The Challenges of the Design-Driven Biotech Resources and Unique Translational Tools to Manage Cardiac Self-Renewal and Regeneration to be utilized in Clinical Practice

Toward Cardiac Regeneration: Combination of PSC-Based Therapies and Design Driven Bioengineering Strategies

At present, the approaches aimed at increasing myocardial regeneration after infarction are not available. The dispute over the existence of cardiac progenitors in the adult heart is still unsolved; on the same trail, the ability of immature cardiac progenitor cells to engraft with pre- existing CMs in vivo has not been irrefutably determined. Due to their unique capacity to pro-duce functional CMs, a wide variety of cells have been evaluated for therapeutic delivery, including BMCs, MSCs, and endogenous CSCs as the most promising sources for cardiac regenerative medicine application. Thus, the key question now is the identity of cells capable of producing functional CMs, replenishing those lost during ischemia.

But despite the encouraging results from these and other in vivo studies on the potential beneficial effects of PSC based cell therapy for heart failure, preclinical experiments, using CMs derived from human PSCs, have given controversial results [23].

Design-driven tissue engineering strategies are probably a relatively more suitable option. And given the limited regenerative capacity of the heart, the design-driven bioengineered scaffolds and tissue engineering approaches are thus ideal for cardiovascular regenerative medicine applications. The main goal is to create a cardiac graft which can be implanted and restore the functionality of the myocardium without major side effects. To this aim, tissue engineering in-tends to recreate the microenvironment of the cardiac tissue, in terms of cell composition, stiffness, geometry, physical and electrical stimuli and, extracellular matrix, and in order to generate constructs with an actual translational value, keeping in mind the native characteristics of the cardiac tissue [24]. And although SC therapy is a promising treatment for myocardial infarction, the minimal functional improvements observed clinically limit its widespread application. Where a need exists to maximize the therapeutic potential of those SCs by first understanding what factors within the infarct microenvironment affect their ability to regenerate the necrotic tissue, and to define a novel mechanism by which the extracellular environment of the infarction regulates the therapeutic potential of MSCs.

With respect to the developments within the biomaterial scaffold space, although they offer an effective solution to myocardium cell retention and cell number, the transplant process is generally more invasive, and challenges with effective electromechanical integration still remain. Meanwhile, the scaffolds could be modified or implemented in combination with molecules that activate the recruited cells (e.g., statins) and amplify their therapeutic potential. In conclusion, it is likely that no single approach to SC membrane reengineering will provide the “magic bullet” for cardiac cell therapies, and that the next generation of therapies will likely utilize combinations of these technologies to fully harness the therapeutic potential of transplanted SCs.

However, regardless of the employed strategy, generation of tissue-like engineered structures suitable for regenerative cardiology applications needs further considerations, specifically in relation to some aspects that may be critical for their efficacy and potential undesired effects in vivo.

Cardiac SCs (CSCs) as Promising Cell Types for Cardiac Regeneration

Myocardial infarction results in an irreversible loss of CMs due to aging or pathophysiological conditions, which is generally considered irreversible, and can lead to lethal conditions with subsequent adverse remodeling and heart failure. But human PSCs, including ESCs and iPSCs, can selfrenew while maintaining their pluripotency to differentiate into all cell types, including CMs. Furthermore, identifying new sources for CMs and promoting their formation rep-resents a goal of cardiac biology and regenerative medicine.

Meanwhile, because of the lack of strong evidence supporting the existence of resident CSCs, efforts are focused on how to mobilize and promote those few CMs that have potential to proliferate in the hearts. Whereas endogenous CMs can proliferate at a low frequency, methods that promote CM proliferation may be a vital research field in the future. The aforementioned pathways could be manipulated, optimized, and synergized to achieve better functional improvement after cardiac injury. In addition to inducing CM proliferation, other alternative approaches could be considered (Figure 3).

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Figure 3: Strategies for treating cardiac repair and regeneration Transplantation of embryonic stem cell (ESC) derived CMs or iPSC derived CMs into myocardial infarction heart. CMs could also be reprogrammed from Fb in vitro or by transcriptional factors (TFs) or chemicals in vivo. Transplantation of MSCs promotes neovascularization and cardiomyocyte survival through paracrine mechanism. Epicardial patch containing growth factors or cardiac cells restores heart function after myocardial infarction. Administration of modified RNA (modRNA) of paracrine factors promotes heart function and drives heart progenitor cell fate. CMs could be promoted to proliferation by regulation of factors or signaling pathways.

Fb, fibroblasts; ESC, embryonic stem cell; CMs, cardiac myocytes; TF, transcriptional factor; iPSC, induced pluripotent stem cell; MSCs, mesenchymal stem cells; modRNA, modified RNA.

Source: Lingjuan He, Ngoc B. Nguyen, Reza Ardehali and Bin Zhou. Heart Regeneration by Endogenous Stem Cells and Cardiomyocyte Proliferation. Controversy, Fallacy, and Progress. Circulation. 2020; 142: 275-291.

In situ reprogramming of fibroblasts to cardiomyocytes by overexpression of specific transcriptional factors represents a promising direction for future study and translational applications to be implemented into clinical practice [25].

Recent studies have also suggested that small molecules can induce reprogramming of fibroblasts to CMs in vivo, which offers a dual pharmaceutical approach to regenerate CMs and reduce scar formation [26].

Human ESCs or iPSCs derived cardiac progenitors and CMs have been successfully transplanted and confirmed the survival of transplanted cells within the host myocardium and an improvement in cardiac function after injury. Furthermore, dual SC therapy, such as human iPSC derived CMs and MSCs, or human ESC-derived epicardium and CMs, synergistically improves cardiac function and augments vascularization in the injured myocardium. Moreover, re- cent advances in engineered epicardial patch containing multiple cardiac cell types are improved heart function and post-infarction neovascularization [27, 28, 29].

In this context, CSCs are considered to be very promising cell types for cardiac regeneration. And within the past decade, among CMs many types of putative CSCs have been reported to regenerate the injured myocardium by differentiating into new CMs. Some of these CSCs have been translated from bench to bed with reported therapeutic effectiveness. However, recent basic research studies on stem cell tracing have begun to question their fundamental biology and mechanisms of action, raising serious concerns over the myogenic potential of CSCs [30].

In particular, c-Kit+ CSCs represent a promising candidate for cardiac-specific SC lineages, which is likely heterogeneous in nature [31]. Moreover, resident CSCs are self-renewing and can give rise to CMs. Their multi-potentiality allows them to differentiate along the three main cardiac lineages: myocytes, endothelial cells, and smooth muscle cells. After their injection in the ischemic heart, the formation of the above-mentioned cell types contributes to the regeneration of myocardium and the improvement of its contractility.

But the approaches securing cardiac regeneration in post-infarction period are not available to be practiced. And the key problem is the identity of cells be born to generate functionally active cardiac myocytes replenishing those being lost during ischemia [32, 33].

Meanwhile, resident CSCs (rCSCs) may be a crucial source to initiate and prompt myocardial self-renewal and regeneration. And along with the latter, endogenous cardiomyogenic SCs (CMSCs) might be better choice for cardiac repair. Ideally, those cells should be directly stimulated in situ, avoiding extraction, purification, culture and reinjection. It is therefore of uttermost importance to understand the identity and function of the cells that constitute the natural environment of cardiac progenitors and support their quiescence, self-renewal and activation [34].

CSC therapy holds great potential to prompt myocardial regeneration in patients with is-chemic heart disease. The selection of the most suitable cell type is pivotal for its successful application. Therefore, different next generation cell types are currently under investigation for the treatment of the diseased myocardium [35].

Targeted Management of Regenerative Cardiomyogenesis

Drug (including SC driven) development continues to move in the direction of Personalized and Precision Medicine (PPM), where ideally the most effective therapy or treatment is determined by the genetic makeup of the patient and thus by a spectrum of biomarkers to be targeted by the therapeutics (including those being based on SCs). Regarding PPM based cardiology practice and the innovations made in the field of SC driven technologies, the focus in the last years has been moved towards a concept of the new wave of cardiac myocyte (CM) formation via a scenario of dedifferentiation and proliferation of mature CMs. The observation that CSCs can be developed inside a pool of immature cardiac cells by formation of “cell-in-cell structures” (CICSs) has enabled us to conclude that CICSs being encapsulated are implicated into mammalian cardiomyogenesis over the entire lifespan [2, 36, 37] (Figures 4A,4B).

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Figure 4A: The CSCs inside CMCs and the formation of CSC- containing CICSs in the cultures obtained from newborn and 20- and 40-day-old rats.

(A) Experimental design. The cells were plated and cultured for up to 30 days, followed by immunostaining or time-lapse microscopy. (B–G) Immunocytochemistry.

The nuclei of the cells have been stained with Hoechst. Transmitted light and fluorescent images are merged. (B) c-kitC CSC inside a CM obtained from a newborn rat (day in vitro). (C) Isl1C CSC inside a CM obtained from a newborn rat (day in vitro). As documented by the expression of Ki67, both the CSC and the host cell exhibit proliferative ability. (D) ScaC CSC encapsulated between the nuclei of the host cell (20-day-old rat, day in vitro). (E) A mature c-kitC CSC containing CICS with a prominent coating (“capsule”) with 3 pores (white arrows, 40 day old rat, day in vitro 6). Optical sectioning shows the host cell nucleus (blue) just above the CICS. (F-G) The CICS capsule in detail. (F) Erosion of the Isl1C CSC-containing CICS capsule (black arrow) obtained from a 40-day-old rat, day in vitro. The pores are also visualized (white arrows). The capsule interior is positive for sarcomeric a actinin, also observed in (G). (G) Erosion of the c-kitC CSC containing CICS capsule (black arrow) obtained from a newborn rat, day in vitro 20. The pores are seen (white arrows). CMs, cardiac myocytes; CSC, cardiac stem cell; CICSs, cell-in- cell structures”

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Figure 4B: Cell-in-cell structures (CICSs) identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats.

Transmitted light and fluorescent images are merged. (A and B) Isl1C CSCs inside cardiomyocytes of 20-day-old rats (Isl1, green), a-Sarcomeric actin, red). (C) c-kitC CICS. (40-day-old rat, c-kit, green; Ki67, red). (D) c-kitC CICS. (40-day-old rat, c-kit, green; a-Sarcomeric actin, red).

It had been demonstrated before that new CMs are generated through formation of CSC derived transitory amplifying cells (TACs) either in the CM colonies or in a process of intracellular development of CICSs being encapsulated.

The data obtained, accumulated and interpreted, provide the basis for the suggestion that CSC mediated regenerative cardiomyogenesis is initiated in the myocardium immediately after the onset of myocardial ischemia. We can only assume that both encapsulated and nonencapsulated CICSs with poorly differentiated TACs inside are undergoing early rupture due to greater sensitivity to hypoxia and pH reduction at the acute stage of ischemia. If this is true, the key proximal question is the estimation of the survival rate of TACs released from encapsulated and non-encapsulated CICSs in the damaged area. In the case of their massive death, the myocardium is deprived of the significant number of TACs, which are in principle able to replace lost mature CMs.

Earlier on syngeneic animals were shown that a local laser apoptotic effect on tissues caused an intensive transition of mesenchymal BMSCs from the bloodstream to the zone of programmed cell death (Figure 5).

Apoptotic bodies (ApB) of cardiomyocytes combine the functions of CSC and HSCs in the areas of myocardial regeneration, and contain a complex of molecules, carriers of “epigenomic memory” about the tissue belonging of a dead cell. When ApB enters the CSC via endocytosis, a specific set of long and short noncoding RNAs express genes that determine the direction of differentiation of resident myocardial stem cells. It can be assumed that this hypothesis is true not only for the heart, but also for other organs and tissues [38, 39].

Those data for the first time suggest that the development of CSCs inside the encapsulated CICSs is important for cardiac self-renewal and maintenance of CSC based pool. At the same time, the development of CSCs inside a population of mature CMs is resulting in the formation of pre-cardiac myocytes, which are able to substitute for irreversibly injured CMs, representing the major mechanism of myocardial regeneration. And that, in turn, would open up a green light to secure the targeted management of regenerative cardiomyogenesis.

One of the main findings is the identification of a new type of CSC containing CICSs in heart samples obtained in the early post-infarction period. Due to the opinion, the above mentioned characteristics of two types of CICSs, that is, encapsulated vs. non-encapsulated CICSs, are responsible for the differences in maturity of TACs which develop inside them. Of note, the greater extent of TAC maturity was observed in non-encapsulated CICSs, which might have been accounted for by the higher level of differentiation of host cell and easier availability of humoral factors diffusing to the membrane-enveloped CSCs. In contrast, less differentiated TACs were released from encapsulated CICSs, which is paralleled by lower differentiation status of the host cell and poor diffusion of paracrine factors stimulating differentiation through the thick capsule. Formation of new CMs through proliferation and differentiation of CSCs within colonies is blocked after infarction and restored only 10–14 days after, the cardiomyogenesis in the early postinfarction period occurs by using population of TACs, which develop inside cardiac cells of different levels of maturity with formation of encapsulated and non-encapsulated CICSs. Therefore, improved understanding of molecular mechanisms giving rise to colonies and CICSs, may contribute to development of new pharmacological therapies targeting the process of cardiomyogenesis [2, 3, 4]. The results from those research and observation provide direct evidence for the replicative division of encapsulated SCs, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple TACs following the capsule rupture.

In conclusion, functional CSCs can reside not only exterior to but also within CMs. The presence of CSC-derived colonies, CICSs and TACs in myocardium proved the previous hypothesis about two pathways that generate new CMs in adult heart. Moreover, we suggest that TACs play a central role in self-renewal of myocardium throughout the lifetime of mammals [40].

The Prospects for the Introduction of Biodesign Inspired Drugs for Cardiac Regeneration

SC-driven translational applications have made significant progress in the field of cardiovascular regenerative medicine over the past few decades. Based on the new mechanisms and unique phenomenon, we are developing improvement strategies to boost the potency of SC repair and to generate the “next generation” of SC based and regulatory biomolecules-based (bimodal) therapeutics. Moreover, our strategies should aim at more personalized SC therapies in which individual disease parameters influence the selection of optimal cell type, dosage and delivery approach. And encouraging preclinical and clinical studies as one and solid entity reporting significant SC mediated cardiac regeneration would rapidly pave the way for clinical translation. Thus, a desire to discover innovative SC based technologies of the next step generation would encourage governments and companies to focus directly on regenerative medicine as a future potential economy and social insurance booster.

In reality, the global (worldwide) SC therapy market is still in an early stage. And the developing translational pipelines for rising applications will build the competition among merchants amid the conjecture time frame. Consequently, the focus of research in the field has since shifted to SC derived paracrine factors, including mRNA, and miRNA, which are entering into the extracellular space either in soluble form or packed into membrane vesicles. The search for chemical signals capable of stimulating cardiomyogenesis is ongoing despite continuous debates regarding the ability of mature cardiac myocytes to divide or dedifferentiate, transdifferentiation of other cells into cardiac myocytes, and the ability of CSCs to differentiate into cardiac myocytes. Future research is aimed at identifying novel cell candidates capable of differentiating into cardiac myocytes. The observation that CSCs can undergo intracellular development with the formation of “cell-in-cell structure” and subsequent release of transitory amplifying cells with the capacity to differentiate into cardiac myocytes may provide clues for stimulating regenerative cardiomyogenesis.

The worldwide SC therapy market is still in an early stage. And the developing translational pipelines for rising applications in the above-mentioned microecosystem will build the competition among merchants amid the conjecture time frame. And thus partnering between SC-related researchers, cell biodesigners and bioengineers, cardiac clinicians and surgeons, business and regulatory bodies and government can help ensure an optimal development program that leverages the Academia and industry experience and FDA’s new and evolving toolkit to speed our way to getting new tools into the innovative SC markets. We guess that the above mentioned would illustrate a unique Micro Ecosystem to be used for SC treatment in the near future to come, and tp provide a unique platform for dialogue and collaboration among thought leaders and stakeholders in government, academia, biopharma, foundations, and disease and patient advocacy with an interest in improving the system of healthcare delivery on one hand and drug discovery, development, and translation, on the other one, whilst educating the policy community about issues where biomedical science and policy intersect.