Hear the beat: decellularized mouse heart regenerated with human induced pluripotent stem cells

Abstract

Heart tissue engineering holds a great potential for human heart disease therapy. Regeneration of whole biofunctional human heart is the ultimate goal of tissue engineering. Recent advances take the first step towards whole heart regeneration. However, a substantial amount of challenges have to be overcome.

Keywords::

• decellularization
• extracellular matrix
• induced pluripotent stem cells
• multipotential cardiovascular progenitors
• recellularization

Heart tissue engineering for heart disease therapy

Heart is a complicated organ with well-organized cellular and extracellular matrix (ECM) architectures that are essential for developing and mediating the contraction force of myocardium. An electrically coupled network, cardiac conduction system, controls the rhythmic beating of heart. Human heart consists of a variety of cell types including cardiomyocytes (CMs), smooth muscle cells (SMCs), endothelial cells (ECs) and cardiac fibroblasts (CFs). CMs provide the contraction power of heart. SMCs and ECs form the vasculature. And ECs contribute to heart valve and endocardium formation. In adult mouse and human heart, approximately 50% cells are CFs. CFs play an important role in regulating heart formation, as well as heart functions, including electrical coupling and cardiac contractility [1,2]. Damaged or malfunctioned heart cells can lead to heart disease, which is the leading death cause in the USA [3]. Myocardial infarction affects 8 million people in the USA and increases at a rate of 800,000 new cases per year [4]. Over 5 million patients in the USA suffer from heart failure, a disabling and deadly condition [4]. Although various drugs and surgical interventions for heart disease therapy have been developed, current drug therapy is not curative. Due to the limited regenerative capability of human heart, cardiac function cannot be spontaneously recovered in myocardial infarction or heart failure patients. Without organ transplantation, end-stage heart failure is 100% fatal. Unfortunately, the number of donor hearts is always less than the demand of transplantation. Therefore, heart tissue engineering offers a promising therapeutic approach for human heart disease by making cardiac tissues, heart valves, as well as whole functional heart for transplantation.

Hurdles for whole human heart engineering

Theoretically, heart engineering requires a resource of heart cells, such as CMs, SMCs, ECs, CFs and a 3D heart scaffold that allows the seeded cells to attach, assemble, synchronize and form 3D structures. Currently, a variety of synthetic matrices and natural-derived biomatrices have been developed and widely utilized for tissue engineering [5]. However, such matrices normally have a uniform composition and are lack of 3D architecture as well as microniches in the native heart. Recent progress of 3D printing technology has enabled the rebuilding of 3D matrix scaffold with similar geometry as the native heart [6]. However, the native heart ECM is composed of very complex structure and biofunctional molecules, such as collagens, fibronectin, laminin, as well as a variety of conjugated growth factors and other functional proteins. Importantly, the most fascinating characteristic of native heart ECM is the dynamic change of ECM structure and composition during organogenesis. All these remain as the major hurdle for rebuilding artificial heart scaffold using evenly processed biometrics to authentically recapitulate the native heart ECM scaffold. In addition, the limited availability of human heart cells remains as another major hurdle for human heart engineering. CMs directly isolated from patients under heart transplantation are the only available cell resource for human heart engineering. However, these patients are normally bearing end-stage heart disease and have been subjected to prolonged pharmaceutical therapy. In addition, adult CMs lose the capability of proliferation. Thus, the diseased adult heart cell is not an ideal cell resource for making transplantable bioartificial heart.

Whole-heart regeneration using whole-organ-specific ECM scaffold

Recently, decellularization of whole organs, such as lung, liver, kidney and heart, has been utilized to obtain intact organ-specific scaffolds for potential whole-organ regeneration [7]. Whole hearts from mouse [8], rat [9] and porcine [10] were decellularized to obtain intact heart scaffolds. Bioartificial rat heart constructs have been generated by recellularizing decellularized rat hearts with neonatal rat CMs, which showed a partial blood pumping function [9]. Recently, we engineered human heart constructs by recellularizing decellularized whole mouse hearts with multipotential cardiovascular progenitors (MCPs) derived from human induced pluripotent stem (iPS) cells [11]. The major advantage of using decellularized heart for whole-heart regeneration over other synthesized or native 2D matrices is that the acellular heart preserves the 3D architecture and the natural ECM components of native heart, which are critical for whole-heart regeneration in vitro. We observed that heart ECM promoted and regulated subtype CM specification, CM proliferation and maturation from the repopulated MCPs [11]. These findings indicated that native heart ECM could significantly impact the cardiovascular development from heart progenitor cells. Furthermore, removal of antigenic epitopes from the decellularized heart ECM scaffold could significantly avoid the triggering of immune rejection by hosts of the ECM material [7]. All these properties make whole-heart ECM an ideal 3D scaffold and biocompatible cell carrier for engineering whole bioartificial heart for transplantation.

Novel cell resource for human heart tissue engineering

Due to the limited availability of healthy human cardiovascular cells, human heart tissue engineering has been largely remaining underdeveloped. In recent years, human embryonic stem and iPS cell-derived CMs have emerged as a new resource for human heart tissue engineering [12]. Compared with CMs directly isolated from patients under heart transplantation with end-stage heart diseases, human iPS cell-derived CMs are renewable and free of disease and drug treatment. However, human iPS cell-derived CMs also have very limited proliferation capability, which could potentially prevent the direct use of iPS cell-derived CMs for whole-heart engineering given the huge number of CMs needed for making a human heart. Additionally, a large number of human ECs and SMCs would also be required for regenerating vasculatures in the bioartificial heart. Thus, one critical question in whole human heart engineering field is that whether a single-cell resource with high proliferation potential could be obtained. To address this question, recently, we at the first time utilized human iPS cell-derived MCPs [13,14], rather than beating CMs, for recellularizing acellular mouse heart [11]. MCPs represent the earliest human cardiovascular progenitors in human cardiogenesis, and MCPs can further give rise to three human cardiovascular lineage cells including CMs, SMCs and ECs [13,14]. In our study, we found the seeded human MCPs in situ migrated and differentiated to reconstruct muscle and vascular architectures of the decellularized mouse hearts [11]. The utilization of MCPs for heart tissue engineering was the first study to test the possibility of simultaneously reconstructing both muscle and vessels with a single human cell resource. In addition, given the significantly higher proliferating potential of MCPs than beating CMs, it is our expectation that human iPS cell-derived MCPs would be an ideal resource for the regeneration of personalized heart tissue as well as whole bioartificial heart organ.

Novel strategy for human heart tissue engineering

The classic heart tissue engineering approach is to mix beating CMs, together with other heart cells, into biomaterials to generate a layer of beating cardiac tissue [5,12]. A previous study delivered rat neonatal CMs into whole acellular rat heart by multiple ventricular injections [9]. In our study [11], we developed a novel strategy to ‘grow’, rather than ‘make’, human heart tissues by delivering MCPs into acellular mouse heart through coronary vessels and allowing MCPs to in situ differentiate and reconstruct the acellular mouse heart. Additionally, by perfusing heart constructs with different combinations of growth factors, we could control the muscularization versus vascularization of MCPs inside of acellular heart. Overall, our strategy took advantage of the in situ proliferation, migration and commitment of MCPs into muscular or vascular lineage cells for tissue engineering and explored the feasibility of rebuilding human heart by following with the heart developmental processes.

Major challenges of whole-heart engineering

The rhythmic beating of human heart is controlled by the cardiac conduction system. Abnormalities of conduction system lead to cardiac arrhythmia and even sudden death. A major challenge of whole-heart engineering is the functional regeneration of whole cardiac conduction system. Previous studies reported that human iPS cell-derived CMs contain atrial, ventricular and pace maker-like subtypes [15]. Thus, theoretically conduction system could be rebuilt by using human iPS cell-derived heart progenitor cells. However, how to induce the specification of MCPs into conduction system cells and reconstruct the whole conduction system inside of acellular heart remains unknown now. Another challenge of whole-heart engineering is the regeneration of adequate mechanical force for blood pumping. Unlike CMs in adult human heart, human iPS cell-derived CMs have fetal-like phenotypes and are not functionally mature [15]. Since the in vivo environmental factors such as the mechanical contraction, electrical stimulation and various factors from blood could all affect the maturation and functional synchronization of CMs, it is expected that bioreactors which could functionally recapitulate the in vivo heart environment would be required for whole-heart engineering. Additionally, these bioreactors should be able to provide adequate nutrition and oxygen supplies for the metabolism of engineered whole heart.

Future perspective

The successful regeneration of whole heart would require new ‘software’ and ‘hardware’. The software lies in the deeper understanding of heart developmental biology and the designing of novel engineering strategies by following with principles of human heart formation. Generation of novel bioreactors and perfusion systems, which mimic in vivo heart environment, is the goal for developing new hardware for heart engineering. It is our expectation that whole-heart regeneration will ultimately incorporate knowledge of bioengineering, stem cell biology, heart developmental biology and animal studies into a new platform for both basic and clinical applications. Heart engineering would eventually bridge basic studies to translational therapy of human heart disease as well as other diseases.

Conclusion

Heart tissue engineering holds a great potential for human heart disease therapy. Regeneration of whole biofunctional human heart is the ultimate goal of tissue engineering. Given the challenges of whole-heart regeneration, the development of novel 3D scaffold, cell resource, engineering strategy as well as bioreactor is desperately required. Our recent study utilized the whole mouse heart scaffold and human early stage heart progenitors to rebuild functional heart constructs, which explored the feasibility of whole-heart engineering by following with principles of early heart development [11]. Such strategy could potentially be used to make patient-specific heart tissue and whole heart, as well as be suitable for making other organs.

Financial & competing interests disclosure

L Yang was supported by the University of Pittsburgh start-up and AHA SDG Grant (#11SDG5580002). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.