Identification of genes involved in reprogramming-induced senescence

Abstract

Senescence is a state of irreversible growth arrest first described in human fibroblasts when they undergo serial passage in vitro. Senescence can also occur in response to different stresses, such as replicative exhaustion, DNA damage or overexpression of oncogenes. The growth arrest associated with senescence is implemented by the activation of the p53/p21Cip1 and p16INK4a/Rb pathways. The expression in somatic cells of a combination of pluripotency-associated transcription factors, Oct4, Sox2, Klf4 and cMyc (OSKM) has been shown to generate embryonic stem cell-like cells termed induced pluripotent stem cells (iPSCs). iPSCs have brought new expectations to the study of stem cell biology, drugs development and regenerative medicine. However, the overall low efficiency of the reprogramming and its slow kinetics suggest that there might be barriers limiting the derivation of iPSCs. The expression of the reprogramming factors in normal somatic cells has been shown to activate the p53 and p16INK4a pathways, triggering a senescence response that limits the cells available to be reprogrammed, in what has been called reprogramming induced-senescence (RIS). Our aim is to identify modulators of RIS. We hypothesized that this could help not just to improve the efficiency of generation of iPSCs but could also help to clarify the link between tumour suppressor pathways, pluripotency and de-differentiation. To this end, we carried out a screen to identify shRNAs whose expression bypass the growth arrest imposed by the four reprogramming factors in IMR90 human primary fibroblasts. We screened a genome-wide shRNA library and took advantage of next generation sequencing (NGS) to detect the relative enrichment of shRNAs. We shortlisted 68 genes to validate their ability to bypass the OSKM-induced arrest. Of those, 8 genes consistently rescued the growth arrest in independent experiments. We performed a preliminary characterisation of IFIH1 and IGSF4D. The long-term goal would be to expand the characterisation of these candidates, assess whether they might also have a role in oncogene and replicative induced senescence and investigate whether they affect the efficiency of reprogramming to iPSCs. In addition, conducting a secondary screen with the top candidates shortlisted from the primary screen could contribute to the identification of additional genes that affect reprogramming and senescence.