The Future of Direct Cardiac Reprogramming: Any GMT Cocktail Variety?

Abstract

Direct cardiac reprogramming has emerged as a novel therapeutic approach to treat and regenerate injured hearts through the direct conversion of fibroblasts into cardiac cells. Most studies have focused on the reprogramming of fibroblasts into induced cardiomyocytes (iCMs). The first study in which this technology was described, showed that at least a combination of three transcription factors, GATA4, MEF2C and TBX5 (GMT cocktail), was required for the reprogramming into iCMs in vitro using mouse cells. However, this was later demonstrated to be insufficient for the reprogramming of human cells and additional factors were required. Thereafter, most studies have focused on implementing reprogramming efficiency and obtaining fully reprogrammed and functional iCMs, by the incorporation of other transcription factors, microRNAs or small molecules to the original GMT cocktail. In this respect, great advances have been made in recent years. However, there is still no consensus on which of these GMT-based varieties is best, and robust and highly reproducible protocols are still urgently required, especially in the case of human cells. On the other hand, apart from CMs, other cells such as endothelial and smooth muscle cells to form new blood vessels will be fundamental for the correct reconstruction of damaged cardiac tissue. With this aim, several studies have centered on the direct reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs) able to give rise to all myocardial cell lineages. Especially interesting are reports in which multipotent and highly expandable mouse iCPCs have been obtained, suggesting that clinically relevant amounts of these cells could be created. However, as of yet, this has not been achieved with human iCPCs, and exactly what stage of maturity is appropriate for a cell therapy product remains an open question. Nonetheless, the major concern in regenerative medicine is the poor retention, survival, and engraftment of transplanted cells in the cardiac tissue. To circumvent this issue, several cell pre-conditioning approaches are currently being explored. As an alternative to cell injection, in vivo reprogramming may face fewer barriers for its translation to the clinic. This approach has achieved better results in terms of efficiency and iCMs maturity in mouse models, indicating that the heart environment can favor this process. In this context, in recent years some studies have focused on the development of safer delivery systems such as Sendai virus, Adenovirus, chemical cocktails or nanoparticles. This article provides an in-depth review of the in vitro and in vivo cardiac reprograming technology used in mouse and human cells to obtain iCMs and iCPCs, and discusses what challenges still lie ahead and what hurdles are to be overcome before results from this field can be transferred to the clinical settings.

Generation of NKX2.5GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

“…Although significant progress has been made in the cardiac reprogramming field in recent years, robust and reproducible protocols are still needed to be set up in vitro, mainly in the case of human cells, before this strategy can be translated into the clinic. (López-Muneta et al, 2020;Garry et al, 2021). In this study, we have generated functional human NKX2.5 GFP cFib to facilitate screening factor research.…”

“…This is important to take into consideration since direct cardiac reprogramming in vivo is considered a more feasible therapeutic approach than in vitro, as it has shown a higher reprogramming efficiency (Qian et al, 2012;Song et al, 2012) and can circumvent one of the major problems of cell therapy, namely poor engraftment. (López-Muneta et al, 2020). Alternatively, enhancement of reprogramming efficiency to obtain human iCM would be critical to produce sufficient cells in vitro for transplantation.…”

Cardiac Regeneration: New Insights Into the Frontier of Ischemic Heart Failure Therapy

“…Generation of multipotent iCPCs may offer additional benefits for heart regeneration as they have the potential to differentiate into endothelial and smooth muscle cells, thereby promoting vasculogenesis. Further, while iCMs rapidly exit the cell cycle upon reprogramming, iCPCs remain proliferative until they differentiate into CMs (reviewed in Klose et al, 2019 ; Lopez-Muneta et al, 2020 ). Overexpression of OCT4, KLF4, SOX2, and C-MYC in MEFs, which is well-known to generate iPSCs (Takahashi and Yamanaka, 2006 ; Takahashi et al, 2007 ), was observed to generate small clusters of contracting colonies by day 18 in the absence of leukemia inhibitor factor (LIF) (Efe et al, 2011 ).…”

Advanced Technologies to Target Cardiac Cell Fate Plasticity for Heart Regeneration

“…Nonetheless, in vivo studies performed in mice have highlighted a number of challenges that remain to be overcome before this approach could be used in the human clinic, like the low efficiency of conversion, the immaturity, at least in vitro, of reprogrammed cells, the absence of safe delivery methods, and the inability to precisely direct differentiation towards the desired cell subtype [7,16]. Generation of multipotent iCPCs instead of iCMs may offer some advantages for heart regeneration as they have the potential to differentiate into different cardiac cell lineages, retaining a certain ability to proliferate [99].…”