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Differentiation and Expansion of Human Extra-Embryonic Endoderm Cell Lines from Naïve Pluripotent Stem Cells.

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In human, endoderm is induced in two waves, with the first being the extra-embryonic primitive endoderm (PrE), otherwise known as hypoblast, induced during blastocyst development, and the second being gastrulation-stage… Click to show full abstract

In human, endoderm is induced in two waves, with the first being the extra-embryonic primitive endoderm (PrE), otherwise known as hypoblast, induced during blastocyst development, and the second being gastrulation-stage definitive endoderm (DE). The PrE gives rise to the primary and secondary yolk sac, and has supportive functions during pregnancy for nutrient provision, with descendants of this extra-embryonic lineage also playing a role in embryonic patterning. As in DE specification, we recently found that PrE could be induced in vitro by Wnt and Nodal-related signaling, but that the critical difference was in the pluripotent starting point for differentiation. Thus, blastocyst-like naïve human pluripotent stem cells retain the unique capacity to differentiate into PrE cultures, a cell type resembling the pre-implantation hypoblast. The PrE cells could then be expanded as stable naïve extra-embryonic endoderm (nEnd) cell lines, capable of indefinite self-renewal. Here, we describe detailed protocols to differentiate naïve pluripotent stem cells into PrE and then expand the cultures as nEnd, including descriptions of morphology, passaging technique, and troubleshooting.

Keywords: endoderm; extra embryonic; stem cells; pluripotent stem; embryonic endoderm

Journal Title: Methods in molecular biology
Year Published: 2022

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