Dissecting Stages of Human Kidney Development and Tumorigenesis with Surface Markers Affords Simple Prospective Purification of Nephron Stem Cells

Abstract

When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms’ tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1+CD133− marks SIX2+ multipotent renal stem cells transiting to NCAM1+CD133+ differentiating segment-specific SIX2− epithelial progenitors and NCAM1−CD133+ differentiated nephron cells. In tumorigenesis, NCAM1+CD133− marks SIX2+ blastema that includes the ALDH1+ WT cancer stem/initiating cells, while NCAM1+CD133+ and NCAM1−CD133+ specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1+ nephron stem cells in normal and malignant nephrogenesis.

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

“…Table S2 summarizes the FACS data of NCAM, CD133, and EpCAM in fresh and cultured hFK from three different sources.

Figure 2NCAM + CD133 − Cells Grown in mNPEM Contain Early Nephrogenic Lineages that Can Be Dissected according to EpCAM ExpressionMid-gestation hFKs were dissociated and cultured in serum-containing medium (SCM), serum-free medium (SFM), and mNPEM. FACS analysis was performed for NCAM1, a marker that was previously shown to enrich for the CM and early epithelial structures, CD133 (PROM1), a marker that was previously shown to enrich for differentiating and mature epithelial nephron tubules in the fetal kidney, and EpCAM, an epithelial marker (Pode-Shakked et al., 2016, Shapiro et al., 2011). (A) Schematic representation of experiments.(B) Representative FACS analysis of fresh hFK and hFK cultured in either mNPEM, SFM, or SCM for 7 days.

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“…Culturing hFK in mNPEM instantly showed morphological heterogeneity, including specific undifferentiated stem cell niches and differentiating epithelial cells, which could be further exploited to show an EpCAM expression gradient (EpCAM − , EpCAM dim , EpCAM bright ) within the NCAM1 + CD133 − hFK cell fraction that was previously suggested to enrich for nephron progenitors (Metsuyanim et al., 2009, Pode-Shakked et al., 2016). This gradient, and especially the NCAM1 + CD133 − EpCAM − hFK cells (a presumed early fraction), were not observed when hFK cells were grown and passaged in SFM or SCM.…”

Concise Review: Kidney Generation with Human Pluripotent Stem Cells

“…Metsuyanim et al and Dekel et al characterized stem cell markers in human fetal kidneys and identified NPC surface markers, namely neural cell adhesion molecule 1 (NCAM1) and frizzled class receptor 7 (FZD7), which enabled the purification of an enriched cell population of human NPCs . Pode‐Shakked et al also demonstrated that purified NCAM1+/CD133– cells cultured in serum‐free media were enriched for SIX2+ NPCs, although those cells quickly differentiated in serum‐free media within a week . A recent study from the same group showed at single‐cell resolution a preservation of uninduced and induced cap mesenchyme as well as a transitioning mesenchymal–epithelial state by dissection of NCAM+/CD133– progenitor cells according to epithelial cell adhesion molecule (EpCAM) expression levels .…”

Peroxisome proliferator‐activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target

“…We next wished to ascertain that AML cells correspond to MSCs in terms of gene expression. Since the kidney harbors several cell lineages (e.g., epithelial progenitors and stromal progenitors) (Pleniceanu et al , ; Dziedzic et al , ), we compared the expression of lineage‐specific renal genes in AML cells to Wilms' tumor [WT, known to arise from epithelial progenitors (Pleniceanu et al , ; Pode‐Shakked et al , ; Shukrun et al , ; Pode‐Shakked et al , )], human fetal kidney (hFK), and human MSCs via qPCR. WT expressed high levels of the entire renal epithelial progenitor gene set, including OSR1 , SIX2 , CITED1 , and PAX2 , compared to hFK (Fig C).…”