New Muscle for Old Hearts: Engineering Tissue from Pluripotent Stem Cells

Abstract

Stem cell–based therapies are considered to be promising and innovative therapeutic strategies for heart repair. Patient-derived induced pluripotent stem cells (iPSCs) are now available, which combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells for proliferation and differentiation. Intense research has driven dramatic progress in various areas of iPSC technology relevant for clinically applicable iPSC-based cellular therapies. At this point, it is already possible to generate transgene-free autologous iPSCs from small blood samples or hair, to scale up the expansion and differentiation of iPSCs to clinically required dimensions, and to obtain highly enriched cardiomyocyte preparations. On the other hand, critical hurdles such as the targeted specification of distinct cardiomyocyte subpopulations, survival and proper functional integration of cellular transplants after myocardial infarction, and in vitro engineering of prevascularized muscle patches have yet to be overcome. Nevertheless, concepts of cellular cardiomyoplasty seem to have come of age and the first clinical applications of iPSC-based heart repair can be expected within the coming years.

Introduction

The world's aging population suffers from an increasing incidence of coronary heart disease. Indeed, the top cause of mortality worldwide is ischemic heart disease with about 7.4 million deaths alone in 2012.¹ Despite pharmacological and interventional therapies, the morbidity and mortality of patients with severely impaired ventricular function after myocardial infarction or other serious heart diseases remain high. Patients suffering from heart failure due to severe myocardial infarction, various cardiomyopathies, or congenital malformations have few options. One avenue is heart transplantation, which is limited by the number of available donor organs; or bridging to heart transplantation through left ventricular assist devices. The option of reconstructing cardiac congenital malformations is restricted to an even greater extent and is currently limited to implanting noncontractile Gore-Tex patches.

In light of these limitations, it is clear that new therapeutic approaches are required, and stem cell therapies are now being considered for the treatment of heart failure after myocardial infarction and of other cardiac insufficiencies and cardiomyopathies. In addition, the implantation of engineered contractile heart tissue is under discussion for the surgical replacement of scar tissue after myocardial infarction, as well as for the reconstruction of congenital malformations.

Stem cell–based concepts are widely considered as promising, but experimental and clinical studies applying various adult stem cells for cardiac repair have yielded for the most part disappointing results, in the majority of cases without evidence of any considerable functional improvement.^2,3 Despite some rare cells carrying single cardiac markers, no development of stem cell–derived cardiomyocytes (CMs) or even de novo formation of structured myocardium could be demonstrated. The observed minor functional effects are most likely of paracrine nature, including accelerated revascularization, enhanced myocyte survival in the infarct border zone, and the modulation of scar formation, resulting in improved mechanical properties.⁴

Another concept for cardiac repair is the targeted transdifferentiation of cardiac fibroblasts (Fbs) into functional CMs. This strategy appears especially interesting in view of the hypothesis that an already formed infarction scar with Fbs as the main cellular component could become reconverted into contractile myocardium. Inspired by an approach using the transcription factor MyoD to drive transdifferentiation of Fbs into skeletal muscle⁵ as well as by the groundbreaking work of Takahashi and Yamanaka,⁶ a set of cardiac transcription factors was overexpressed for the targeted differentiation of mouse cardiac and tail-tip Fbs into CMs in vitro.⁷ In contrast to the transdifferentiation of Fbs into neurons, however, the efficient conversion of Fbs into cardiac cells is obviously more difficult to achieve. Although cardiac marker expression could be demonstrated in single cells,⁸ the efficiency of such direct conversion experiments is still controversial.^9,10 On the other hand, the efficiency of the conversion of Fbs in vivo was remarkably high. In light of the difficulties experienced by other researchers in confirming these findings, it is clear that much more work is required to develop robust protocols for the transdifferentiation of Fbs into real, functional CMs.¹¹

Although more recent data on adult stem cells have been disappointing, other kinds of stem cells have become available. So-called induced pluripotent stem cells (iPSCs) provide, for the first time, a source for the production of patient-derived CMs for heart repair that is not ethically controversial.¹² iPSCs combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells (ESCs) for proliferation and differentiation.

Pluripotent Stem Cells for Clinical Heart Repair

Meanwhile, many hurdles and limitations for the production of clinically applicable iPSC derivatives have already been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood,^13
–15 and highly efficient protocols for the site-specific (and thus relatively safe) introduction of transgenes by homologous recombination have been developed, based on engineered nucleases including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) as well as the clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system.¹⁶ Using these novel tools, it has been possible to develop protocols for footprintless gene editing without the need for antibiotic selection.¹⁷ Such techniques are of key importance for preclinical animal studies and future cellular therapies as they allow, for example, the correction of disease-related mutations or the well-defined expression of reporter transgenes that facilitates the monitoring of graft survival, functional integration, and distribution in small and large animals.¹⁸ Moreover, the targeted introduction of transgenes into safe harbor sites¹⁷ is possible, enabling genetic enrichment of pure CMs before transplantation,^19,20 or to open up the possibility of killing the cellular graft in the case of tumor formation through inducible expression of suicide genes.²¹

iPSCs can now be expanded in scalable suspension culture²² and large numbers of human iPSCs can be produced in fully controlled bioreactors.²³ Moreover, sequential inhibition and activation of molecular differentiation pathways has, for the first time, allowed a targeted, more robust and efficient differentiation of human ESCs and iPSCs.^24,25 In the meantime, small molecules instead of recombinant proteins are used in such protocols, which has greatly improved the efficiency of cardiac differentiation.²⁶ The introduction of small molecules as an activator or inhibitor of molecular key pathways has facilitated the development of scalable protocols that are relatively inexpensive and more robust. Highly improved cardiac differentiation efficiencies of up to 90% now enable the production of the vast numbers of CMs required for clinical cell therapy or the engineering of large contractile muscle patches in stirred tank bioreactors.²⁷ Further enrichment is possible through genetic strategies but also by using nongenetic approaches including fluorescence-activated cell sorting (FACS)^28,29 or metabolic enrichment.³⁰ The functionality of human PSC-derived CMs, also in terms of proper reaction to various pharmacological stimuli, has been proven by various groups in vitro. Moreover, promising reports strongly suggest that various approaches such as long-term culturing, three-dimensional tissue engineering, mechanical or electric stimulation, and treatment with neurohormonal factors, or combinations thereof, will lead to mature human PSC-derived CMs.³¹

More difficult still is the targeted production of specific CM subtypes. However, if proper protocols are applied, the majority of PSC-derived CMs are typically of a ventricular phenotype,²⁷ which is the desired CM subtype for cellular therapy for myocardial infarction or the in vitro engineering of cardiac muscle patches. Moreover, potential key factors responsible for CM specification, for example, into pacemaker cells, have been identified^32,33 and attempts to develop protocols for subtype specification are ongoing.³⁴ Nevertheless, real breakthroughs have yet to be reported.

Further cell types of interest for cardiac cell therapy are endothelial cells, pericytes, and smooth muscle cells, all of which may help to improve revascularization after myocardial infarction, as well as fibroblasts, which are an essential component of engineered myocardial patches as a matrix producer. In the case of endothelial cells, protocols are now available that allow the efficient generation of functional cells with high capacity for further expansion.³⁵ Here, it is still under discussion as to whether it is necessary to achieve specification into endothelial subtypes, for example, microvascular endothelial cells,³⁶ or whether the generated endothelial cells express sufficient plasticity to appropriately adapt in the given niche in vivo. Pericytes are considered important to support vascular sprouting. Although the identification, enrichment, and characterization of these cells are still difficult because of the lack of truly specific pericyte markers, there are now protocols for the targeted generation of pericytes.³⁷ Smooth muscle cells, as the third important vascular cell type, can also be differentiated together with ECs from iPSCs via a common vascular progenitor.³⁸ Also, similar to CMs, the first protocols have been developed that allow the generation of endothelial cells and smooth muscle cells based on the application of small molecules.³⁹

Fibroblasts are important for engineering contractile heart tissue. If no fibroblasts are present in the myocardial constructs, there is no matrix consolidation, only low survival of CMs, and the constructs do not reach sufficient stability and stiffness.²⁰ On the other hand, it should be emphasized that not much is known about the required characteristics of fibroblasts for application in cardiac tissue engineering. Fibroblasts play a critical role in maintaining extracellular matrix (ECM) homeostasis in the heart, and a switch toward myofibroblasts appears to result in excessive collagen accumulation, contributing to impaired cardiac function. The understanding of fibroblast function in healthy and diseased hearts, and in myocardial tissue engineering, is hampered by the lack of specific markers and the heterogeneity of fibroblast precursors.⁴⁰ Actually, various protocols have been published on deriving mesenchymal cells from pluripotent stem cells.⁴¹ However, the exact definition and discrimination of the different mesenchymal cell lineages including mesenchymal stem cells, pericytes, fibroblasts, and myofibroblasts remain difficult. Not only is there a considerable overlap of markers between these cell types, but the degree of plasticity within these mesenchymal cell lineages is also unclear. Therefore, further research is required to better define cardiac fibroblasts and the manner in which mesenchymal cells with corresponding characteristics can be generated from iPSCs.

Besides the development of robust and scalable differentiation protocols, research on safety issues concerning iPSC-based cell transplants, including the appearance of genetic abnormalities,⁴² is probably the most critical aspect for future clinical application. It is necessary to carefully investigate to what extent mutations are already present in the source cells,⁴³ are generated during reprogramming, or are enriched during subsequent human iPSC (hiPSC) expansion, which is mandatory for many therapeutic applications. In addition, standard operation procedures are required to routinely check iPSCs and their derivatives for abnormalities before clinical use. However, the tumor risk can be considered much lower for terminally differentiated CMs than for other hiPSC-derived cell types. In fact, rhabdomyosarcomas, as the only malignant muscle-derived primary heart tumors, are rare.⁴⁴ Most of these tumors are found in children and are presumed to arise from persisting immature embryonic cells.⁴⁴

Despite these unresolved safety issues, the first patient has already undergone treatment for macular degeneration using hiPSC-derived retinal cells, following approval by the Japanese authorities.⁴⁵

In Vivo Application of iPSC Derivatives for Cardiac Cell Therapy

Clearly, the availability of human iPSCs and CMs derived from iPSCs has solved only some of the most severe limitations in current concepts for myocardial repair (Fig. 1). Further hurdles apply, in particular to the method of application of cellular grafts for heart repair.

FIG. 1.

Induced pluripotent stem cells (iPSCs) for myocardial repair. (A) Generation of patient-specific iPSCs. (B) Genetic engineering for safe introduction of selection genes and potential correction of disease-related mutation and clinical scale expansion of undifferentiated iPSCs. (C) Targeted differentiation and enrichment of cardiovascular cell lineages. (D) Engineering of bioartificial heart muscle and vascularized tissue patches. (E) Intramyocardial cell injection or implantation of an iPSC-derived muscle patch. CMs, cardiomyocytes; ECs, endothelial cells; Fbs, fibroblasts; SMCs, smooth muscle cells; TALEN, transcription activator-like effector nuclease.

The direct injection of cells to improve heart function was investigated in various animal models with a variety of mouse and human cell sources, including undifferentiated cells, stem cell–derived cardiovascular progenitors,^46,47 and CMs.^19,48 Remarkably, only a minor portion of the transplanted cells remained in the heart, regardless of the cell type or application method (intramyocardial or intracoronary injection). The early loss of transplanted cells obviously has several underlying causes, including insufficient transcoronary migration, contraction-related rapid loss through the injection channel, venous drainage to the lungs, and low survival within the ischemic myocardium.⁴⁹ Another critical aspect is the choice of the cell type to be transplanted. Besides CMs, cardiovascular progenitors are considered to be a preferred cell type because of their proliferative capacity, which may compensate for transplantation-related cell loss, and their ability to differentiate not only into CMs but also into endothelial cells and smooth muscle cells. However, although survival and cardiovascular in vitro differentiation were demonstrated, the injection of murine iPSC-derived cardiovascular progenitors into severe combined immunodeficient (SCID) mice did not result in the formation of structured myocardium.⁴⁴ Despite the elimination of undifferentiated cells through FACS enrichment, the transplanted cardiovascular progenitors formed largely unstructured tumor tissue consisting of CMs, smooth muscle cells, and endothelial cells, which brought into question the usefulness of early cardiovascular progenitors compared with more mature CMs.

The transplantation of murine ESC-derived CMs into mice resulted in structural and functional coupling of surviving grafts.⁵⁰ In contrast, injecting human PSC-derived CMs into the hearts of immunodeficient mice never did yield significant integration or long-term survival of the cell transplant (Van Laake et al.,⁵¹ reviewed by Nunes et al.⁴⁸). Although this has not been proven, it has been hypothesized that physiological incompatibilities, in particular differences in contraction frequency, present an underlying reason.⁵² Consequently, Shiba and colleagues chose guinea pigs with their relatively low heart rate as another small animal model for the transplantation of human ESC-derived CMs. Indeed, they observed that, after intramyocardial transplantation into the guinea pig heart, hESC-derived CMs formed functional de novo myocardium and led to considerable functional improvements after myocardial infarction (MI).⁵² However, the problem of low graft retention and survival was not solved in this study. In fact, it was necessary to inject extremely high numbers of myocytes (i.e., 10⁸ cells, which correlates to ∼2×10¹⁰ or approximately four times the total number of myocytes in the adult human adult left ventricle, when projected from the guinea pig to the human heart, which is 20 times larger).

So far, another problem for the clinical translation of stem cell–based heart repair has been the lack of suitable preclinical large animal models for the transplantation of iPSC derivatives. Although various reports have claimed to have generated iPSCs from pigs and sheep, there are still no true pluripotent stem cells available from these animals, and the reported iPSC-like cells typically depend on the introduced transgenes.⁵³ On the other hand, immunological rejection of human iPSC derivatives is obviously difficult to prevent in pigs and sheep. Although no engrafted CMs could be detected, at least engraftment and long-term survival of vascular hiPSC-derivatives has been demonstrated in a large animal model of MI,¹⁸ using novel imaging technologies. Interestingly, improved intramyocardial graft survival after the coinjection of human mesenchymal stem cells (MSCs)¹⁸ was observed, which might indicate an immunomodulatory effect of these cells. Another report has raised hopes that the survival of iPSC-derived myocytes may be achievable in pigs, using suitable protocols of pharmacological immunosuppression.⁵⁴ Interestingly, the authors observed that injection through a fibrin patch containing insulin growth factor (IGF)-encapsulated microspheres significantly improved cell retention and survival in the treated heart.⁵⁴

A rhesus monkey model of MI was used to investigate the effects of transplanted allogeneic early stage-specific embryonic antigen-1 (SSEA-1) cardiovascular progenitors.⁴⁷ Immunohistological analyses 2 months after injection of these cells into the scar area revealed engraftment of green fluorescent protein (GFP)-labeled donor cells and provided evidence of their differentiation into cardiomyocytes, apparently with a mature phenotype. These findings more recently led to a first clinical trial in 2014,⁵⁵ with no complications observed in the patient so far.

In another extremely promising approach, the survival and functional coupling of large human ESC-derived heart muscle islands was demonstrated in a preclinical nonhuman primate (NHP) model of MI,⁵⁶ although no conclusive data concerning potential increased contractility and improved heart function were shown.

Engineering iPSC-Based Bioartificial Cardiac Tissue in Vitro

If significantly improved retention and survival of infused donor cells can be achieved, the question still remains as to whether, how, and to what extent simple CM injection, even if it leads to de novo formation of contractile heart muscle, can overcome the substantial functional consequences of previously formed noncontractile scar tissue. Addressing this considerable limitation, the transplantation of in vitro–engineered heart tissue may be an alternative approach to actually replace fibrous scar tissue after myocardial infarction. On the basis of natural or synthetic biocompatible or biodegradable materials, contractile tissue constructs can be engineered in vitro either by seeding cells on matrices or by mixing soluble matrix components and cells.

Despite substantial progress in engineering heart tissue, the resulting bioartificial muscle still has relatively small dimensions and its structure is much simpler than the structure of the native heart with its complex spirally oriented muscle fibers. Ott and colleagues addressed the current limitations via another approach. They demonstrated that seeding neonatal rat CMs onto a completely acellularized rat heart can lead to a contractile tissue construct.⁵⁷ Remarkably, however, no follow-up studies demonstrating the further development of this concept have been published so far. Whereas reseeding the acellular vascular structures of natural matrices with endothelial cells appears possible, as has been shown for other matrices such as small intestinal submucosa,⁵⁸ it remains to be shown that larger tissue structures, such as the human heart, can be efficiently reseeded with the cell types of interest. Given the large size and the rather low migratory potential of terminally differentiated CMs, it is highly questionable whether a dense functional myocardium can be achieved by reseeding an acellular heart matrix.

Most other myocardial tissue engineering approaches are based on layers of thin biocompatible matrix sheets that are seeded with CMs⁵⁹ or on cells that are mixed with animal-derived or synthetic hydrogels, as initially introduced by the group of T. Eschenhagen.⁶⁰ For many years, such approaches were limited by the lack of a suitable human cell source and were based either on neonatal rat CMs⁶¹ or on murine ESC-derived CMs.⁶² More recently, the availability of improved differentiation protocols for human ESCs and iPSCs based on relatively inexpensive small molecules as described previously⁶³ facilitated the production of the required numbers of CMs and the development of contractile muscle patches from human pluripotent stem cells.²⁰

On the basis of such developments, several groups have reported bioengineered human myocardium consisting of human pluripotent stem cell derivatives. Miniaturized, fibrin-based constructs were engineered from human ESC- derived CMs as a novel means for drug screening and safety pharmacology.⁶⁴ Also, iPSC-based CM sheets have been applied to model human mitochondrial disease.⁶⁵ Tulloch and colleagues demonstrated that engineered human tissue constructs based on CMs derived from human ESCs and iPSCs became connected to the host vasculature 1 week after transplantation onto rat myocardium.⁶⁶ In another study, it was shown that engineered cell sheets consisting of human iPSC-derived CMs can improve cardiac function in a porcine model of ischemic cardiomyopathy.⁶⁷ Our group has shown that human iPSCs enable the generation of functional bioartificial cardiac tissue (BCT), which develops contractile forces almost similar to native myocardium.²⁰ Further developments have led to comparable constructs based on defined human and partially synthetic matrix components, which may facilitate clinical applications in the future.⁶⁸

Conclusions

Despite considerable progress, there are still many open questions and hurdles to be overcome before the clinical application of iPSC-derived CMs or engineered heart muscle. In the case of cell injection, it is still unknown whether this can lead to substantial functional improvement despite scar formation after myocardial infarction. Also, the current low survival and retention rates of transplanted CMs must be improved and a proper functional integration and coupling of transplanted cells must be achieved at least in the infarct border zone, or in case of other indications, such as genetic cardiomyopathies, in the noninfarcted myocardium. Although in vitro–engineered myocardial tissue may indeed provide a means to replace noncontractile scar tissue that has formed after myocardial infarction, these technologies require further development in terms of larger tissue dimensions and proper vascularization.

Of course, the elucidation and reduction of risks associated with the chromosomal abnormalities that become apparent after reprogramming and iPSC expansion⁴² are of utmost importance, and the risk of teratoma formation must be further assessed. However, the extremely low incidence of CM-derived tumors in human hearts suggests a low risk factor for a malignant transformation of terminally differentiated iPSC-derived CMs, and potential implant-related arrhythmias could be controlled through technical pacemaker devices. Therefore, CMs and bioartificial cardiac tissue derived thereof may be among the first iPSC-based transplants to be applied in the clinic and could eventually be broadly applied in the field of cellular heart repair.

Footnotes

Acknowledgment

The author is grateful to Nina McGuinness for revising the manuscript.

Author Disclosure Statement

No competing financial interests exist.

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