Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions

• TSCs can be grown in defined serum-free TX media on Matrigel-coated surfaces
• In TX culture, expression of stem cell markers and a stable karyotype are maintained
• Expression and methylation profiles in TX are highly similar to standard conditions
• TSCs retain full differentiation potential in TX media in vitro and in vivo

SummaryTrophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.