WCRB2014 POSTER PRESENTATIONS (1) (335 abstracts)
Serum-free culture of mouse trophoblast stem cells
Shuai Sun 1 , Shota Yano 1 , Momo O Nakanishi 2 , Michiko Hirose 3 , Kazuhiko Nakabayashi 4 , Kenichiro Hata 4 , Atsuo Ogura 3 , Kunio Shiota 1 & Satoshi Tanaka 1
1The University of Tokyo, Bunkyo-ku, Japan; 2Developmental and Stem Cell Biology Hospital for Sick Children, Toronto, Ontario, Canada; 3RIKEN BRC, University of Tsukuba, Tsukuba, Japan; 4National Research Institute for Child Health and Development, Setagaya-ku, Japan.
Introduction: Trophoblast stem cells (TSC) maintain their undifferentiated status under the presence of fibroblast growth factor 4 (FGF4), heparin and feeder cell-conditioned medium (CM). It has been reported that activin A can replace CM. However, even in such stem cell condition, a portion of TSC spontaneously undergoes differentiation. In addition, when TSC are induced to differentiate, all trophoblast subtypes autonomously appear without the addition of any exogenous factors. In order to ask at what degree various unidentified factors contained in fetal bovine serum (FBS) affect differentiation of TSC, we sought to establish serum-free culture conditions (SFC) for TSC.
Methods: FBS in the conventional TSC medium was replaced by knockout serum replacement (KSR). Growth of TSC was assessed by counting number of viable cells during serial passages. Integrity of TSCs in SFC was examined by RNA-sequencing. Furthermore, differentiation potency of TSCs maintained under SFC was investigated in vitro by expression analysis of marker genes and in vivo by the chimera analysis.
Results and discussion: Fibronectin or laminin was necessary for adhesion of TSCs in SFC. Presence of FGF4, heparin and activin A was not sufficient to achieve proliferation rate comparable to FBS-containing conditions. Addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor was found to promote proliferation in SFC. TSCs cultured in such SFC had a gene expression pattern characteristic to TSCs and exhibited a differentiation potential both in vitro and in vivo. Thus, we established SFC for maintenance of TSC, which should be useful for future study.
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ISSN 2052-1472 (online)
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