Abstract: One of the main problems of induced pluripotent stem cell (iPSC) production is that iPSCs often lack the capacity for differentiation when they are reprogrammed partially. Using Sendai virus vector, our lab has isolated homogenous populations of partially reprogrammed iPSCs, termed paused iPSCs, by reducing exogenous KLF4 level, which can resume reprogramming to acquire high pluripotency when the KLF4 level is increased. We had made Rex1-reporter paused iPSCs which can monitor Rex1 expression, which is increased only in fully reprogrammed iPSCs. Using these pluripotency-reporter cells, I tried to find potential roadblock genes that may prevent the reprogramming from acquiring pluripotency to obtain fully reprogrammed iPSCs efficiently. To find the roadblock genes, I introduced Genome-scale CRISPR-Cas9 knockout library into the reporter cells to screen for genes that may be functionally related to acquisition of full pluripotency. I isolated cells that showed higher pluripotency upon introduction of the sgRNA library and identified 43 candidate genes based on the sgRNA sequences integrated into the cells. To validate the relationship between the candidate genes and acquisition of pluripotency, I knocked out or knocked down each of them in the paused iPSCs and confirmed that knockdown of Lnx1 gene, encoding E3 ligase, increased the pluripotency significantly, indicating its potential function as a roadblock gene during acquisition of pluripotency. In addition, Lnx1 downregulation facilitated pluripotency acquisition during Sendai virus-based and Retrovirus-based reprogramming, suggesting that Lnx1 prevented cells from acquiring pluripotency during reprogramming.
Funding Source: JSPS KAKENHI No. JP23H02407 JST SPRING No.HJI06015
