Major amino acids in collagen hydrolysate regulate the differentiation of mouse embryoid bodies

To take advantage of the therapeutic potential of embryonic stem cells (ESCs), it is necessary to regulate their differentiation in response to defined factors. In this study, in order to explore novel molecules that regulate the differentiation of ESCs, we investigated whether collagen hydrolysate, collagen-characteristic amino acids, glycine (Gly), l-proline and trans-4-hydroxy-l-proline (l-Hyp); or dipeptides, proline-hydroxyproline and hydroxyproline-glycine regulate the differentiation of mouse embryoid bodies (EBs). We identified that treatment with collagen hydrolysate or Gly repressed the expression of the mesendodermal markers, Brachyury and Foxa2 in EBs and maintained the undifferentiated state of mESCs in a feeder-free monolayer culture. In contrast, l-Hyp promoted the expression of Brachyury, Mixl1, Gsc and Foxa2 in EBs. And the treatment with l-Hyp promoted cardiac differentiation within EBs, which was proven by the spontaneous contraction of cardiomyocytes and the expression of the cardiac markers, α-MHC, MLC-2v and Nkx2.5. Results suggest that l-Hyp is a promising new inducer for reproducible and efficient differentiation of mesendoderm lineages.