Fig 1: mESCs differentiate to express endometrial precursor cell markers CD9 and CD13 by three weeks in standard culture. By one, two, and three weeks in culture, mESCs display both human endometrial epithelial CD9 (b, f, j) and stromal CD13 cell markers (c, g, k). Co-expression is illustrated in (d, h, and l). As shown in (m), colonies of mESCs showed progressive differentiation of CD9 immunoreactive cells over a three-week period (58%, 60%, and 31% in weeks one, two, and three, respectively), while populations of differentiated CD13 cells increased over this same duration (57%, 34% and 62% in weeks one, two, and three, respectively). Bottom right scale bar in panel (l) indicates 20 μm for all panels
Fig 2: Mouse EBs express human endometrial markers, confirmed by PCR. EBs express human endometrium glandular cell marker CD9 (green, a), stromal marker CD13 (red, b). Minimal CD9/CD13 co-expression is observed at this point (c). HOXa10 co-expression (green) with CD9 and CD13 are shown in (d and e), respectively. Mesenchymal stem cell markers CD146/PDGFR-β are shown in (f) (yellow). White arrows indicate positive expression. Scale bars indicate 20 μm. RT-PCR confirmed expression of CD9 and CD13 as well as Foxa2, Hand1, Hoxa10, ER-A and -B, and PR-A and -B (g; ladder in left column, positive and negative controls shown in far right columns)
Fig 3: FACS sorting of CD13+ and CD146+/PDGFR-β + cells from mouse EBs. Total EB cells gated as DAPI+/CD45−/CD13+ (a) and DAPI+/CD45−/CD146+/PDGFR-β + (b) are shown. Graph legends indicate percent of parent population. c shows the proportion of CD13+ and CD146+/PDGFR-β + cells isolated from both the total cell population and viable CD45- population in EB medium by flow cytometry. Quantitative RT-PCR revealed significantly up-regulated expression of Hoxa10, Foxa2, ER-A, PR-A and PR-B, in a CD13+ (stromal) subpopulation (d). The x-axis represents the gene and the y-axis represents the relative fold change to the Beta-actin gene control. Standard deviations of means are shown with brackets
Fig 4: Mouse endometrium expresses markers of human endometrium. Gross murine uterus is shown in (a) (1 cm scale) followed by H&E staining of mouse endometrium (b; black arrow indicates glandular architecture and black arrow indicates stroma). Immunofluorescence staining for extracellular matrix markers cytokeratin, vimentin, and E-Cadherin is shown in (c, d and e), respectively. Expression of ER-A (green) and ER-B (red) (f), PR-A (green) and PR-B (red) (g), CD9 (green) and CD13 (red) (h), PDGFR-β/CD146 cell markers (i), and HOXa10 (green) (j) is shown in mouse endometrium. A schematic of the distribution of cell populations of interest is shown in (k). White arrows indicate positive expression; scale bars at bottom right of panels B-J correspond to 20 μm
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