Insights into Differentiation of Pluripotent Stem Cells to Cardiac Progenitors and Cardiomyocytes

Abstract

The mammalian heart is the first organ formed during development. It forms the crux of the circulatory system of the body and pumps blood (and therefore, nutrients) to all parts of the body. The continuous functioning of the heart is indispensable to the life of the organism. However, such an important organ, that undergoes constant wear and tear, has been shown to have a limited regenerative potential. This, along with a number of genetic, metabolic and environmental factors has resulted in the diseases of the cardiovascular system to become the leading cause of death globally. In spite of decades of research, no effective treatments for cardiovascular diseases especially, ischemic heart diseases, has so far been developed. The conventional strategies are more palliative in nature and do not provide for a long-term solution. On the other hand, the cell-based approaches (mainly adult stem/progenitor cells) that have been developed face limitations in terms of availability of cells having transplantable quality and quantity. Pluripotent stem cells (PSCs) have the ability to self-renew, unlimited proliferative potential and the ability to differentiate to any cell type belonging to the three germ lineages. On account of these properties, PSCs could be used to develop strategies for treatment of ischemic heart diseases. However, the use of PSCs for cell transplantation has limitations in terms of: (a) availability of suitable markers for identification of PSC-derived progenitors or cardiomyocytes, (b) efficient strategies for enrichment of PSC-derived cell types, (c) strategies for scaling up the number of enriched cells and (d) efficient strategies for successful cell transplantation. In our laboratory, we have previously derived and established two transgenic mouse PSC lines, GS-2 embryonic stem cells (ESCs, Singh et al., 2012) and N9 induced pluripotent stem cells (iPSCs, Verma et al., 2017) from the constitutively EGFP-expressing transgenic ‘green’ GU-2 mouse (Devgan et al., 2003). Using these two mouse PSC lines and the well-established wild-type mouse D3 ESCs, we tried to develop strategies for the enrichment and expansion of mouse PSC-derived cardiac progenitors (CPs) which could be used for experimental cell transplantation studies. In addition to this, we also extended our studies to the human iPSCs, using the SCVI840 iPSC line (Stanford University Cardiovascular Institute Biobank, Stanford University, USA) to develop strategies to differentiate human iPSCs to CPs and cardiomyocytes using 2D and 3D culture systems. Aim and Scope of the Present Investigation The aims of the current study were: (a) to establish a culture system for differentiation of mouse PSCs to CPs and cardiomyocytes; (b) to develop strategies for enrichment and scale-up of mouse PSC-derived CPs and (c) to develop a culture system for differentiation of human iPSCs to CPs and cardiomyocytes in 2D and 3D cultures. The thesis is divided into four chapters. The first chapter is a general introduction followed by three data chapters describing the results from our study. The first chapter, “General Introduction” provides an up-to-date review of literature pertaining to our overall study. A basic understanding of the process of commitment and differentiation pluripotent cells to cells of the cardiac lineage, which occurs in the inner cell mass of the blastocyst (in vivo) or in PSCs (in vitro), is described. Further, a brief overview of the conventional and new generation approachesfor treatment of ischemic heart diseases (IHD), their advantages and limitations, is provided. The potential of PSCs and their derived progenitors and cardiomyocytes for the treatment of IHDs is discussed in detail. In addition to this, different strategies that could be used for enrichment, scale-up and transplantation of PSC-derived cardiac cell types are elaborated. The second chapter, “Differentiation of Mouse PSCs to CPs and cardiomyocytes” is divided into two parts. Part A describes the culture and characterization of the three mouse PSC lines, GS-2 ESCs, N9 iPSCs and D3 ESCs using morphological assessment, SEM analysis, gene expression analysis and immunocytochemistry. Further, the spontaneous differentiation of the three mouse PSC line to cells of the three germ layers is described. The in vitro differentiation of the three mouse PSCs to CPs and functional cardiomyocytes is elaborately explored. Lastly, Sca-1+ CPs were identified in the spontaneous differentiation cultures of all the three mouse PSC lines. Part B describes a serendipitous observation previously made in our laboratory, regarding the cessation of contractility in mouse ESC-derived cardiomyocytes following exposure to monochromatic light. This section describes the mechanism underlying this novel finding and the role of various extrinsic and intrinsic parameters in the observed phenomena. Following identification of Sca-1+ CPs in the spontaneous differentiation of culture of mouse PSCs, efforts were made to develop strategies for their efficient enrichment and expansion. The third chapter, “Strategies for Enrichment and Expansion of CPs Differentiated from Mouse Aim and Scope of the Present Investigation PSCs” describes the data pertaining to this. Firstly, the use of extended culture of embryoid bodies (EBs) in suspension for enrichment of Sca-1+CPs in spontaneously differentiating GS-2 ESC cultures is described. The efficiency of enrichment was assessed on morphological observations, immunostaining and flow cytometry. Next, the use of magnetic activated cell sorting (MACS) to enrich Sca-1+ CPs, following the depletion of CD31+ endothelial cells and endothelial progenitor cells, differentiated from GS-2 ESCs and N9 iPSCs is discussed. Further, the strategies for in vitro expansion of mouse PSC-derived Sca-1+CPs to obtain higher numbers of enriched CPs (for possible use in cell transplantation) are described. Lastly, the characterization of the PSC-derived Sca-1+ CPs before and after expansion based on their gene expression profile, and in vitro cardiac differentiation potential is discussed. The fourth chapter, “Differentiation of human iPSCs to CPs and cardiomyocytes” provides detailed insights regarding the extrapolation of our studies performed using the mouse PSCs to human iPSCs. The first part of the chapter deals with the establishment of a culture system to differentiate the human iPSCs (SCVI840 line) to functional cardiomyocytes. It describes the identification and characterization of different stages of cardiac commitment and differentiation. Following establishment of a defined culture system for differentiation of human iPSCs to CPs and cardiomyocytes, extension of the study to 3D culture systems is discussed. The culture and differentiation of human iPSCs in PCL-gelatin nanofibrous scaffolds to obtain a functional contracting “cardiac patch” which could be used for transplantation of PSC-derived CPs and cardiomyocytes into ischemic hearts is described in the second part of the chapter. Lastly, following the three data chapters, separate sections for the thesis summary and bibliography cited, are provided. Overall, the scope of the study was to develop strategies for efficient enrichment and expansion of PSC-derived CPs for their subsequent use in experimental cell transplantation in the future. The data presented in the thesis is in accordance with the above theme