Fig 1: Upon removal of FGF4, mouse TS cell cultures were initially proliferative but increased in number only until day 4 of differentiation (A). Sca-1+ trophoblast cells isolated from non-proliferating, differentiated TS cultures and plated at decreasing densities in conditions to support TS proliferation formed colonies however Sca-1− and non-sorted, differentiated TS cells did not (B). When these cells were compared to the Sca-1− subpopulation of trophoblast cells by qRT-PCR, Sca-1+ positive cells displayed higher expression of Sca-1 and the trophoblast stem and progenitor markers, Esrrb and Epcam, Rhox4, respectively, while Ascl2 was not different (C).
Fig 2: Cartoon depicting proposed position of Sca-1+ cells within the mouse trophoblast stem cell lineage. P-TGC, parietal trophoblast giant cell; SpA-TGC, spiral artery-associated trophoblast giant cell; Gly-T, glycogen trophoblast; Sp-T, spongiotrophoblast; S-TGC, sinusoidal trophoblast giant cell; Syn-T, syncytiotrophoblast.
Fig 3: LR-MSC in bleomycin-induced pulmonary fibrosis (BLM) mirrors the behavior of which in IPF. A Immunofluorescence of BLM and normal lungs show the colocalization of mouse LR-MSC marker Sca1 and myofibroblast marker a-SMA. Arrows indicate positive staining of a-SMA in LR-MSC. Arrowheads indicate the parabronchial distribution of LR-MSC during homeostasis. B Immunofluorescence of BLM and normal lungs show the expression of CHOP in LR-MSC. C Gating strategy for analyzing the expression of EpCam in Sca1+CD45-CD31- cells by FACS. D Isolated LR-MSC morphology was revealed by conventional light microscopy after 3 days of culture. E Schematic of the workflow used to isolate LR-MSC during homeostasis (Saline) and fibrosis (Bleomycin) for subsequent experiments. F Counts of isolated LR-MSC in each normal or BLM C57BL/6 mouse. G Western blot shows the expression of ER stress and fibrosis-associated proteins in isolated LR-MSC
Fig 4: Sca-1 was determined to be highly expressed in undifferentiated TS cells in culture by Northern blot and qRT-PCR (A) and FACS analysis (B). Day 0/0 represents undifferentiated, proliferating TS cells while 2, 4, 6 and Day 2, Day 4 and Day 6 represent cultures differentiated for 2, 4 or 6 days. Sca-1 was also expressed in TS cells in culture as detected by immunofluorescence (C; +FGF) however became undetectable following differentiation (C; -FGF). Immunofluorescence in cells (C) in the presence and absence of FGF matched the pattern of expression observed by FACS (B), however was not obvious in as a high frequency (+FGF) which is likely due to the more sensitive detection of Sca-1 expression by FACS. Scale bar = 100 um. Northern blot results shown in (A) are cropped and organized to be presented as a composite from multiple blots/films.
Fig 5: TS cell markers, Cdx2 and Esrrb were more highly expressed in Sca-1+ trophoblast isolated from differentiated TS cell cultures than in non-sorted, proliferating TS cell cultures (A). Sca-1+ trophoblast isolated from differentiated TS cultures gave rise to proliferating trophoblast cell lines that when differentiated by the removal of growth factors, expressed genes indicative of syncytiotrophoblast (Gcm1, Syna), spongiotrophoblast (Tpbpa) and TGCs (Prl3b1) (B). These differentiated cells were also positive for alkaline phosphatase activity (C, blue) and PAS (D, magenta/arrowhead) indicating differentiation of sinusoidal TGCs and glycogen trophoblast, respectively. D0 represents undifferentiated, proliferating TS cells while, D2, D4, D6 represent cultures differentiated for 2, 4 or 6 days. In C, scale bar = 500 um. In D, scale bar = 100 um.
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