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Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids

ABSTRACT: Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identi-fying novel genes associated with kidney development and disease. A rapid, efficient, and scala-ble organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE251862 | GEO | 2024/03/14

REPOSITORIES: GEO

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Project description:Bulk ATAC_seq on GFP expressing FACS-isolated cells from E16.5 and P0 Six2TGC and compound Ezh1 and Ezh2 mutant kidneys( Six2TGC_Ezh2-/- , Ezh1+/-; Six2TGC_Ezh2-/-; and Ezh1-/- ; scRNA-seq analysis of NPCs (Six2/GFP+ cells) from E16.5 as well as P2 kidneys. Genotypes analyzed: Six2TGC (E16.5&P2), Six2TGCEzh2-/- (E16.5), Ezh1+/-;Six2TGCEzh2-/- (E16.5), and Ezh2-/-;Six2TGCEzh2-/- (E16.5) Six2/GFP+ nephron progenitor cells (NPCs) give rise to all epithelial cell types of the nephron, the filtering unit of the kidney. NPCs have a limited lifespan and are consumed near the time of birth. Pre-term birth or prenatal stress further shorten the lifespan of NPCs and result in nephron deficit and chronic kidney disease. Accordingly, there is a pressing need to better understand the factors that regulate NPC lifespan in order to develop novel regenerative strategies. Epigenetic factors are implicated in maintenance of organ-restricted progenitors such as NPCs, but the chromatin-based mechanisms are not well understood. In this study, we examined the role of the H3K27 methyltransferases, Enhancer of zeste (Ezh1 and Ezh2), in NPC maintenance using a combination of gene targeting, chromatin profiling, and single-cell RNA analysis. We find that Ezh2 expression correlates with NPC growth potential, and that Ezh2 is the dominant H3K27 methyltransferase in NPCs and epithelial descendants. Surprisingly, NPCs lacking H3K27me3 maintain their progenitor state albeit cycle slowly leading to formation of fewer nephrons. Unlike Ezh2 loss-of-function, dual inactivation of Ezh1 and Ezh2 triggers overexpression of the transcriptional repressor Hey1 and downregulation of Six2 and result in unscheduled activation of Wnt4-driven differentiation, early termination of nephrogenesis and severe renal dysgenesis. Double-mutant NPCs also overexpress the Six family member, Six1. However, in this context, Six1 is unable to access the closed Six2 enhancer and fails to maintain NPC stemness. At the chromatin level, Ezh1 and Ezh2 act by restricting accessibility of AP1 factors to their genomic binding motifs and their absence provokes a regulatory landscape akin of differentiated and non-lineage cells. We conclude that Ezh2 is required for NPC renewal potential, while maintenance of NPC lifespan and tempering the differentiation program require cooperation of both Ezh1 and Ezh2.

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Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids

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