Additional Supporting Information may be found in the online version of this article.

sc-12-0961_sm_SupplFigure1.tiff997KSupporting Information Figure 1. Markers and conditions used for isolation of all endometrial epithelia. (A) All mouse endometrial epithelia (glandular and luminal) express EpCAM (a&b) and their basal surface is marked with ITGA6 and ITGB1 (a&c). All endometrial epithelia are bounded by a laminin-marked basement membrane (b). (B) To isolate endometrial epithelia the uterus was first cut into donuts. The luminal epithelium was expelled and digested into single cells. The remaining uterine horn (containing the glandular epithelium) was also minced and digested. Dissociated cells from both fractions were combined, stained with EpCAM, Thy1 and lineage markers PECAM1, PTPRC and Ter119. The epithelial cells of the uterus, EpCAM+Thy1-Lin- cells, were isolated from this cellular fraction. (C) The majority of EpCAM+Thy1- uterine cells were Lin-. Given that a small fraction of EpCAM+Thy1- uterine dissociated cells were Lin+ (2%), EpCAM+Thy1-Lin- cells were utilized in all experiments to select for only endometrial epithelia. (D) Mice housed in groups of five underwent estrous cycling and were non-synchronous based on estrous staging performed via daily vaginal cytology. (E) Grafts regenerated from two groups of cells: DsRed positive EpCAM+Thy-Lin- combined with WT neonatal stroma (a-c) and DsRed positive EpCAM-Thy+/-Lin- combined with WT neonatal stroma (d-f) are shown. EpCAM+Thy-Lin- cells could clearly regenerate into RFP marked round hollow structures based on immuno-fluorescent imaging (b&c). Conversely, these structures were not visualized in grafts regenerated from EpCAM-Thy+/-Lin- cells. Only diffuse RFP signal not associated with a clear structure could be seen in tissue regenerated from EpCAM-Thy+/-Lin- cells. Scale bars equal 50 μm.
sc-12-0961_sm_SupplFigure2.tiff75KSupporting Information Figure 2. Hormonal deprivation resulted in a significant enrichment for endometrial epithelia capable of regenerative activity in vivo. Logarithmic dilutions of epithelia isolated from hormonally intact or hormonally deprived uteri were grown in vivo and regeneration was scored based on the presence of pankeratin positive epithelial structures/glands. The number of regenerated glands in each dilution was assessed. Hormonal depletion resulted in a significant enrichment (p<0.0001) in endometrial epithelia capable of regeneration in vivo (1 in 69 in hormonally deprived vs. 1 in 862 in hormonally intact endometrial epithelia).
sc-12-0961_sm_SupplFigure3.pdf216KSupporting Information Figure 3. Isolation of EpCAM+CD44+ITGA6hi endometrial epithelia. (A) Experimental approach for FACS isolation of EpCAM+CD44+ITGA6hi cells from DsRed transgenic whole uterine dissociated cellular preparations. The DsRed signal was confirmed by FACS (a). Lineage positive cells were removed while epithelial cells were selected from dissociated whole uterine preparations by gating on the EpCAM+Thy1-PTPRC-PECAM1-Ter119- population (b). The CD44+ITGA6hi gate was set using the fluorescence minus one (FMO) approach which entails staining with all antibodies except anti-CD44 (c). Based on this gating EpCAM+CD44+ITGA6hi cells were detected on fully stained samples (anti-CD44 antibody included) (d). (B) Enrichment for CD44 in the EpCAM+CD44+ITGA6hi cellular fraction was confirmed via immunocytochemistry. Messenger RNA levels for CD44 detected by QPCR were enriched in this cellular fraction as well. As a control, EpCAM levels were examined and showed no significant difference between the EpCAM+CD44+ITGA6hi and EpCAM+CD44- ITGA6hi/EpCAM+CD44+/-ITGA6lo populations. (C) Pankeratin marked all endometrial epithelia (EEPC and non-EEPC) in both the native uterus and regenerated endometrium.
sc-12-0961_sm_SupplFigure4.tiff142KSupporting Information Figure 4. Many EEPC are cycling and proliferating. (A) Analysis of FACS isolated EEPC and non-EEPC populations by Hoechst staining reveals an increased proportion of cells in the G2/M and S phases of the cell cycle in EEPC compared to non-EEPC fractions. (B) A higher proportion of proliferating cells was also found in the EEPC fraction compared to the non-EEPC fraction as determined by intracellular Ki67 FACS analysis. In this experiment, isotype staining was performed as a control to determine gating for detection of Ki67 positive cells. (C) A higher level of TERT transcript, relative to GAPDH, was detected in EEPC compared to non-EEPC by Q-PCR. (D) EEPC bound more readily to fibronectin and collagen coated surfaces compared to non-EEPC.
sc-12-0961_sm_SupplFigure5.tiff1266KSupporting Information Figure 5. Co-administration of estrogen and progesterone resulted in an increase in the number of endometrial epithelial progenitor cells (EEPC). (A) The efficacy of hormonal supplementation with pellets was confirmed by measuring serum estrogen and progesterone levels in experimental mice. (B) The percentage of EEPC was measured by FACS in placebo and hormonally supplemented mice. The percentage of EEPC remained unchanged when estrogen or progesterone was administered singly but doubled in mice co-treated with estrogen and progesterone. (C) Examples of the distribution of CD44 positive cells in the endometrium of hormonally treated mice compared to placebo as determined by IHC. Increased numbers of basally located CD44 positive cells were detected in the endometrial epithelia of estrogen and progesterone treated mice. (D) A significant increase in the number of endometrial epithelia capable of in vivo regeneration was observed when estrogen and progesterone were coadministered (1 in 198 in estrogen and progesterone treated uteri vs. 1 in 1166 in placebo p=0.006). All scale bars equal 50 μm and results are mean ± SD.
sc-12-0961_sm_SupplFigure6.tiff641KSupporting Information Figure 6. Clonal in vitro growth of isolated murine endometrial epithelia. (A) EpCAM+Thy1- cells (endometrial epithelia) could give rise to spheres in a 3-D culture assay. (B) Spheres resembled endometrial glands (a) and expressed epithelial (b) but not stromal markers (c). (C) The majority of endometrial epithelia capable of forming spheres were the progenitors (EEPC) and not the differentiated epithelia (non-EEPC). (D) Blocking CD44 with a neutralizing antibody decreased the sphere forming capacity of endometrial epithelia in vitro. (E) The number of in vitro spheres formed was proportional to the number of input cells. (F) Mixtures of DsRed and GFP marked endometrial epithelia produced only single colored spheres in the 3D assay (a-d) suggesting a single cell gives rise to each sphere. In this assay chimeric spheres were not observed. The linear relationship between number of cells and regenerated spheres was maintained when color marked endometrial epithelia were mixed and grown in vitro. (G) Endometrial epithelial cells culture in vitro could be passaged serially. All scale bars equal 100 μm and results are mean ± SD.
sc-12-0961_sm_SupplFigure7.tiff313KSupporting Information Figure 7. Isolated EEPC do not express estrogen or progesterone receptors. FACS isolated EEPC did not express ERα (A) or PR (C) (96.9±2.5% ERα negative and 96.0±6.9% PR negative) while abundant expression of both hormone receptors was detected in the non-EEPC fraction (B&D) (85.2±7.5% ERα positive and 72.9 ± 15.3% PR positive).
sc-12-0961_sm_SupplFigure8.tiff1702KSupporting Information Figure 8. Subsets of human endometrial epithelia marked with EpCAM/CD44/ITGA6 have progenitor activity. (A) EpCAM, E-Cadherin (CDH1) and Keratin 8 (KRT8) mark endometrial epithelia and MME and Vimentin (VIM) mark endometrial stroma based on immunohistochemistry and Q-PCR. (B) Basally located CD44/EpCAM and CD44/ITGA6 dually positive epithelial cells are detected in the proliferative (a-d) and secretory (e-h) normal human endometrium. (C) EpCAM+CD44+ITGA6hiLin- endometrial cells are enriched for cells with in vitro sphere forming capacity suggesting that this cell pool contains the human endometrial epithelial progenitors. (D) Basally located CD44 positive cells in the human endometrial epithelium are predominantly ERα and PR negative. Scale bars equal 50 μm
sc-12-0961_sm_SupplTable1.tiff25KSupplementary Table 1
sc-12-0961_sm_SupplTable2.tiff312KSupplementary Table 2
sc-12-0961_sm_SupplTable3.tiff27KSupplementary Table 3
sc-12-0961_sm_SupplTable4.tiff7KSupplementary Table 4
sc-12-0961_sm_SupplTable5.xls140KSupplementary Table 5

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