CSK Negatively Regulates Human Definitive Endoderm Cell Specification From Pluripotent Stem Cells

Pluripotent stem cells(PSC),which resemble the inner cell mass or epiblast cells in the early embryos,are capable of differentiating into ectoderm,mesoderm and endoderm cells.Therefore,they can be used to model the embryonic developmental trajectory in vitro and produce sufficient cells for disease modeling,drug screening and cell therapy.In the past,scientists relied on xenopus,zebrafish,chickens,and mice to elucidate the basic principles of signaling pathways and gene regulation underlying the process of vertebrate embryonic development.It is difficult to conduct similar experiments on human embryos due to the lack of human samples and ethical issues.However,with the successful establishment of human PSCs based differentiation technology,researchers come to realize that in vitro differentiation model based on human PSCs could serve as a powerful tool for us to understand mechanisms of signaling pathways,gene expression,and epigenetic regulation which guide cell specification and diversification during human development.In this study,we set up a human PSCs based differentiation platform to reveal how the non-receptor tyrosine kinase gene CSK regulates fate determination from embryonic stem cells(ESC)into definitive endoderm(DE)as well as pancreatic precursor(PP)cells.First,human embryonic stem cells(hESC)were successfully differentiated into SOX 17 and CXCR4 double positive DE cells by activating the Wnt and Nodal/Activin signaling pathways.The expression pattern of CSK and its downstream target gene SFK(SRK family kinase,SFK)was analyzed during endoderm and pancreas differentiation process.We knocked out the CSK by CRISPR/Cas genome editing tool in hESC and found it was a negative regulator during hESC differentiation toward DE.The differentiation efficiency of CSK knockout hESC to form SOX17 and CXCR4 double positive cells reached up to 95%,which is about 30%higher than that of wild-type stem cells.Application of a small molecule inhibitor PP1 of SFK almost completely blocks the differentiation ability of both CSK wild-type and knockout hESC into DE cells,suggesting that activation of the SFK signaling pathway is essential for definitive endoderm differentiation.Reversal of enhanced differentiation phenotype of CSK knockout hESC by SFK inhibitor is consistent with previous findings that CSK deposits inhibitory phosphorylation on SFK to prevent SFK activation.Next,we conducted ATAC-seq analysis to characterize dynamic changes of genome-wide chromatin accessibility during the process of hESC differentiation towards DE.A Hippo/YAP pathway specific signature of open chromatin regions was found in CSK wild-type differentiated cells but not in CSK knockout differentiated cells.It implies that CSK knockout promotes hESC differentiation toward DE probably through inhibiting the Hippo/YAP pathway.Indeed,decreasing the entry of YAP into the nucleus by the ROCK inhibitor Y27632 could significantly increase the DE differentiation efficiency of CSK wild-type hESC.In contrast,the use of the GPCR agonist LPA to promote the entry of YAP into the nucleus greatly inhibits the DE formation efficiency regardless of CSK wild-type or knock-out hESC,demonstrating that SFK activation after CSK knockout is effective for inhibiting Hippo/YAP pathway to promote the lineage fate conversion from hESC to DE.Finally,to further identify the key regulatory transcription factors downstream of Hippo/YAP activation and inhibition,some key transcription factors specifically expressed in hESC and DE cells were analyzed.It was revealed that after YAP inhibition,the endoderm-specific transcription factors EOMES,GATA6,and SOX17 are rapidly activated,whereas the expression of the pluripotency factor OCT4 is rapidly shut off in the differentiated cells.When YAP was persistently activated during the differentiation,the expression of EOMES,GATA6,and SOX17 could hardly be turned on,but instead,high expression of OCT4 was maintained.These findings indicate that the effective shutdown of pluripotency gene expression plays a critical role in fully activating the regulatory network of DE specific transcription factors.Given the inhibitory effect of ROCK inhibitor on Hippo/YAP pathway,we successfully optimized differentiation conditions in this study to significantly improve the efficiency of hESC differentiation towards definitive endoderm and pancreatic precursor cells,which provides new evidence for optimizing in vitro differentiation protocol of hPSC.In summary,the successful establishment of PSC based in vitro differentiation model in this study helped reveal the unknown mechanism of signaling pathways,transcription factors,and chromatin remodeling regulated by CSK during differentiation of hESC to definitive endoderm cells.