Identification of culture adaptation-related pathways in human pluripotent stem cells using genome-wide screening

Human pluripotent stem cells (hPSCs) acquire chromosomal aberrations and mutations during prolonged culturing. This process allows them to grow better in culture, in a process called “culture adaptation”. These genetic aberrations hinder the usage of cells at high passage. Despite the identification of several pathways that enable culture adaptation, a thorough examination of such pathways has not been conducted yet. To this end, we utilized a CRISPR/Cas9-based genome-wide screening using haploid human embryonic stem cells. The screening included a loss-of-function library, with sgRNAs targeting more than 18,000 genes. To analyze genes involved in culture adaptation we grew the cells continuously for more than 100 days. In order to identify culture adaptation-related genes, we compared the high-passage population to the population at the starting point of the experiment by examining the abundance of the mutated genes in the library. Using this procedure, we have been able to identify enrichment of genes from several signaling pathways like the mTOR signaling pathway and the RhoA/ROCK pathway, as well as additional pathways that were not known to play a role in the growth of hPSCs. To validate our results, we used chemical modulators of proteins in the pathways that we identified and examined their effect on the growth of the cells using several hPSC lines. Moreover, we examined the effect of these modulators on apoptosis, cell cycle and differentiation. In summary, our work established a platform for improved growth media for hPSCs, hopefully, reducing the accumulation of genetic aberrations in these cells.