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

• Published in: Journal of Reproduction and Development Vol. 67; no. 3; pp. 197 - 205
• Main Authors: SUN, Shuai, YANO, Shota, NAKANISHI, Momo O, HIROSE, Michiko, NAKABAYASHI, Kazuhiko, HATA, Kenichiro, OGURA, Atsuo, TANAKA, Satoshi
• Format: Journal Article
• Language: English
• Published: Japan The Society for Reproduction and Development 01.01.2021; Japan Science and Technology Agency
• Subjects: Activin; Cell culture; Cell differentiation; DNA methylation; Embryo fibroblasts; Extracellular matrix; Fetal calf serum; Fibroblast growth factor 4; Fibroblasts; Fibronectin; Gene expression; Heparin; Inverse agonists; KnockOutTM Serum Replacement; Laminin; Original; Placenta; Stem cell transplantation; Stem cells; Suspension culture; Trophoblast stem cell; Vitronectin; KnockOutTM Serum Replacement; Extracellular matrix; Suspension culture; Trophoblast stem cell
• ISSN: 0916-8818; 1348-4400
• DOI: 10.1262/jrd.2020-119

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.