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Myocardial infarction–induced heart failure is a prevailing cause of death in the United States and most developed countries. The cardiac tissue has extremely limited regenerative potential, and heart transplantation for reconstituting the function of damaged heart is severely hindered mainly due to the scarcity of donor organs. To that end, stem cells with their extensive proliferative capacity and their ability to differentiate toward functional cardiomyocytes may serve as a renewable cellular source for repairing the damaged myocardium. Here, we review recent studies regarding the cardiogenic potential of adult progenitor cells and embryonic stem cells. Although large strides have been made toward the engineering of cardiac tissues using stem cells, important issues remain to be addressed to enable the translation of such technologies to the clinical setting.
Heart disease is a significant cause of morbidity and mortality worldwide. In the United States, heart failure is ranked number one as a cause of death, affecting over 5 million people and with more than 500,000 new cases diagnosed each year.1 The health care expenditures associated with heart failure were $26.7 billion in 2004 and are estimated to $33.2 billion in 2007. Although significant progress has been made in mechanical devices and pharmacological interventions, more than half of the patients with heart failure die within 5 years of initial diagnosis. Wide application of heart transplantation is severely hindered by the limited availability of donor organs. To this end, cardiac cell therapy may be an appealing alternative to current treatments for heart failure.
Recent investigations focusing on engineering cells and tissues to repair or regenerate damaged hearts in animal models and in clinical trials have yielded promising results. Considering the limited regenerative capacity of the heart muscle, renewable sources of cardiomyocytes are highly sought. Cells suitable for myocardial engineering should be nonimmunogenic, should be easy to expand to large quantities, and should differentiate into mature, fully functional cardiomyocytes capable of integrating to the host tissue. Adult progenitor cells (APCs) and embryonic stem cells (ESCs) have extensive proliferative potential and can adopt different cell fates, including that of heart cells. The recent advances in the fields of stem cell biology and heart tissue engineering have intensified efforts toward the development of regenerative cardiac therapies. In this article, we review findings pertaining to the cardiogenic potential of major APC populations and of ESCs (Fig. 1). We also discuss significant challenges in the way of realizing stem cell–based therapies aiming to reconstitute the normal function of heart.
Bone marrow (BM) is a heterogeneous tissue comprising of multiple cell types, including minute fractions of mesenchymal stem cells (MSCs; 0.001–0.01% of total cells2) and hematopoietic stem cells (HSCs; 0.7–1.5cells/108 nucleated marrow cells3). The heterogeneity of BM makes challenging the identification of a subpopulation of cells capable of cardiogenesis, and studies of BM cell–to–cardiac cell transdifferentiation should be examined through this prism.
The notion that BM-derived cells may contribute to the regeneration of the heart was first illustrated when dystrophic (mdx) female mice received BM cells from male wild-type mice.4 More than 2 months after the transplantation, tissues of the recipient mice were histologically examined for the presence of Y-chromosome+ donor cells. Besides the skeletal muscle, donor cells were identified in the cardiac region, suggesting that circulating BM cells contribute to the regeneration of cardiomyocytes.
Further supporting evidence was provided by Jackson et al.5 in studies using a side population (SP) of cells characterized by their intrinsic capacity to efflux Hoechst 33342 dye through the ATP-binding Bcrp1/ABCG2 transporter. The cells were isolated from the BM fraction of HSCs of Rosa26 mice constitutively expressing the β-galactosidase reporter gene (LacZ). After SP cells were injected into mice with coronary occlusion–induced ischemia, cells coexpressing LacZ and cardiac α-actinin were identified around the infarct region with a frequency of 0.02%. Endothelial engraftment was more prevalent (3.3%). The observed improvement in myocardial function may thus be attributed to the potential of BM cells to give rise to a rather endothelial progeny. This may be a parallel to cardiovascular progenitors from differentiating ESCs giving rise to cardiomyocytes, and endothelial and vascular smooth muscle lineages.6,7
Orlic et al.8 also reported the regeneration of infarcted myocardium after transplantation of lineage-negative (LIN−)/C-KIT+ BM cells from transgenic mice constitutively expressing enhanced green fluorescent protein (eGFP). Cells were injected in the contracting wall close to the infarct area. Nine days after transplantation, an impressive 68% of the infarct was occupied by newly formed myocardium with eGFP+ cells displaying cardiomyocyte markers such as troponin, MEF2, NKX2.5, cardiac myosin, GATA-4, and α-sarcomeric actin. Similar outcomes were reported by the same group9 when mouse C-KIT+ (but not screened for LIN) BM cells were transplanted.
Although these findings led to the conclusion that BM cells can repopulate a damaged heart, work by other investigators has casted doubt on this assertion. Balsam et al.10 noted that mice with infarcts receiving BM LIN−/C-KIT+, C-KIT-enriched or THY1.1low/LIN−/stem cell antigen-1 (SCA-1+) cells exhibited improved ventricular function. However, donor cells expressed granulocyte but not heart cell markers 1 month after injection. In another study,11 HSCs carrying a nuclear-localized LacZ gene flanked by the cardiac α-myosin heavy chain promoter were delivered into the periinfarct zone of mice 5h after coronary artery occlusion. One to 4 weeks later, LacZ+ cells were absent in heart tissue sections from 117 mice that received HSCs. Similarly, no eGFP+ cells were detected in the infarcted hearts of mice infused with BM cells constitutively expressing eGFP. Finally, Nygren et al.12 in similar transplantation experiments observed only blood cells (mainly leukocytes) originating from BM HSCs in the infarcted myocardium without evidence of transdifferentiation of donor cells to cardiomyocytes.
Occurrence of transdifferentiation is also not supported by the low frequency of transplanted BM cells detected in the infarct border zones. Moreover, BM-derived cells were shown to fuse with various mature cells, including cardiomyocytes.13,14 Such cells resulting from the fusion of cardiac cells with BM progenitors do not necessarily adopt a bona fide heart cell phenotype. This became evident when SP cells from the BM of wild-type male mice were injected into sarcoglycan-deficient female mice.15 Y-chromosome+ cells were detected in the myocardium of recipient mice but without expressing γ-sarcoglycan. Thus, fusion may not lead to reprogramming of BM progeny to fully functional cardiomyocytes.
A subpopulation of BM cells was recently reported to self-renew for more than 140 population doublings while retaining their potential for trilineage differentiation.16 These cells appear to be different from HSCs and MSCs because they do not exhibit many of the known markers. Additionally, the cells are negative for the pluripotency marker OCT4. Upon coculture with neonatal rat cardiomyocytes, the cells become myocardial-like by both fusion and differentiation. After injecting these cells in the ventricular wall of nude rats that underwent coronary ligation, the left ventricle end-diastolic and -systolic dimensions were improved, myocardial fibrosis was decreased, and the capillary density in the infarct was augmented. The authors concluded that the BM cells promoted angiogenesis and cardiomyocyte proliferation possibly by the observed increase in paracrine factors with proliferative and protective effects.
Besides the hypothesized “paracrine effect,” two additional attributes of BM-derived MSCs have strengthen their potential for heart therapies. First, undifferentiated MSCs appear to home to the infarcted heart after their injection in pigs,17 rats,18 and dogs.19 The homing of MSCs to areas of injury including the infarcted heart may be linked to the level of stromal cell–derived factor 120,21 although the physiological importance of this dependence remains unclear. Second, there is evidence that MSCs can induce local immune permissiveness.22 If this is the case, the risk for inflammation and graft rejection after allogeneic MSC transplantation will be minimal.
Despite the divergence in findings about the potential of BM cells for heart repair or regeneration, clinical trials (reviewed by Wollert et al.23 and Dimmeler et al.24) involving the delivery of BM cells to patients with heart maladies have been reported or are currently in progress. Intracoronary injection of unfractionated23 or mesenchymal BM cells25 in ischemia patients resulted in functional improvement characterized by an increased ejection fraction and reduced ventricular dilation compared to a control group of patients receiving sham infusions. Small but significant improvement (~5%) in left ventricular ejection fraction was also noted in a more recent trial26 after intracoronary delivery of BM cells to myocardial infarcts. This is in stark contrast to the 0.6% increase in injection fraction observed by Lunde et al.27 in cardiac patients receiving BM mononuclear cells via intracoronary injection.
Overall, delivery of BM cells appears to be safe and may have beneficial effects on damaged myocardium, at least over the relatively short time span of most studies. However, clinical investigators must examine the long-term safety (e.g., possible increased risk for atherosclerosis) and efficacy of BM cell therapies before these become part of mainstream treatments of heart maladies. Addressing these issues will require elucidation of the mechanisms by which reported functional improvements are brought about by BM cells.
Finally, various groups have attempted to mobilize endogenous BM cells with cytokines for infarct repair. The benefits stemming from treatment with cytokines such as the granulocyte colony-stimulating factor for heart disease patients are debatable, considering the conflicting results that have been obtained from animal studies.28–32 The topic of cytokine mobilization of BM cells for heart repair is not discussed here but has been reviewed elsewhere.33,34
Recent reports of the isolation of resident myocardial progenitors have challenged the long-standing paradigm of the heart being an organ with no regenerative capacity. Cardiac progenitor populations are identified based on a negative selection for mature cardiomyocyte, endothelial, or smooth muscle markers, and/or a positive selection for primitive cell markers such as C-KIT35,36 or SCA-1.14
Like the BM37 and other adult tissues,38 the heart also hosts SP progenitor cells. These cells are ~1% of the total cell population in the adult mouse heart39 and can fuse with skeletal muscle cells. Martin et al.40 also reported the isolation of mouse cardiac SP cells displaying SCA-1high, C-KITlow, CD34low, and CD45low. Differentiation of the SP cells toward α-actinin+ cells is achieved by coculture with mature cardiomyocytes. Functional attributes including contractility and intracellular calcium transients of such differentiated cardiac SP cells (CD31−/SCA-1+) were described more recently.41
Differentiation of other heart-residing progenitors to myocardium cells has also been reportedly promoted only after coculture with native cardiomyocytes. For example, cardiac stem cells, which form aggregates termed “cardiospheres,”42 isolated from human atrial/ventricular specimens also generate beating structures only when cultured with rat cardiomyocytes (in contrast to mouse cardiospheres that start to beat spontaneously without coculture). These cells proliferate in vitro and express cardiac markers such as cardiac troponin I (cTnI) and atrial natriuretic peptide (ANP). When the cells were injected in SCID mice, myocyte and vascular cell (expressing α-smooth muscle actin) progenies were observed, but there was no difference in the infarct sizes between mice receiving human cardiospheres and those in the control group (phosphate buffer saline-injected). Interestingly enough, potential fusion between the host cells and cardiospheres was not investigated. This is an important issue given that fusion of implanted heart-residing progenitor cells with adult heart cells has been reported.14 In addition, the requirement for coculture of these progenitor cells with native cardiomyocytes is not practical from a future clinical application viewpoint.
To that end, more recent studies suggest that isolated human cardiac progenitor cells can be coaxed toward functional cardiomyocytes without coculture. Goumans et al.43 showed that human cardiac stem cells cultured in differentiating medium with 5-azacytidine (5-aza-dC; a DNA demethylation agent) and ascorbic acid form elongated aggregates that beat spontaneously and ~35% of the entire cell population express α-actinin. This fraction increases to ~98% when the differentiating medium is supplemented with transforming growth factor-β (TGF-β). The differentiated cells also express connexin-43 (Cx-43) and connexin-40 in a typical gap-junctional pattern, and display chronotropic responses amenable to pharmacological modulation. It will be interesting to investigate how these cells perform in vivo.
Bearzi et al.44 also demonstrated that a population of C-KIT+ cells (1.1% of total cells) residing in the human myocardium can give rise to myocytes (as well as smooth muscle and endothelial cells) when cultured in serum-containing medium with dexamethasone. These progenitors, which are negative for hematopoietic and endothelial antigens, contribute to the actual heart muscle and to a lesser extent to the arterioles and capillaries when injected to rodents with cardiac infarcts. The human and rodent cells show structural coupling (Cx-43) and synchronicity in calcium tracings. The investigators also excluded the possibility of fusion between the grafted and the host cells as shown after (i) delivering Cre-recombinase expressing progenitors to the infarcted heart of mice expressing loxP-flanked eGFP and (ii) analyzing the number of sex chromosomes in donor and recipient cells. Regeneration of the myocardium was accompanied by an increase in the ejection fraction and an improvement of ventricular function.
Cardiac progenitor cells have also been identified based on the expression of the LIM homeodomain transcription factor Isl1. Isl1−/− mouse embryos exhibit growth retardation with severely abnormal hearts at approximately E9.5 and die at E10.5.45 ISL1+ cells isolated postnatally from mouse myocardium express sarcomeric α-actinin and cardiac troponin T (cTnT) in a partially or completely organized pattern46 after a coculture with neonatal cardiac myocytes. The cells exhibit contractile activity, and electromechanical coupling to neighboring cells as evidenced by the presence of Cx-43, and display action potentials similar to nodal and atrial cells. Differentiation of ISL1+ cells has also been carried out by coculturing with fixed cardiac cells, suggesting that the process is independent of cell fusion. When the ISL1+ progenitors are cultured alone in medium conditioned by myocytes, they do not express α-actinin. As with cardiac SP cells, there seems to be a requirement for cell–cell contact with native heart cells for successful induction of ISL1+ cells toward cardiac muscle lineages. Despite their apparent multilineage potential,47 ISL1+ cells are present in the mouse heart only until the perinatal period challenging the notion for extensive contribution to cardiac regeneration in adults.
Finally, fetal/neonatal48,49 heart cells have also been shown to proliferate and mature to fully functional cardiomyocytes, which form nascent intercalated discs with host cardiomyocytes upon engraftment. It is virtually certain that human fetal donor cardiomyocytes cannot be obtained in quantities useful in a clinical setting. However, these studies provide a proof of concept that other cells resembling fetal cardiac cells (e.g., ESC-derived cardiomyocytes) may be an effective alternative for heart therapies.
Skeletal muscle satellite cells (also known as myoblasts) fuse with muscle fibers to regenerate injured muscle. Besides their autologous nature, myoblasts derived in small quantities from biopsies can proliferate extensively in vitro, making them attractive for clinical use. Skeletal muscle progenitor cells were shown to differentiate into cardiomyocytes,50,51 but more recent evidence refutes such transdifferentiation. Differentiated myoblasts do not express junction proteins (e.g., Cx-43 or N-cadherin) and therefore cannot integrate electromechanically to the host cardiac tissue.52
Nevertheless, skeletal muscle progenitors appear to acquire some of the attributes of cardiomyocytes upon transplantation to animals,53 resulting in improved heart function.50 More importantly, myoblasts were used in patients with severe ischemic cardiomyopathy in a phase I clinical trial.54 The cells were detected in the infarcted hearts months after their ex vivo growth and implantation concomitantly with an increase from 24% to 32% in ejection fraction. Of note, both this clinical trial and animal studies55 revealed an increased risk for arrhythmias stemming from the failure of the implanted myoblasts to become functionally coupled with the native cardiomyocytes. Nonetheless, the mechanisms underlying the documented enhancement of ejection fraction and contractility, increased wall thickness, and in some cases proliferation of donor cells in the infarcted area after skeletal muscle cell transplantation are currently unclear and warrant further investigation.
ESCs with their unrestricted proliferation and potential to differentiate into multiple cell lineages can serve as an inexhaustible source of cells for cardiac tissue engineering. Both mouse ESCs (mESCs) and human ESCs (hESCs) have been shown to differentiate into cells exhibiting cardiac-specific markers and corresponding functional attributes. Cardiomyocyte differentiation is typically carried out with ESCs56,57 organized in aggregates either on low attachment surfaces58 or during coculture with cells such as visceral endoderm-like END-2 cells or HepG2 carcinoma cells.59,60 Human ESCs can also be coaxed to heart cells while cultured in monolayers.61
Many details about the exact signals directing the differentiation of ESCs to heart cells are still unclear. Nevertheless, several agents (Table 1) are reported to stimulate or enhance the commitment of ESCs to heart cells in vitro, including serum, 5-aza-dC,58 retinoic acid,57 and ascorbic acid,62 which have rather pleiotropic effects. On the other hand, molecules engaged in embryonic heart development such as TGF-β ligands63,64—mainly bone morphogenetic proteins (BMPs)63,65—fibroblast growth factors (FGFs),65 and Wnt signals or antagonists66,67 are attractive candidates for inducing ESC cardiogenesis (Fig. 2).
Myocardial differentiation of mESCs was initially demonstrated in embryoid body (EB) suspension culture.68 Cells within EBs differentiate into cardiomyocytes in addition to yolk sac and blood island cells. More recently, Kattman et al.6 showed that a population of brachyury+/flk1− cells within mouse EBs gives rise to a second group of FLK1+ cells that are progenitors of cardiomyocytes and vascular smooth muscle and endothelial cells.
Mouse ESC–derived cardiomyocytes have many of the morphological, biochemical, and functional characteristics of native cardiac cells. Cells organize into contractile areas (Fig. 3) with cell–cell junctions and exhibit sarcomeric organization similar to those of cardiomyocytes in vivo.69 Moreover, they express transcription factors such as NKX2.5, GATA-4, and MEF2. Structural components including sarcomeric myosin heavy chains (α- and β-MHC), myosin light chains (MLC-2a and −2v), α-actinin, cTnT, and cTnI are also detected in differentiated ESC progeny. The action potentials of mESC-derived heart cells depending on their stage of differentiation are akin to those of pacemaker cells (early-stage differentiated cells) or atrial and ventricular cells (terminally differentiated cells).70
The cardiac differentiation of mESCs has been studied longer than that of hESCs. These studies have expanded our view of events and factors that play key roles during heart cell differentiation. To that end, mESC-derived heart cells will remain an important tool for research on heart development. Although hESCs are physiologically more relevant, mESCs can be propagated faster, and in most instances their differentiation to cardiac cells is based on protocols that are fairly straightforward to implement. This facilitates streamlining their use in diagnostic applications, including high-throughput assays for the discovery of drugs71 against cardiac maladies.
The findings from mESC studies have served as starting points for attempts to differentiate hESCs into heart cells. Similar to mESCs, hESCs form beating areas of cardiac muscle–like cells when cultured as EBs. Some of the factors known to enhance the cardiogenic commitment of mESCs (e.g., 5-aza-dC14,58,72) have similar effects on hESCs, while others (e.g., retinoic acid57,58) do not. Still, the differentiation efficiency in EB cultures is generally low, and as other reports (Table 1) and observations in our laboratory suggest, such differentiation depends on parameters including the hESC line used, the starting cell concentration, the size of the forming EBs, the type of surface used for adhering EBs to organize into beating foci, the concentration of exogenous agents, and timing of their application.
Species differences between mESCs and hESCs should also be considered. Spontaneously beating foci appear within 8–10 days in differentiating mESC cultures, whereas the corresponding time for contractile regions to emerge in hESC cultures reportedly varies from as early as 7 days73 to over a month.74 Moreover, mESC-cardiomyocytes appear growth-arrested, whereas cardiac progeny of hESCs gradually withdraws from cell cycle as differentiation proceeds but proliferation is still evident both in vitro and in vivo even when markers of mature cardiac cells emerge.75 Laflamme et al.76 reported the proliferation of hESC-derived cardiomyocytes after their delivery to the uninjured heart of nude rats. Cells harvested from EB outgrowths were enriched in cardiac cells by Percoll gradient centrifugation (which does not preclude the presence of undifferentiated cells though) and were heat shocked briefly to enhance their survival. Four weeks after transplantation, ~14.4% of human cells within the graft expressed Ki67 and 2.7% incorporated BrdU leading to a sevenfold increase in graft size. This was concomitant with angiogenesis within the human cardiac implants. The cells expressed β-MHC, MLC-2v, and ANP, but not Cx-43. Therefore, it may be advantageous for nonterminally differentiated cardiac cells generated from hESCs to exhibit moderate proliferation while differentiating since a small number of cells will be sufficient for repairing extended areas of injury. Expanding implanted cells may also facilitate vascularization of the new tissue.
More recently, hESC-derived cardiomyocytes were transplanted in rodents after myocardial infarction, but the animals were monitored for more than 3 months.77 Similar to the above study, the transplanted cells survived selectively, and improved heart function was observed for 4 weeks. However, this improvement diminished at 12 weeks despite the formation of substantial grafts in the infarcted hearts. Even when three times more cells were injected,78 resulting in proportionally increased graft sizes, the initial preservation of cardiac function became insignificant after 12 weeks compared to control animals injected with noncardiac hESC–derived cells.
Improvement of the long-term functionality of hESC-derived cardiomyocytes may require directing their differentiation with physiologically relevant factors instead of by coculture or EB culture methods. Factors such as activin A and BMP4 may be used to direct the commitment of hESCs to cardiac lineages.61 Similar to mESC commitment to cardiovascular progenitors,6 Yang et al.7 demonstrated that hESCs forming EBs in serum-free medium with activin A, BMP4, bFGF, vascular endothelial growth factor (VEGF), and a canonical Wnt signaling inhibitor (Dickkopf homolog 1 [DKK1]) differentiate into cardiac, endothelial, and vascular smooth muscle cells. These progenitors, which are C-KITneg/KDRlow (also known as FLK1), generate more than 50% contracting cardiomyocytes when plated in monolayer cultures and exhibit electrophysiological attributes resembling those of human atrial/ventricular myocytes. When C-KITneg/KDRlow-derived cells were injected to the hearts of nonobese diabetic/SCID mice, populations of human cells emerged coexpressing α-actinin, CD31, and smooth muscle MHC. Two weeks after injection, a 31% higher ejection fraction was observed in mice receiving progenitor cells than those injected with media alone. Of note is the fact that there were no teratomas observed.
Caspi et al.79 also reported the absence of tumors upon grafting proliferating hESC-cardiomyocytes into immunosuppressed rats with either uninjured or infarcted hearts. Others,80 however, observed teratomas after delivery of hESC-cardiac cells to rat hearts. Similar conflicting findings have been published81–86 about the formation of tumors after transplantation of committed mESCs to mice with ischemic hearts. Such discrepancies may be attributed to the different methodologies employed for ESC differentiation and isolation of their progeny. Further research is necessary to determine the proliferation and tumorigenic potentials (see next section Tumor formation and immunogenicity of ESCs and ESC-derived cells) of hESC-derived heart cells, especially over longer time spans than those reported thus far.
Although APCs do not appear to be tumorigenic, the documented formation of teratomas by ESCs87 points to a potential danger if undifferentiated ESCs are inadvertently present in the cell population intended for transplantation. This problem can be compounded further by potential karyotypic abnormalities that have been observed in some hESC lines after prolonged culture.88–90 Eliminating the risk for teratoma formation is essential before any hESC-based therapy becomes reality.
Genetic tagging with antibiotic resistance genes (e.g., Hygror) or reporter genes (e.g., eGFP) downstream of cardiomyocyte-specific promoters (e.g., Nkx2.5) may be an effective method for removing noncardiac cells. This concept was illustrated by Klug et al.91 with the generation of cardiomyocytes from mESCs carrying the aminoglycoside phosphotransferase gene (neor conferring resistance to G418) under the cardiac-restricted α-MHC promoter. After in vitro differentiation, the cells were subjected to G418 selection, and the resulting cardiomyocyte population exhibited greater than 99% purity. When these cells were delivered into the left ventricular free wall of dystrophic mdx mice, noncardiomyocyte outgrowths from the engrafted cells were absent. More importantly, the cells formed stable intracardiac grafts aligning well with host cardiomyocytes.
A similar approach has been applied to select hESC-derived cardiac cells.92,93 Human ESCs expressing the Hygror–eGFP fusion protein downstream of the human MLC-2v promoter were subjected to cardiac differentiation and selection.92 Approximately, 94% of the resulting cells stained positively for cardiac-specific markers. After grafting these cells to rat hearts, immunostaining did not reveal OCT4+ (undifferentiated) cells although specific data on teratoma formation were lacking. Also, gap junctions were observed between the grafted cells and the host myocardial cells after 4 weeks. These findings suggest that transplanted ESC-derived cells undergoing selection may integrate functionally to the heart without forming tumors.
However, the long-term effects of introducing genes in ESCs need further scrutiny. Moreover, screening based on antibiotic resistance may still result in the selection of false positive clones92 or karyotypically abnormal subpopulations adopting a heart cell fate. On the other hand, the throughput of current cell sorting methods (e.g., flow cytometry) is still inadequate for processing ESC-derived cardiomyocytes in clinically relevant quantities. Advanced high-throughput technologies in conjunction with improved protocols for cardiomyocyte differentiation of hESCs will contribute toward diminishing or eliminating the risk for tumorigenesis in hESC-derived grafts.
Besides the risk for tumor formation, the immunogenicity of stem cells intended for therapies should also be examined. Human ESCs and hESC-derived grafts appear less susceptible to immune rejection than adult cells.94 Evidence94,95 suggests that hESCs have low immunogenicity, a property that depends strongly on the transplantation setting (e.g., allogeneic and xenogeneic).96 Reduction of host reactivity to allogeneic ESC-derived transplants could be achieved through immunosuppression, which unfortunately is associated with serious side effects. Alternatively, the cells can be engineered to minimize their immunostimulatory potential. For example, cell banks can be established for the storage of hESC lines modified to display particular HLA profiles although generating a sufficient number of HLA-isotyped hESC lines will require enormous effort and resources. Immune rejection of implanted cells may also be avoided by eliminating immune attack–triggering agents such as proteins of the major histocompatibility complex,97 but the long-term impact of such knockouts is unknown.
Generation of autologous hESCs may be possible through the transfer of somatic nuclei of the patient to enucleated human eggs, an approach known as therapeutic cloning. Although current technologies for successful and efficient transfer of human cell nuclei require further development, the use of human oocytes for the generation of autologous donor cells encumbers nontrivial ethical and legal problems. For this purpose, alternative methods (e.g., nuclear reprogramming,98,99 parthenogenetically derived embryos,100 and induced pluripotent stem cells101,102) for deriving hESCs or hESC-like cells are under intense investigation and may ultimately eliminate the need for human embryos and oocytes.
Similar ethical issues do not pertain to the derivation and use of APCs, but the issue of culture-induced antigenicity remains. A frequent argument for the use of APCs in cell therapies is their autologous nature. However, similar to ESCs, APCs manipulated in vitro for their expansion and differentiation are exposed to culture medium supplements (e.g., serum) that alter their antigen profile. This can lead to rejection of transplanted cells as shown in animal studies.103 This issue needs to be addressed in the context of heart therapies utilizing stem/progenitor cells.
Although no evidence exists of tumor formation in APC grafts, relying on adult stem cells as a source for heart cells presents additional challenges as well. First, tissue-residing stem cells are typically isolated in very small numbers as part of heterogeneous populations. Apparently, each laboratory develops and implements different protocols for the isolation and purification of these cells rendering difficult the interpretation and comparison of studies on cardiogenic APCs. Second, unlike ESCs, most APCs (with exceptions, e.g., multipotent APCs104) have limited proliferation potential hindering greatly efforts for scaling up APC cultivation systems. Culture conditions allowing for prolonged proliferation of these cells are highly desirable, but factors promoting APC expansion are not fully known. In many instances, the presence of mitogens (e.g., EGF and platelet-derived growth factor [PDGF]) in the culture media used for the maintenance of progenitor cells may contribute to the appearance of genetically abnormal strains over extended cell passaging. Third, many tissue-residing progenitors possess limited plasticity that makes their differentiation to distant cell types uncertain. In fact, evidence to support the proposal for transdifferentiation, that is, the adoption of a substantially different fate by cells already committed to a specific lineage, has been controversial, and for some studies alternative explanations are more plausible.105,106 Fourth, aging of APCs could impact their use for regenerative therapies. Progenitor cells isolated from older patients show impaired proliferation, adhesion, and incorporation into vascular structures.107 Fifth, safety issues have been raised regarding the use of the some types of APCs for myocardial repair, including arrhythmias with skeletal myoblasts,108 myocardium scars with MSCs,109 and cardiac calcification with BM-derived cells.110 Lastly, claims of endogenous cell proliferation due to paracrine mechanisms (e.g., APC-secreted mitogens) are still under investigation although the extent of such proliferation reported is substantially lower than that required for heart repair.
Because of their extensive proliferative capacity, the generation of a large number of stem cells, especially hESCs, for downstream applications, including cardiac tissue engineering, is possible in principle. However, realizing this possibility will require the development of bioprocesses suitable for large-scale expansion and directed differentiation of stem cells. Currently, stem cells are maintained in static cultures such as dishes and multiwell plates. This mode of cultivation has inherent shortcomings which impede the production of cells in clinically relevant masses. Such quantities will require an impractically large expanse considering that the number of cells grown in static cultures is proportional to the available substratum area. Further, continuous monitoring and control of the physical and chemical environment within a dish culture are challenging. This problem becomes increasingly important when ESCs reach high densities where local concentration gradients of factors and nutrients contribute to the observed uncontrolled differentiation of these cells.
Culture of stem cells in stirred vessels may facilitate overcoming some of these issues. Stirred-suspension bioreactors have a simple design and are amenable to scale-up. More importantly, this culture setup allows for automated surveillance and control of the process variables that are crucial for stem cell pluripotency and expansion. Stirred-tank bioreactors are based on a mature technology platform and are the workhorses for the production of cellular products in the biotechnology industry. Stem cell culture systems developed around this bioreactor design may therefore be easier to translate to an industrial production setting than entirely novel bioreactors. Thus, cultivation of stem cells in stirred suspension vessels represents a major alternative to current stem cell culture systems.
In conventional stirred-suspension vessels, concentrations of 106–107 cells/mL are common. To put this in perspective, 1g of adult myocardium contains approximately 2–4×107 myocytes.111 A typical myocardial infarction that induces heart failure leads to a loss of approximately 50g of heart muscle.112 Then, an engineered myocardial tissue should contain at least 1–2×109 myocytes to compensate for the lost tissue. Therefore, suspension culture vessels with working volumes of a few hundred milliliters to a few liters will be sufficient, although issues related to the respective efficiency of differentiation should be considered as well. As a proof of concept, Schroeder et al.113 used a 2-L stirred-tank bioreactor with automated control for the differentiation of mESCs carrying the αMHC promoter-neor gene to heart muscle cells. After 9 days of EB culture and 9 days of selection with G418, approximately 1.28×109 essentially pure cardiomyocytes were generated. The resulting cell clusters were contracting, and the cells displayed striated patterns for αMHC, α-actinin, and cardiac troponins T-C. Others have also reported the generation of cardiomyocytes from mESCs in scalable setups.114–116 Application of such selection schemes in a large-scale bioreactor should be viewed with caution. Selection entails the death of noncardiac cells that represent a substantial fraction of the total cell population. The resulting cell debris can have a significant impact not only on the quality of the derived cardiomyocytes but also on the downstream processing (e.g., separation) of these cells.
To our knowledge large-scale (e.g., bioreactor) cardiomyocyte differentiation of hESCs has not been reported to date although their culture as human EBs (hEBs) has been described.117–119 Cultivation of hEBs in a high-aspect rotating vessel resulted in massive cell death due to hEB excessive agglomeration. Human ESCs cultured in a slow turning lateral vessel formed hEBs without further agglomeration, but no information was available about lineage-specific markers displayed by these cells.119 Cameron et al.117 reported the propagation of hESCs as hEBs in spinner flasks and evaluated their potential for hematopoietic differentiation. A 15-fold expansion in the total number of EB-derived cells cultured for 21 days in spinner flasks was observed compared to only a 4-fold expansion in static cultures.
Maintenance and directed differentiation of stem cells in stirred vessels will require further improvements before such systems can be brought to commercial use for the preparation of cell therapeutics. For example, cardiac differentiation of ESCs as EBs involves their transfer onto adhesive surfaces for the formation of beating areas with mainly cardiac cells. Isolation of cells from these contractile areas is not straightforward, and methods such as microdissection120 may not be readily scalable, necessitating the development of efficient strategies for cardiac cell culling. Also, online assessment of differentiating cell phenotype linked to high-throughput selection platforms for cardiac cells will be essential. Moreover, the formulation of chemically defined media121,122 for propagating and coaxing stem cells to heart cells123 will be necessary. This is a critical issue considering the problems associated with the use of serum, including great batch-to-batch variability, high cost, and the risk for zoonosis. Obviously, better understanding of the determinants underlying stem cell self-renewal and cardiogenesis will help to advance the development of scalable systems for the production of cardiac muscle cells for therapies.
Efficient differentiation of stem/progenitor cells per se may not be sufficient for translating stem cell–based heart therapies to the clinical setting. Another important aspect is the organization of the cells in tissues with properties of native cardiac muscle. This also relates to the method of delivery of the cells to the damaged heart tissue for its repair. Direct injection of differentiated cells (intracoronary or catheter-based infusion) is problematic because cell homing to the scarred tissue and its repair are seriously challenged by the local hemodynamic conditions and the pulsating muscle. Further, up to 95% of injected cells may become nonviable during injection or shortly after.124 Issues pertinent to the transplantation of stem cells in the damaged myocardium, including the electromechanical integration of donor cells, time and mode of delivery, and dose and outcome measures, are reviewed elsewhere.125,126
Nonetheless, cells for myocardial repair may be engineered into functional tissues (e.g., patches) before their transplantation. Cardiac tissue constructs127 should be contractile, electrophysiologically stable, mechanically robust and flexible, amenable to vascularization, and nonimmunoreactive. An approach pioneered by Langer and Vacanti128 involves the use of matrices onto which cells are seeded. Various materials used in the construction of these matrices, which include polylactic acid, alginate, and polyethylenglycol, are reviewed elsewhere.129,130 Cells within scaffolds conduct action potentials,131 exhibit synchronous contractile activity, and become hypertrophic under electrical stimulation.132 However, current scaffold designs require substantial improvements. Cells within scaffolds experience low oxygen tension posing a serious problem for cardiomyocytes that are metabolically very active. Further, many of the scaffolds used in engineered tissues are fairly rigid, hindering the free self-assembly of cells into structures resembling the in vivo myocardial architecture and limiting the transmission of contractile activity. Decellularized scaffolds derived from native pericardium have been proposed as an alternative (as in the case of engineered aortic valves133), but adequate sourcing and immunogenicity134 impose considerable impediments for their use.
Soluble scaffold materials as demonstrated by Eschenhagen et al.135 may help to overcome some of these problems. When freshly isolated heart cells are mixed with collagen type I and extracellular matrix proteins, they form a strongly contracting tissue. Successful reconstitution of contracting tissue requires the culture of the construct under mechanical load.136 Also, the presence of nonmyocyte heart cells along with the cardiomyocytes in the engineered tissue contributes to the formation of capillaries and endoepicardial surface lining. Transplantation of such tissue equivalents to immunosuppressed rats with myocardial infarcts led to a functional improvement without induction of arrhythmias.137 Grafts were vascularized and electrically coupled to the host heart. Although these findings are encouraging, scaling these engineered tissues to constructs suitable for larger animals and human patients will be a major challenge.
Besides the use of rigid and soluble scaffolds, a third approach was taken by Shimizu et al.138 based on thin films of poly-N-isopropylacrylamide (PIPAAm), a thermoresponsive polymer. Neonatal rat cardiomyocytes are seeded on PIPAAm films and cultured at 37°C until they reach confluence. Upon lowering the temperature to 20°C, the polymer film with attached cells is peeled off intact. When sheets are stacked together, they pulsate simultaneously. These sheets were also beating for up to 12 weeks after their transplantation into dorsal subcutaneous tissues of nude rats. Extensive Cx-43 coupling was observed among cells of the different layers along with vascularization of the graft, well-differentiated myofilaments, and desmosomes. Notably, there were no changes due to inflammation of the construct. Although this approach for engineering cardiac tissue equivalents appears promising, thick stacks of cell sheets (in the order of at least ~1cm) will be needed for restoring the function of an ailing human heart. Inevitably, issues are raised pertinent to adequate oxygen (as well as nutrient) transport within such tissue constructs. Considering that cardiomyocytes constitute only 30% of the total myocardial mass139 and other heart cells are also essential for proper cardiac function, it will be interesting to see if sheets can be generated with mixed populations of heart cells.
Cardiomyocyte-like cells generated by differentiation of ESCs and APCs exhibit phenotypes resembling native heart myocytes. Regeneration of cardiac muscle from endogenous proliferation of heart-residing progenitors still remains debatable. Even if heart APCs can give rise to mature cardiac progeny in vivo (e.g., via a pharmacological intervention), the extent of such expansion may not be adequate for effective clinical therapies. However, findings reviewed here undoubtedly prove that stem/progenitor cells will play a key role as renewable sources of cardiomyocytes. There are still significant challenges before stem cell–based therapies for heart disease will become a clinical reality. Conceivably, the intense ongoing research in this field and the resulting advances in recent years suggest that such challenges can be tackled effectively sooner than previously believed.
This work was funded by a J.D. Watson Award from the New York State Office of Science, Technology and Academic Research (to E.S.T.), and by grants HL55324 and HL61610 (to J.M.C.) and HL092398 (to E.S.T.) from the National Institutes of Health.
No competing financial interests exist.