Regulation of 3-O-Sulfation of Heparan Sulfate During Transition from the Naïve to the Primed State in Mouse Embryonic Stem Cells.

Autor:	Ota H; Department of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo, Japan., Nishihara S; Department of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo, Japan. shoko@soka.ac.jp.; Glycan & Life System Integration Center (GaLSIC), Soka University, Hachioji, Tokyo, Japan. shoko@soka.ac.jp.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2303, pp. 443-452.
DOI:	10.1007/978-1-0716-1398-6_35
Abstrakt:	Mouse embryonic stem cells (mESCs), which are established from the inner cell mass of pre-implantation mouse blastocysts, rapidly expand and form dome-shaped colonies. The pluripotent state of mESCs has been defined as the "naïve" state. On the other hand, characteristics of mouse epiblast stem cells (mEpiSCs), which are derived from the epiblast of mouse post-implantation blastocysts, has been described as the "primed" state. Human embryonic stem cells/induced pluripotent stem cells (hESCs/iPSCs) are also defined as primed state cells because their gene expression pattern and signal requirement are similar to those of mEpiSCs. Both mEpiSCs and hESCs/iPSCs proliferate slowly and form flat colonies. It is therefore difficult to genetically modify primed state cells and apply them to regenerative medicine. Therefore, stable methods of reversion from the primed to the naïve state are required. Clarifying the molecular mechanisms that underpin the primed-to-naïve transition is essential for the use of such cells in basic research and regenerative medicine applications. However, this is a challenging task, since the mechanisms involved in the transition from the naïve to the primed state are still unclear. Here, we induced mEpiSC-like cells (mEpiSCLCs) from mESCs. During induction of mEpiSCLCs, we suppressed expression of 3-O-sulfated heparan sulfate (HS), the HS4C3 epitope, by shRNA-mediated knockdown of HS 3-O-sulfotransferases-5 (3OST-5, formally Hs3st5). The reduction in the level of HS 3-O-sulfation was confirmed by immunostaining with an anti-HS4C3 antibody. This protocol provides an efficient method for stable gene knockdown in mESCs and for the differentiation of mESCs to mEpiSCLCs. (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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