evt differentiation medium  (Cell Signaling Technology Inc)

Journal: Stem cell research

Article Title: Establishment of human induced trophoblast stem-like cells from term villous cytotrophoblasts

doi: 10.1016/j.scr.2021.102507

Figure Lengend Snippet: Detection of hTSC markers in iTSC and STB and EVT markers after iTSC differentiation. (A-J) Immunostaining for TSC markers GATA3, TFAP2C, TEAD4, ELF5, ITGA6, TP63, CDH1, CK7 and Ki67 (Green) in 5F-G3 iTSC at passage 10–15. Nuclei were stained with DAPI (Blue). Similar results were obtained with three independent cell lines generated using the six factors (sup ). (K, K’) Immunostaining of STB marker hCG (green) in iTSC cultured in TSC medium (K) or STB differentiation medium (K’) at 3 days in vitro demonstrating the capacity of iTSC to generate of multinucleated syncytiotrophoblasts under appropriate differentiation conditions. Similar results were obtained with two independent cell lines. (L, L’) Immunostaining of HLA-G (green) in iTSC cultured in TSC medium (L) or EVT differentiation medium (L’) at 7 days in vitro demonstrating the capacity of iTSC to generate HLA-G + cells with bipolar morphology under appropriate differentiation conditions. Similar results were obtained with two independent cell lines. (M-N’) Representative images of CK7 (M’) and HLA-G (N’) expressing cells detected on the bottom surface of the Transwell membrane after iTSC cultured in TSC medium (M, N) or EVT medium (M’, N’) on the top surface of Transwell membrane for 7 days. Only iTSC cells in EVT differentiation medium penetrated Transwells membrane. scale bar = 100 μm.

Article Snippet: EVT differentiation was performed through the modification of a protocol described previously ( ). iTSC were seeded at a density of 0.75 × 10 5 per well in Col-IV-coated ibidi μ-dishes in EVT differentiation medium (EVTM: advanced DMEM/F12, 0.1 mM 2-mercaptoethanol (Gibco 31350), 0.3% BSA, 1% ITS-X supplement (Gibco 51500–056), 100 ng/ml NRG1 (Cell Signaling 5218SC), 7.5 μM A83–01, 4% knockout serum replacement (ThermoFisher 10828010), and 2% Matrigel.

Techniques: Immunostaining, Staining, Generated, Marker, Cell Culture, In Vitro, Expressing, Membrane