Maintenance of mouse trophoblast stem cells in KSR-based medium allows conventional 3D culture

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  • SUN Shuai
    Department of Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
  • YANO Shota
    Department of Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
  • NAKANISHI Momo O
    Department of Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
  • HIROSE Michiko
    RIKEN BRC, University of Tsukuba, Tsukuba, Japan
  • NAKABAYASHI Kazuhiko
    Department of Maternal-Fetal Biology, Research Institute, National Center for Child Health and Development, Tokyo 157-8535, Japan
  • HATA Kenichiro
    Department of Maternal-Fetal Biology, Research Institute, National Center for Child Health and Development, Tokyo 157-8535, Japan
  • OGURA Atsuo
    RIKEN BRC, University of Tsukuba, Tsukuba, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan
  • TANAKA Satoshi
    Department of Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

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<p> Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOutTM Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.</p>

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