Fig 1: Induced trophoblast stem cells (iTSCs) generated from umbilical cord show trophoblast stem cell (TSC)-like marker expression and morphology: (A) Schematic representation of iTSC generation from umbilical cord using a direct reprogramming approach. During conversion of reprogramming intermediates (passage number (p) = 0) into iTSCs, the supplements A-485, BMP4, or EPZ were added for 1–3 days; (B) mRNA expression of GATA3 and TFAP2C (trophoblast markers), EPCAM (epithelial cell marker), and NANOG (pluripotency marker) at different stages during conversion into iTSCs with and without the supplements, measured by qPCR and normalized to expression of housekeeping genes GUSB and EEF2. Expression levels in TSC_1, derived from first-trimester placental tissue, are shown for reference. Data points represent log2 fold-change (FC) relative to iTSC p = 0; (C) representative phase-contrast and immunofluorescence images of source umbilical cord mesenchymal stem cells (MSCs) (negative control for trophoblast markers), the iTSCs with different supplements (p = 33–35), and TSC_1 (positive control for trophoblast markers) stained for CD90 (fibroblast marker, magenta), EpCAM (epithelial cell marker, green), TFAP2C, KRT7, or GATA3 (trophoblast markers, magenta), with 4′,6-diamidino-2-phenylindole (DAPI) (nuclei, cyan). Scale bars: 200 µm.
Fig 2: The optimization and characterization of granulosa-like cell differentiation protocols from human induced pluripotent stem cells (iPSCs).A Schematic representing of a three-stage approach (mesoderm, intermediate mesoderm (IM), granulosa cells (GCs)) to generate patient-specific models of GC-like cells by guided differentiation of iPSCs. B mRNA expression levels of key genes in stem cell pluripotency (differentiation day 0), mesoderm (differentiation day 2), and intermediate mesoderm cells (differentiation day 4–6) by using qRT-PCR analysis under different combinations of SB4 compound and/or BMP4. C A representative table depicting small molecular chemicals and recombinant proteins used in four tested conditions of GC differentiation in this study. D Brightfield representative images showing the intermediate mesoderm embryonic bodies (EBs) before attachments (day 6) and as differentiated GC (day 12). E Comparative examination of mRNA expression levels of mesoderm, IM, GC marker genes between four different differentiation conditions. F Immunofluorescence (IF) staining characterizations of mesoderm marker (GATA4) and GC markers (AMHR2, CYP19A1) in differentiation condition #2. The luteinized cumulus granulosa cells served as IF positive control cells. Scale bar, 50 μm. G The quantification of fluorescence images of (F) (left panel). Bars indicate the mean ± SD (n = 4).
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