Direct reprogramming of murine fibroblasts to blood

Abstract

Transdifferentiation is the direct conversion of somatic cells to another functionally distinct cell type with bypassing the pluripotency state. This rapid cellular fate change can be achieved by overexpression of lineage specific transcription factors, as recently shown for reprogramming to neuronal or muscle cells. The aim of this project was to evaluate whether this approach could be applied to the generation of transplantable haematopoietic cells for regenerative medicine. As a result of my investigation, I have defined a limited set of haematopoietic regulators that convert murine fibroblasts to blood. Expression of only five genes (Scl, Lmo2, Gata2, Runx1c, Erg) in fibroblasts led to the generation of round grape-like blood colonies within 6 days of culture. These emerging cells, when cultured with cytokines, gave rise to myeloid, erythroid, megakaryocytic and T-lymphoid lineages. A similar procedure performed on fibroblasts lacking p53 led to even more robust reprogramming with additional generation of mature B-lymphocytes. The timely investigation of early stage reprogramming cultures revealed that transdifferentiation is mediated through two intermediates: transient endothelial VE-cadherin positive haemogenic endothelium and multipotent cKIT positive haematopoietic progenitors. Importantly, the latter exhibited robust in vitro clonogenic potential and short-term in vivo repopulation capabilities. The major focus is now to confer long-term multilineage repopulation potential on reprogrammed cells mostly by amending culture conditions and improving existing protocol. Achieving this goal will open new avenues for the generation of patient-specific haematopoietic cells for therapeutic applications.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Valerie Kouskoff (Supervisor) & Georges Lacaud (Supervisor)