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ABSTRACT
The extra-embryonic endoderm lineages, visceral endoderm (VE) and parietal endoderm (PE), comprise part of the yolk sac and support embryonic development. Mouse extra-embryonic endoderm stem (XEN) cells were established from primitive endoderm (PrE), the founder cells of VE and PE, from preimplantation blastocysts. The differentiation of XEN cells is usually induced in adherent conditions without medium conditioned by mouse embryonic fibroblasts (MEFCM), which maintains self-renewal of the cells. In this study, we analyzed the effects of suspension culture and retinoic acid (RA) treatment on the differentiation of XEN26 cells, a mouse XEN cell line, toward PrE derivatives. The suspension culture without both MEFCM and RA promoted the expression of α-fetoprotein (Afp) mRNA, a specific marker for VE, at 3- to 4-fold higher levels (P < 0.05) than those in adherent culture. In these conditions, the cellular content of hepatocyte nuclear factor-4α (HNF4α) protein, a crucial transcription factor involving Afp expression, increased, while that of HNF1α was relatively constant. On the other hand, RA treatment induced upregulation of the mRNA for tissue plasminogen activator (Plat), a specific marker for PE, in both adherent and suspension culture conditions. From these results, our new attempts, including the application of RA treatment and suspension culture, for the induction of differentiation in XEN26 cells led to significant upregulation of Plat and Afp, respectively.
