Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell‐derived definitive endoderm cells

Abstract

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+) c-Kit(+) , Cxcr4(+) E-cadherin(+) cells or Cxcr4(+) PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine.

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

“…Previous studies reported that the TGF-␤/Smad, Wnt/␤-catenin, and FGF signaling pathways can regulate the formation of the definitive endoderm (2,4,7). To test which signal that activates ER stress is associated with definitive endoderm differentiation, we performed Western blotting assays with the whole lysate from ESCs treated with TG/TM and found an active translation of Bip, which was not evident in the control cultures (Fig.…”

“…Several signaling pathways and cascades of transcription factors have been reported to have roles in the induction and specification of the definitive endoderm at different stages of development (2)(3)(4)(5)(6). TGF-␤ signaling has been thought to be essential for the formation of the definitive endoderm (7,8).…”

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

“…Distinct levels and combinations of signals through pathways such as Wnt, Nodal/TGFβ, BMP, and FGF/MAPK direct the exit from pluripotency and govern the induction of lineage-specific transcription factors, which ultimately dictate cell type. For example, high flux through the Wnt and Nodal pathways cooperatively engages master transcriptional regulators of endoderm formation, including Sox17 and GATA4 (Gadue et al 2006;Grapin-Botton and Constam 2007;Li et al 2011a). These factors subsequently induce more terminal differentiation makers, such as Hnf4α and albumin, to generate the specialized cells associated with this germ layer (Bossard and Zaret 1998;Kanai-Azuma et al 2002).…”

Histone deacetylation promotes mouse neural induction by restricting Nodal-dependent mesendoderm fate

“…mESC lines E14tg2a, 46C, HDAC1 lox/lox , Rosa26Cre-ER mESC and HDAC2 lox/lox , Rosa26Cre-ER mESC were used in this study and cultured in feeder-free medium 64 . Monolayer culture for neural differentiation was performed as previously described 40 .…”