Generation of induced cardiospheres via reprogramming of skin fibroblasts for myocardial regeneration

Abstract

Cardiovascular disease (CVD), especially myocardial infarction (MI), is the leading cause of death worldwide. Despite recent advances in the treatment of MI, a significant proportion of patients still died of progressive heart failure. However, these therapies are only palliative without addressing the fundamental pathogenic process of MI, for example, the loss of functional cardiomyocytes. It has been demonstrated that endogenous cardiospheres (eCS), a form of cardiac progenitor cells (CPCs) that can be isolated from the adult heart, can be differentiated into functional cardiomyocytes in vivo, and thus represent a more effective cell-based therapy for treatment of MI. Unfortunately, the therapeutic application of eCS for MI is limited by low yield of cell harvesting, declining quality and quantity during the ageing process, and the need for highly invasive heart biopsy. Therefore, there is an emerging interest in generating cardiosphere- like stem cells from somatic cells with somatic reprogramming as an alternative source for CPC-based therapy. This novel approach may provide an unlimited source of stem cells with cardiac differentiation potential without the need for any invasive procedures. In this project, I infected the mouse embryonic and adult skin fibroblast cells with retrovirus-expressing Sox2, Klf4, and Oct4, and then subjected them to different cocktails of growth factor, small molecular cardiac transcription factors, and extracellular matrix to induce their transdifferentiation into cardiosphere. During the process of reprogramming, the expression of CPC marker genes (Mesp1, Isl, and Nkx2.5) was upregulated and further enhanced by the administration of Gsk3beta inhibitor BIO and Oncostatin M. I successfully induced the formation of cardiosphere-like stem cells through the sphere formation approach (induced cardiosphere, iCS). The iCS derived from mouse embryonic and adult skin fibroblasts contained CPC that can be differentiated into cardiomyocytes in vitro, and they were comparable to eCS from the perspective of action potential, calcium transient, contractility, and whole-genome gene expression profile. Transplantation of iCS into a mice model of acute MI provided therapeutic efficacy similar to eCS in improving cardiac function, decreasing infarct size, preserving left ventricular wall thickness, and increasing capillary density. Those engrafted cells had the potential to differentiate into cardiomyocytes and endothelial cells in vivo. Furthermore, human fibroblasts can also be reprogrammed into human iCS (hiCS) using the same strategy and have the capability of differentiating into cardiomyocytes in vitro. In conclusion, the results showed that donor-specific iCS can be generated through the somatic reprogramming process from skin fibroblasts without passing through a pluripotent stem cell stage by systematic screening of a panel of cardiotrophic growth factors. This novel approach can provide a source for cell therapy for treatment of MI. However, the iCS contains mixed cell populations, the same as eCS, and more specific CPC surface markers for purification are needed. Moreover, hiCS lose their ability to aggregate as spheres once dissociated; therefore, further optimisation of their culture conditions is required to support hiCS maintenance and expansion.