De Lazaro Del Rey, I; (2015) In vivo cell reprogramming to pluripotency: generating induced pluripotent stem cells in situ for tissue regeneration. Doctoral thesis , UCL (University College London).

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Abstract

Artificially induced changes of cell identity are increasingly attracting attention as potential strategies to regenerate diseased or injured tissues, but still rely heavily on ex vivo culture with the exception of a small number of in vivo transdifferentiation studies. The reprogramming of somatic cells to pluripotency in vivo is even less explored, partly due to fears of teratoma formation. In this thesis, we hypothesised that such twist in cell fate can be safely achieved in vivo provided that sufficient but transient levels of reprogramming factors are locally expressed. We also speculated that transiently pluripotent cells can be generated in different tissues, thanks to the universality of the Yamanaka reprogramming factors, and that they may contribute to replenish the injured site after an insult. In vivo induction of pluripotency was first described in the liver and later in the skeletal muscle of wild-type mice. In both scenarios, the fast but transient upregulation of pluripotency markers and downregulation of tissue-specific genes did not progress to teratoma formation. The in vivo reprogrammed hepatocytes were established as a cell line in vitro, the so-called in vivo induced pluripotent stem (i2PS) cells, and their pluripotency was confirmed at the molecular and functional levels. Clusters of in vivo reprogrammed cells within the skeletal muscle tissue were found to express pluripotency and myogenic progenitor markers and to re-integrate in the normal tissue architecture after a transient proliferative stage recapitulating events of normal postnatal myogenesis. Finally, in vivo reprogramming to pluripotency resulted in a modest enhancement of regeneration and functional rehabilitation in a model of skeletal muscle injury. In conclusion, this work not only provides proof-of-concept of safe in vivo cell reprogramming to pluripotency but also presents a thorough characterisation of the in vivo reprogrammed cells and supports the potential of such strategy to improve regeneration after injury.

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