Leucine-rich repeat–containing G-protein–coupled receptor 5–positive cells in the endometrial stem cell niche Irene Cervelló, Ph.D., Claudia Gil-Sanchis,

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Leucine-rich repeat–containing G-protein–coupled receptor 5–positive cells in the endometrial stem cell niche Irene Cervelló, Ph.D., Claudia Gil-Sanchis, Ph.D., Xavier Santamaría, Ph.D., M.D., Amparo Faus, B.Sc., Julia Vallvé-Juanico, Ph.D., Patricia Díaz-Gimeno, Ph.D., Oriana Genolet, Ph.D., Antonio Pellicer, Ph.D., M.D., Carlos Simón, Ph.D., M.D. Fertility and Sterility Volume 107, Issue 2, Pages 510-519.e3 (February 2017) DOI: 10.1016/j.fertnstert.2016.10.021 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Molecular phenotypic profile of the LGR5-expressing cells in human endometrium. (A) Representative fluorescence-activated cell sorter plot showing endometrial LGR5 staining. (B) Flow cytometric analysis of LGR5+ cells in the epithelial and stromal fractions of the human endometrium. Histograms showing specific coexpression of LGR5+ cells with several mesenchymal (CD90 and CD73), hematopoietic (CD34, CD45, and CD163), endothelial (CD31), and stem cell (CD133) markers. Bars indicate positive staining for each marker compared with isotype control samples. (C) Table summarizing the percentage of positive cells obtained by means of flow cytometry (n = 3–7 samples) for LGR5+ cells with different surface markers: CD9, CD13, CD31, CD133, CD34, CD45, CD73, and CD90. Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Representative sections from the left kidneys of three xenotransplanted mice are shown with (A) Prussian blue staining and (B) CD9 and (C) vimentin (Vm) immunohistochemistry. Control samples are shown in the last column: spleen for Prussian blue and endometrium for CD9 and Vm. Images were taken at ×40 and ×100 magnification. A dashed line delineates the kidney structure from the endometrial-like newly formed tissue. Positive staining for Prussian blue, shown by blue spots, suggests that newly formed endometrial tissue was developed from the human injected cells (arrows). Immunohistochemical analysis for human endometrial markers CD9 (epithelium) and Vm (stroma) was performed to test if the newly formed tissue had endometrial-like characteristics. Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 LGR5+ cell gene set enrichment analysis (GSEA). Enriched KEGG functions in LGR5+ cells isolated from (A) endometrial epithelial cells and (B) endometrial stromal cells. LGR5+ cells were associated with hematopoietic cell lineage KEGG pathway (Supplemental Fig. 4; www.genome.jp/kegg-bin/show_pathway?hsa04640). The most enriched pathways (adjusted P<.05) and enrichment scores are presented. Enriched pathway in both epithelium and stroma is indicated by blue and yellow, respectively. Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Schematic illustration of proposed hypotheses to describe the function of human endometrial LGR5+ macrophage-like cells with hematopoietic origin. (A) LGR5+ macrophage-like cells are recruited via bone marrow; and (B) LGR5+ perivascular macrophages modulate the niche. Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Diagram showing the specific phenotypes of LGR5+ cells related to hematopoietic lineage markers. Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Results from the in vitro assays, showing the standardization of the superparamagnetic iron oxide nanoparticle (SPIO) protocol. Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 List of genes up- and down-regulated in the epithelial and stromal fractions of the LGR5+ cell populations of the human endometrium versus their whole epithelial and stromal counterparts (LGR5−). Fertility and Sterility 2017 107, 510-519.e3DOI: (10.1016/j.fertnstert.2016.10.021) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions