The future of direct cardiac reprogramming: any GMT cocktail variety?

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 NKX2.5(GFP) Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5(GFP) reporter cardiac fibroblasts. We first established a new NKX2.5(GFP) reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5(GFP) hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5(GFP) cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.This work was supported by PID 2019-107150RB-I00/AEI/ 10.13039/501100011033 to XC-V; by the “Ramón y Cajal” State Program, Ministry of Economy and Competitivenes

Generation of Functional Human NKX2.5GFP Reporter Cell Lines for Direct Reprogramming into Proliferative Cardiac Progenitors

The mammalian heart is the first organ formed in the embryo and is composed of four chambers: right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV), and three layers: the endocardium, the myocardium, and the pericardium. The endocardium is formed by simple squamous epithelium known as endothelium, which lines the chambers and is joined to myocardium by a thin layer of connective tissue. The pericardium is comprised of two distinct sublayers: the inner serous pericardium, which is formed by visceral pericardium or epicardium and parietal pericardium separated by pericardial cavity, and the outer fibrous pericardium made of dense connective tissue. The myocardium is the muscular layer of the heart, it is the thickest one and is located between the endocardium and the pericardium. With regard to cellular composition, the heart is principally formed by cardiomyocytes (CM), cardiac fibroblasts (cFib) , vascular smooth muscle cells (localized fundamentally within the myocardium), vascular endothelial cells (located within the myocardium and endocardium) and mesothelial cells present in the pericardium 1–3 . CM are specialized cells with a complex filament structure responsible for the control of the rhythmic beating of the heart and present heterogeneity depending on the location, morphology, and function, including atrial, ventricular, sinoatrial nodal, atrioventricular nodal, His bundle, and Purkinje fibers 4,5

Generation of Functional Human NKX2.5GFP Reporter Cell Lines for Direct Reprogramming into Proliferative Cardiac Progenitors

The mammalian heart is the first organ formed in the embryo and is composed of four chambers: right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV), and three layers: the endocardium, the myocardium, and the pericardium. The endocardium is formed by simple squamous epithelium known as endothelium, which lines the chambers and is joined to myocardium by a thin layer of connective tissue. The pericardium is comprised of two distinct sublayers: the inner serous pericardium, which is formed by visceral pericardium or epicardium and parietal pericardium separated by pericardial cavity, and the outer fibrous pericardium made of dense connective tissue. The myocardium is the muscular layer of the heart, it is the thickest one and is located between the endocardium and the pericardium. With regard to cellular composition, the heart is principally formed by cardiomyocytes (CM), cardiac fibroblasts (cFib) , vascular smooth muscle cells (localized fundamentally within the myocardium), vascular endothelial cells (located within the myocardium and endocardium) and mesothelial cells present in the pericardium 1–3 . CM are specialized cells with a complex filament structure responsible for the control of the rhythmic beating of the heart and present heterogeneity depending on the location, morphology, and function, including atrial, ventricular, sinoatrial nodal, atrioventricular nodal, His bundle, and Purkinje fibers 4,5

The future of direct cardiac reprogramming: any GMT cocktail variety?

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 four Isl1 reporter iPSC lines from cardiac and tail-tip fibroblasts derived from Ai6IslCre mouse

Islet-1 (Isl1) is a transcription factor essential for life expressed in specific cells with different developmental origins. We have generated iPSC lines from fibroblasts of the transgenic Ai6 x Isl1-Cre (Ai6IslCre) mouse. Here we describe the complete characterization of four iPSC lines: ATCi-Ai6IslCre10, ATCi-Ai6IslCre35, ATCi-Ai6IslCre74 and ATCi-Ai6IslCre80

Generation of four Isl1 reporter iPSC lines from cardiac and tail-tip fibroblasts derived from Ai6IslCre mouse

Islet-1 (Isl1) is a transcription factor essential for life expressed in specific cells with different developmental origins. We have generated iPSC lines from fibroblasts of the transgenic Ai6 x Isl1-Cre (Ai6IslCre) mouse. Here we describe the complete characterization of four iPSC lines: ATCi-Ai6IslCre10, ATCi-Ai6IslCre35, ATCiAi6IslCre74 and ATCi-Ai6IslCre80

Generation of four Isl1 reporter iPSC lines from cardiac and tail-tip fibroblasts derived from Ai6IslCre mouse

Islet-1 (Isl1) is a transcription factor essential for life expressed in specific cells with different developmental origins. We have generated iPSC lines from fibroblasts of the transgenic Ai6 x Isl1-Cre (Ai6IslCre) mouse. Here we describe the complete characterization of four iPSC lines: ATCi-Ai6IslCre10, ATCi-Ai6IslCre35, ATCiAi6IslCre74 and ATCi-Ai6IslCre80