Evidence of In Vitro Preservation of Human Nephrogenesis at the Single-Cell Level

• Published in: Stem cell reports Vol. 9; no. 1; pp. 279 - 291
• Main Authors: Pode-Shakked, Naomi, Gershon, Rotem, Tam, Gal, Omer, Dorit, Gnatek, Yehudit, Kanter, Itamar, Oriel, Sarit, Katz, Guy, Harari-Steinberg, Orit, Kalisky, Tomer, Dekel, Benjamin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.07.2017; Elsevier
• Subjects: AC133 Antigen - analysis; cancer stem cells; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epithelial Cell Adhesion Molecule - analysis; Human Embryonic Stem Cells - cytology; Humans; Kidney - cytology; kidney stem/progenitor cells; Nephrons - cytology; Nephrons - growth & development; Organogenesis; renal development; single cell gene expression analysis; Single-Cell Analysis; stem cell markers; stem cells; Wilms' tumor; stem cells; single cell gene expression analysis; kidney stem/progenitor cells; renal development; stem cell markers; cancer stem cells; Wilms' tumor
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2017.04.026

During nephrogenesis, stem/progenitor cells differentiate and give rise to early nephron structures that segment to proximal and distal nephron cell types. Previously, we prospectively isolated progenitors from human fetal kidney (hFK) utilizing a combination of surface markers. However, upon culture nephron progenitors differentiated and could not be robustly maintained in vitro. Here, by culturing hFK in a modified medium used for in vitro growth of mouse nephron progenitors, and by dissection of NCAM+/CD133− progenitor cells according to EpCAM expression (NCAM+/CD133−/EpCAM−, NCAM+/CD133−/EpCAMdim, NCAM+/CD133−/EpCAMbright), we show at single-cell resolution a preservation of uninduced and induced cap mesenchyme as well as a transitioning mesenchymal-epithelial state. Concomitantly, differentiating and differentiated epithelial lineages are also maintained. In vitro expansion of discrete stages of early human nephrogenesis in nephron stem cell cultures may be used for drug screening on a full repertoire of developing kidney cells and for prospective isolation of mesenchymal or epithelial renal lineages for regenerative medicine. [Display omitted] •mNPEM enables in vitro preservation of human renal embryonic CM and epithelia•EpCAM allows further dissection of expanded NCAM+CD133− early nephric population•Single-cell analysis unveils a continuous lineage hierarchy in nephrogenesis and WT•Splice isoform switching confirms a unified MET hierarchy in nephrogenesis and WT In this article, Dekel and colleagues use flow cytometry and single-cell analysis to show that human fetal kidney cultured in modified nephron progenitor expansion medium (mNPEM) preserves all major nephric developmental lineages (including the Cap mesenchyme, early epithelial progeny, differentiated epithelium, and mesenchymal-to-epithelial-transition states). The biomarker system they developed allows distinguishing between early nephric lineages in expanded nephron stem cell cultures affording prospective isolation.