Adenoviral Vector Mediated Transient Reprogramming of Cardiac Cells Towards Pluripotency

Abstract

Transient dedifferentiation of cardiomyocytes has been established as a key mechanism behind the enhanced proliferation and cardiac regenerative capacity observed in lower vertebrates. In contrast, mature mammalian cardiomyocytes exist in a terminally differentiated state such that regeneration is significantly impaired. It has been previously demonstrated that somatic cells can be reprogrammed temporarily to a proliferative, dedifferentiated state in vitro and in vivo through transient overexpression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM). However, the response of cardiomyocytes to transient OSKM expression has not previously been explored. In this thesis, the effects of adenoviral vector mediated OSKM delivery to cardiac cells in vitro and in vivo were investigated. Forced expression of OSKM in cultures of neonatal rat and mouse cardiomyocytes induced rapid dedifferentiation as evidenced by sarcomere dis-assembly and downregulation of cardiomyocyte-specific genes (Myh6, Myh7). This induced dedifferentiation was associated with an increase in cell cycle activity consistent with an enhanced proliferative capacity. The initiation of reprogramming was further confirmed by the expression of early stage reprogramming markers (ECad) however, late-stage reprogramming markers remained silenced and the generation of pluripotent stem cells was not observed. Furthermore, parallel to decreases in exogenous OSKM expression, partially reprogrammed cells regained a contractile cardiomyocyte-like morphology and a gene expression profile consistent with their redifferentiation towards functional cardiomyocytes. The effect of ectopic OSKM expression was also investigated in vivo through direct intramyocardial injection of the same adenoviral vector. Within 6 days after injection, upregulation of endogenous pluripotency genes (endogenous Oct3/4 and Gdf3) was observed indicating the activation of a reprogramming-like response within the adult mouse myocardium. The increased expression of such genes was maintained only temporarily consistent with a transient reprogramming response which was further evidenced by the absence of teratomas. Finally, the capacity of an OSKM expressing adenoviral vector to induce this transient reprogramming response in a mouse model of myocardial infarction was confirmed. In conclusion, this work reports the first evidence of adenoviral mediated transient reprogramming of cardiac cells both in vitro and in the healthy and injured myocardium in vivo. Furthermore, this study was the first to explore adenoviral vectors for in vivo OSKM delivery and highlighted challenges associated with this approach. Further work is necessary to understand the source and fate of partially reprogrammed cells as well as determine whether this strategy can provide any therapeutic benefit in the heart. However, it is hoped that the results presented here can help inform future research into the development and applications of transient reprogramming-based strategies.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Kostas Kostarelos (Supervisor), Giulio Cossu (Supervisor) & Irene De Lazaro Del Rey (Supervisor)