Abstract
Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high‐throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein‐coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell‐induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA‐ROCK‐Cofilin‐PAXILLIN‐actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell‐induced reprogramming resulted in alteration of cytoskeleton, loss of human‐induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells 2019;37:318–331
Highlights
Direct reprogramming of human somatic cells by ectopic expression of OCT4, SOX2, KLF4 and c-MYC (OSKM) transcription factors results in generation of induced pluripotent stem cells, which are similar to human embryonic stem cells in many of their properties (Okita et al, 2007)
To identify new candidate genes involved in human induced pluripotent stem cell (hiPSC) generation, we developed a highthroughput assay adapted to a 384-well format for genome-wide small interfering RNA screening using the Dharmacon library (Figure 1A)
Numerous studies which reported identification of a new effectors or barriers of reprogramming process have broaden our knowledge about complex interaction of the different signalling pathway and reprogramming mechanisms (Ebrahimi, 2015; Omole and Fakoya, 2018; Toh et al, 2016) and have highlighted an important role for MET, metabolism, apoptosis, cytoskeletal rearrangements, autophagy, immune response, cell cycle alterations, epigenetics and many others for the effective hiPSCs generation
Summary
Direct reprogramming of human somatic cells by ectopic expression of OCT4, SOX2, KLF4 and c-MYC (OSKM) transcription factors results in generation of induced pluripotent stem cells (hiPSCs), which are similar to human embryonic stem cells (hESCs) in many of their properties (Okita et al, 2007). A number of molecular and cellular barriers of reprogramming have been identified to date (Feng et al, 2009; Fritz et al, 2015; Qin et al, 2014), resulting in an overall 2-5% efficiency, indicating that the majority of cells are unable to complete reprogramming towards pluripotency (Ebrahimi, 2015; Neganova et al, 2017; Neganova et al, 2016). To identify novel regulators of reprogramming, we developed a high-throughput RNAi screening assay. This strategy allowed us to perform knockdowns of 784 members of the different kinases and phosphatases at the initiation stage of reprogramming. Six members of the G protein-coupled receptors (GPCRs) family, namely GPR42, GPR20, EDG5, GPR123, GPR116 and GPR37L1 were identified highlighting a putative role for GPCRs in the initiation stage of reprogramming
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