Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells

Abstract

Significance Statement Recent progress in stem cell biology has enabled researchers to induce nephron progenitor cells (NPCs) and kidney organoids from pluripotent stem cells (PSCs). However, shepherding NPC differentiation toward a specific nephron segment remains a challenge. The authors observed the effects of various growth factors in cultured mouse embryonic NPCs during three phases of the differentiation process, demonstrating that phase-specific manipulation of Wnt and Tgf- β signaling is critical for podocyte differentiation. Using this insight into the nephron-patterning process, they were able to selectively induce human PSC-derived podocytes with molecular, morphologic, and functional characteristics of human podocytes. This novel protocol will facilitate accessibility to human podocytes, and these PSC-derived podocytes are expected to serve as a valuable resource in kidney research. Background Previous research has elucidated the signals required to induce nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling the generation of kidney organoids. However, selectively controlling differentiation of NPCs to podocytes has been a challenge. Methods We investigated the effects of various growth factors in cultured mouse embryonic NPCs during three distinct steps of nephron patterning: from NPC to pretubular aggregate, from the latter to epithelial renal vesicle (RV), and from RV to podocyte. We then applied the findings to human PSC-derived NPCs to establish a method for selective induction of human podocytes. Results Mouse NPC differentiation experiments revealed that phase-specific manipulation of Wnt and Tgf- β signaling is critical for podocyte differentiation. First, optimal timing and intensity of Wnt signaling were essential for mesenchymal-to-epithelial transition and podocyte differentiation. Then, inhibition of Tgf- β signaling supported domination of the RV proximal domain. Inhibition of Tgf- β signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resultant protocol enabled successful induction of human podocytes from PSCs with >90% purity. The induced podocytes exhibited global gene expression signatures comparable to those of adult human podocytes, had podocyte morphologic features (including foot process–like and slit diaphragm–like structures), and showed functional responsiveness to drug-induced injury. Conclusions Elucidation of signals that induce podocytes during the nephron-patterning process enabled us to establish a highly efficient method for selective induction of human podocytes from PSCs. These PSC-derived podocytes show molecular, morphologic, and functional characteristics of podocytes, and offer a new resource for disease modeling and nephrotoxicity testing.