Heparan sulfate (HS) is essential for animal development. This polysaccharide plays central roles in a variety of developmental pathways including FGF, Wnt, Hedgehog, and BMP signaling. Though HS roles in mouse embryonic stem (mES) cells have been determined, its functions in human ES (hES) cells have not yet been delineated. To determine the roles of HS in development, we used CRISPR-Cas gene editing to engineer hES cell lines deficient in the EXT1 gene. EXT1 is an essential gene for the biosynthesis of HS. The EXT1-/- cells lack HS, and as a result, cell surface binding of basic fibroblast growth factor (bFGF) is significantly reduced. This result is significant because bFGF signaling is critical for hES cell propagation in the pluripotent state (self-renewal). FGF signaling occurs under hES cell culture conditions with high concentrations of bFGF but cells differentiate in media with low FGF concentrations. Thus EXT1-/- cells are capable of maintaining self-renewal and express pluripotency markers under bFGF-rich culture conditions. Using RNA-Seq analysis, we identified key developmental pathways and processes that are differentially regulated in these HS-deficient cells. Specifically, when EXT1-/- hES cells were assessed for their ability to differentiate into all three germ layers, they failed to express markers of mesoderm and endoderm, suggesting that HS is required for differentiation. Taken together, these results suggest that HS is dispensable for self-renewal under normal hES cell culture but plays critical roles in differentiation. Our cell line is poised to provide insight into HS in human development and HS-related diseases.

Heparan sulfate in self-renewal of human embryonic stem cells

256th National Meeting (2018)

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