in thepreclinical and clinical setting. Of course, sorting the effects of the cellsfrom the growth factor delivery will require additional control groupswhere one of the combined factors is omitted.

2.2.4. Comments on CDC therapyTaken together, the achievementsmadewith CDCs support some ad-

vantages over previous bone marrow-derived cell therapies. The needfor ex vivo cell expansion of CDCs has provided insight to a later post-MI delivery timeline, and it is promising that cell delivery intomature in-farct scars 1–4months post-MI has resulted in detectable improvementsin clinical endpoints (primarily a reduction in infarct size). How such areduction in scar volume is achieved remains mysterious because scarsize is typically quite stable by 3 months post-MI. We speculate thatthe cellsmay reactivate innate immunemechanisms, particularly relatedto themacrophage. It is important to note that long-term cell retention isalmost nil with both CDCs and BMMNCs; thus, any benefitsmust requireonly the transient presence of the cell. Although double-blinded studieswith CDCs have been precluded by the need for myocardial biopsy,such trials in bone marrow-derived cell therapy have demonstratedthe importance of using proper controls, and this will be necessary inmoving forward with larger-scale trials. Since some groups are nowmoving toward allogeneic CDCs, it should be feasible to have placebo-controlled trials and to test these cells in acute MI patients [52].

3. Pluripotent stem cell-derived cardiomyocyte delivery

Considering themodest benefits fromheart regeneration clinical tri-als to date, there has been some debate over cell source — is there amore potent cell type to use for transplantation into the heart? Denovo cardiomyocytes meet many of the desired characteristics outlinedearlier, but finding a reliable cell source for cardiomyocytes was pre-cluded until the last decade. Methods to derive cardiomyocytes fromhuman pluripotent stem cells (hPSCs) have progressed tremendouslysince the first report of mouse embryonic stem cell-cardiomyocyte der-ivation [53], and there are now several efficient protocols to achieve car-diac differentiation that mimic developmental pathways (reviewed inRef. [54]). These differentiation advancements have brought hPSC-cardiomyocytes to the forefront as a promising next-generation cellsource, and their transplantation into the heart has been studied exten-sively in preclinical experiments. The leading strategy for cell deliveryhas been the intramyocardial injection of dispersed cardiomyocytes,which mirrors the delivery methods established in many cardiac-derived and bone marrow-derived cell transplantation clinical trials.Using this approach, various groups have demonstrated that hPSC-cardiomyocytes engraft in the infarct region of numerous animal modelsand result in an increase in cardiac function (reviewed in Ref. [55]).

In contrast to bone marrow derivatives and CDCs, humancardiomyocytes give stable, long-term grafts in infarcted hearts [56]. In-herent cell properties give transplanted cardiomyocytes the capabilityto electrically integrate with the host tissue, which is a prerequisite forsynchronous contraction with the host myocardium. The fluorescent cal-cium reporter proteinGCaMP3 [57] has been auseful tool to study the gapjunction coupling between graft and host tissue, and geneticallymodifiedhuman embryonic stem cell (hESC)-cardiomyocytes expressing GCaMP3have been found to electrically integrate with ischemia/reperfusion in-jured rat hearts (Gerbin et al., in revision) aswell as in cryoinjured guineapig hearts [58,59]. Unlike previous cell transplantation studies that areparacrine driven, this electrical coupling indicates that the engrafted

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Fig. 2. hPSC-derived cardiomyocytes remuscularize the infarctedmacaque heart [63]. (A) hESC-cardiomyocytes robustly engraft in the infarctedmyocardium, outlined by the dashed line,as indicated by confocal immunofluorescence at day 14 after transplantation. Engrafted cardiomyocytes express GFP (human, green) and both hESC-cardiomyocytes and hostcardiomyocytes express the contractile protein alpha-actinin (human and monkey, red) with nuclear DAPI counterstain (blue). Scale bar represents 2 mm. (B) The in vivo maturationof engrafted hESC-cardiomyocytes is evident from 14 days to 84 days postengraftment by costaining for alpha-actinin (human and monkey, red) and GFP (human, green). At latetimepoints, engrafted hESC-cardiomyocytes display a marked increase in cell size, sarcomere alignment, and myofibril content compared to early timepoints. Scale bar represents20 μm. (C) Host vasculature perfuses the hESC-cardiomyocyte graft at 84 days postengraftment, as visualized by 3D rendered microcomputed tomography. Large host arteries andveins are shown in red and blue, respectively, and small vessels perfusing the graft are shown inwhite. Other vessels in themyocardium are shown in gray. (D)Host-derived blood vesselsare found within the hESC-cardiomyocyte grafts at 84 days postengraftment. Host endothelial cells express CD31 (human and monkey, red) and infiltrate the GFP-positive graft area(human, green). Scale bar represents 20 μm. (E) Ex vivo fluorescent GCaMP3 imaging indicates that engrafted human cardiomyocytes are electrically coupled to the infarcted macaqueheart at 14 days postengraftment. GCaMP3 fluorescence intensity (green) and host ECG (red) are plotted versus time and demonstrate 1:1 coupling at spontaneous rate as well as duringatrial pacing at 3 Hz.

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cardiomyocytes are electrically integrating with the host myocardiumand are directly contributing to force generation (Fig. 1B). Indeed, im-provements in systolic function have been reported in various injurymodels after hPSC-cardiomyocyte transplantation [60–62].

Successful studies demonstrating long-term cardiomyocyte engraft-ment and functional integration in rodents have motivated the transla-tion of this approach into a nonhuman primate injury model [63]. Pig-tailedmacaques (Macaca nemestrina) received an ischemia/reperfusioninjury by inflating a balloon catheter into the distal left anterior de-scending coronary artery for 90 min followed by reperfusion. Twoweeks later, after initiating immunosuppression, 1 billion hESC-derived cardiomyocytes were transplanted through transepicardial in-jections into the infarcted myocardial wall. This study was the first todemonstrate large-scale myocardial remuscularization (Fig. 2A), andlarge cardiomyocyte grafts were found in the infarct region 3 monthsafter transplantation. Engrafted human cardiomyocytes demonstratedin vivo maturation from 14 days to 84 days, as indicated by an increasein cell diameter, sarcomere alignment, and myofibril content (Fig. 2B).Grafts were perfused by the host vasculature, which was shown bythe presence of CD31+ endothelial cells in the GFP+ graft and furthersupported by 3D rendered microcomputed tomography to visualizevessels within the graft region (Fig. 2C and D). Furthermore, GCaMP3fluorescence imaging showed that engrafted cardiomyocyteswere elec-tromechanically coupled to the host (Fig. 2E), as had been previouslydemonstrated in rodents. A notable concern from these studies,

however, was the detection of nonfatal ventricular arrhythmias in thecardiomyocyte-engrafted hearts. These arrhythmias were not observedinmice, rats, or guinea pigs [58], demonstrating the importance of usingrelevant large animal models. The ventricular arrhythmias will need tobe managed for safe translation of human cardiomyocytes to the clinic.

4. Translating hPSC-cardiomyocyte delivery to the clinic

While preclinical therapies with hPSC-derived cardiomyocytes haveshown promise and are progressing quickly, questions regarding car-diomyocyte engraftment, phenotype, and large-scale production mustbe addressed in order to promote successful translation from bench tobedside. Firstly, cell survival after transplantation is low regardless ofthe cell type and injury model used. Despite the improved engraftmentafter adopting ‘prosurvival’ cell treatments prior to implantation [60],current methods are not sufficient to achieve long-term high cell reten-tion. The use of tissue engineering approaches such as the implantationof cell sheets, epicardial patches, or cardiomyocytes delivered in bioma-terials may help increase the engraftment rate (reviewed in Refs.[64–66]), although the development of minimally invasive deliverytechniques will be important for clinical translation.

Secondly, the optimal maturation state of cardiomyocytes for trans-plantation is not fully understood. Previous studies suggest that an inter-mediary maturation state may be ideal: mature adult cardiomyocytes donot survive transplantation [67]; immature hESC-cardiomyocytes have

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automaticity, have slow conduction, andmay be proarrhythmic [63]; andmesodermal cardiac progenitor cells do not outperform definitivecardiomyocytes in terms of engraftment or efficacy (Fernandes andChong et al., in press). Current in vitro approaches may maturecardiomyocytes from the ‘early-fetal’ state typically achieved after differ-entiation into a late-fetal or neonatal stage. While in vitro maturation toan adult phenotype will be difficult, as indicated above it also is undesir-able for transplantation purposes (reviewed in Ref. [68]). Experimentsthat directly compare the engraftment of aged hPSC-cardiomyocytes tothe current standard are needed, as well as studies designed to track im-planted cells and characterize their maturation in vivo.

Lastly, one of the major hurdles for successful translation of hPSC-cardiomyocytes into the clinic is developing an “off-the-shelf” cardiomyo-cyte cell product: will these come from hESCs or from human-inducedpluripotent stem cells (hiPSCs)? The biggest advantage of using hiPSCsis their ability to provide an autologous cell source, but unfortunately,this is also one of the major limitations when it comes to clinical and fi-nancial feasibility. The process of obtaining patient-specific somaticcells, reprogramming to induced pluripotent stem cells, differentiatinginto cardiomyocytes (perhaps requiring individual protocol optimiza-tion), and performing quality control would take over 4 months [9].This precludes their use in an acute or subacute MI setting, and converse-ly, we have shown that hPSC-derived cardiomyocytes have no beneficialeffect on cardiac function when delivered into chronic MIs in rats andguinea pigs [56,59]. An additional limitation of the autologous hiPSC ap-proach is the cost. At present, there are very expensive quality control ex-periments that are required before the release of a product derived frompluripotent stem cells, and doing this for each patient is cost prohibitiveunless the regulatory path is changed. There is also an inherent risk of re-sidual undifferentiated stem cells giving rise to teratoma formation, andproper quality control measures must be taken tominimize this risk [69].

Most preclinicalmodels thus far have used hESC-cardiomyocytes, al-though immunosuppression after an allogeneic hESC-cardiomyocyteimplantation is a potential downfall of the therapy. More research isneeded to elucidate proper immunosuppression strategies that addressthe differential immunogenicity and rejection of autologous vs. allogen-ic cells (reviewed in Ref. [70]). Research strategies to engineer HLA-homozygous hESC subclones or “universal donor cells” that are HLAclass 1 negative will be useful in addressing this problem, and excitingprogress has beenmade on this front in recent preclinical work [71]. Re-gardless of cell source, it remains unclear if this number of cells can bemass produced for clinical use in a way that is financially manageableand biologically controlled. Cardiomyocyte production will need to bescaled up significantly tomeet the current demand, although the devel-opment of methods to increase cardiomyocyte proliferation for in vitroscale-up may help alleviate this concern.

5. Concluding thoughts

As thefield of cell-based cardiac repair hasmatured, there has been anatural shift from basic studies toward more clinical and translationalgoals. Nevertheless, it is important to continue with studies focusingon the underlying biology of heart regeneration; understanding the bi-ological mechanisms of cardiac repair will be critical in the field’s suc-cess regardless of therapeutic approach used. Simply put, unless weunderstand the mechanisms through which cell therapies work, thereis noway to rationally improve upon them. A promising alternative ap-proach to heart regeneration is stimulating endogenous repair after in-jury, which takes advantage of cues learned from regenerationexperiments in lower vertebrates and the discovery of the mammalianregenerative window after birth (reviewed in Ref. [72]). Although thisapproach is far from the clinic, exciting progress has beenmade to iden-tify factors that promote cardiomyocyte dedifferentiation and prolifera-tion after injury, including the overexpression of cyclins [73–75], FGFand NRG signaling pathways [76,77], Notch signaling [78,79], andmicroRNAs [80]. Lessons learned here will provide important insight

to the cell therapy field and may guide the development of dual geneand cell delivery therapies.

There are multiple parameters to consider when working towardclinically meaningful regeneration including functional recovery, atten-uation offibrosis, preventing adverse remodeling, cardiomyocyte prolif-eration (and subsequent increase in viable myocardium), and thematuration of regenerated cardiomyocytes. Because an ideal therapywould be suitable for patients with recent cardiac injury or withestablished heart failure, preclinical studies will require careful evalua-tion of how to translate benefits to a more chronically affected patientpopulation. The approaches discussed in this review were limited to is-chemic heart disease; however, the advancements in the field will havebroad implications for other heart failure patients, such as those suffer-ing from dilated cardiomyopathy, hypertrophic cardiomyopathy, orcongenital heart disease. Better understanding the biology governingthe heart’s response to injury and to regenerative cues will provide in-sight to better direct gene therapy, drug delivery, and tissue engineeringapproaches that target nonischemic heart disease.

In conclusion, the past few decades of heart regeneration researchhave been exciting and informative. Considerable progress has beenmade to establish cell transplantation techniques with bone marrow-derived cells and CDCs, and hPSC-derived cardiomyocytes have beenwell established in preclinical studies as a promising cell type movingforward. While many challenges lie ahead before successfullyregenerating the human heart, we are optimistic that thefield ismovingforward on a promising path.

Acknowledgments

This work was supported in part by National Institutes of Healthgrants R01 HL084642 (C.E.M.), P01 HL094374 (C.E.M.), P01 GM81619(C.E.M.), U01 HL100405 (C.E.M.), and T32 EB001650 (K.A.G.) and theNational Science Foundation Graduate Research Fellowship Programunder grant numbers DGE-1256082 and DGE-0718124 (K.A.G.).

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