Functional genomics to identify regulators of pancreatic commitment from human pluripotent stem cells

The use of pluripotent stem cells (PSCs) opened up a wide range of possibilities for medical research, ranging from disease-modelling and drug-screening to regenerative medicine approaches. Virtually every cell line of the body can be generated from PSCs by recapitulating the embryonic development in vitro. However, some developmental processes remain poorly understood, thus differentiation into pure populations remains challenging. While some cell types can be easily generated with high purity, the differentiation into endodermal and especially pancreatic cells remains difficult due to a lack of understanding of the early development. In order to better understand the underlying mechanisms of definitive endoderm (DE) differentiation, an shRNA knockdown screen was performed in human PSCs driven towards DE as well as mesoderm/ectoderm followed by deep sequencing. shRNA abundance in the respective groups was analysed and shRNAs were considered a hit when they were lost in one and enriched in the counteracting sample group. The most promising hits were selected for further investigation: using the CRISPR-Cas9 system, homozygous and heterozygous knockout lines were generated. In a well-established differentiation setup, knockout and control lines were differentiated towards DE and stage-specific differentiation markers were analysed by flow cytometry and quantitative PCR. For two of the hits, DSC2 (Desmocollin 2) and Col4A1 (Collagen Type IV Alpha 1 Chain), an impaired differentiation towards DE was observed, matching the results of the primary screening. Endodermal differentiation markers like GATA6, FOXA2 and SOX17 were expressed on a lower level in the knockout cell lines compared to the respective controls, as observed in qPCR and flow cytometry experiments. This might result in a lineage bias during pancreatic development. However, subsequent differentiation towards pancreatic endoderm and pancreatic progenitors was not significantly disturbed. This endodermal screening approach might reveal novel key genes for pancreatic development unknown until now. Further investigation into their molecular function is necessary to uncover novel mechanisms underlying DE development and enable a deeper understanding of the complex signalling network of differentiation.

Publication History

Article published online:
08 September 2020