Differentiation, characterization and expansion of murine pluripotent stem cells to cardiomyocytes
thesisposted on 02.03.2017, 00:50 by Jain, Kanika
Pluripotent stem cells (PSC) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) represent a potential source for cell therapy, as they are known to differentiate into multiple cell lineages. Since mature cardiomyocytes are known to have extremely limited proliferative capacity, PSC derived adult cardiomyocytes may provide a reliable source of cells for transplantations if large-scale production can be achieved in bioreactors. However, generation of mature cardiomyocytes from PSCs remain a major challenge due to the heterogeneous population of cells obtained following differentiation, therefore limiting their utility. Another challenge that needs attention is obtaining a clinically significant number of cardiomyocytes that could be used for cardiac transplantations. We need to remember that the differentiation process in vitro is never 100% pure, and the heterogeneous pool of cells obtained post differentiation poses a major hurdle to obtain specific cell types relevant for transplantation studies. In order to address this issue, chapter 3 is dedicated to target the endogenous progenitor cardiac gene Nkx2.5 with a selection cassette comprising of IRES-mCherry-IRES-CMV-IRES-hyg3 using the newly developed TALEN approach. The chapter concludes with successful generation of the left, right TALEN along with the Obligate Ligation-Gated Recombination (ObLiGaRe) donor plasmid that can be used to transfect the ESCs/iPSCs eventually generating cardiac reporter cell lines. The generation of the reporter knock-in cell lines would allow easy identification of the generated cardiomyocytes at their progenitor and mature stages and provide a very simplified method of their purification and enrichment. Furthermore, the ability of a novel micro bioreactor “Liquid Marble” (LM) to generate cardiomyocytes in vitro was studied intensively in chapter 4. The marble strategy is based on the principle that the culture media suspension drop (comprising ESCs) rolled over a powder bed can prove to be an efficient system to generate embryoid bodies (EBs). Here, in the LM, murine ESCs were allowed to aggregate in suspension for 5 days. Beating cardiac bodies were generated from the EBs obtained from the LM system. Furthermore, the differentiation process was progressively monitored for the expression of cardiac lineage markers by immunocytochemistry and real-time PCR. Cells obtained were analysed for the expression of Gata4, Flk1 and Mef2C (mesendoderm), followed by the expression of Nkx2.5, Mlc-2a, Mlc-2v, cTnT and α-actinin; indicating progression to the progenitor and mature cardiac cells. Immunocytochemistry analysis for Nkx2.5 and cTNT confirmed the generation of cardiac progenitors and mature cardiomyocytes. This study has the potential to set paradigm for future studies by investigating the conditions for developing cardiomyocytes as a starting population for transplantation and also for research and drug discovery. This study was published in a peer review journal Advanced Healthcare Materials, under the title “Cardiogenesis of Embryonic Stem Cells with Liquid Marble Micro-Bioreactor.” Further, I envisaged using murine iPSCs in the similar LM micro-bioreactor to generate cardiomyocytes. Being derived from adult cells, e.g. skin fibroblasts; iPSCs bypass the ethical issues regarding the use of embryonic tissue to cure disease because the starting material can be obtained from the patients themselves. The cardiomyocytes obtained from this system holds a greater promise for transplantation studies as they overcome the major issue of immune-histocompatibility. The results obtained in chapter 5 clearly indicate that LM is not only supporting the culture of iPSCs but it also allows the formation of functional EBs. This was supported by the reverse transcrption (RT-PCR) analysis, where the EBs expressed markers of all the three germ layers, ectoderm (Nestin), mesoderm (Brachyury, Nkx2.5) and endoderm (Gata4, FoxA2). Upon further differentiation, these EBs expressed proteins of cardiac progenitor (Nkx2.5) and mature (cTnT) genes as well as neural progenitor (Nestin) genes as was revealed by the immunocytochemical analysis. Thus, this study holds immense therapeutic potential as it can be used to differentiate patient specific iPSCs that can generate either cardiomyocytes/neurons and these cells once introduced into the patients will help to overcome the immune rejection issues. Keeping in mind that the differentiated mature cells will be used for clinical applications, it becomes imperative to develop defined and efficient in vitro protocols, which would then provide the stringent levels of safety and quality control making stem cell transplantation therapy realizable. My research outcomes will provide a step up the ladder in the area of cardiac therapy.