Nephron progenitor cells (NPCs) have a central role in kidney organogenesis: they self-renew and differentiate into nephrons, the functional units of the kidney. Human pluripotent stem cells (hPSCs) can transiently produce induced nephron progenitor-like cells (iNPCs), which then differentiate into nephron organoids. Here, we describe a protocol to purify and expand the hPSC-derived iNPCs in a regular monolayer culture format with an optimized iNPC culture medium. Under this culture condition, iNPCs are programmed to a state with their transcriptome much closer to primary human NPCs than the transient hPSC-derived iNPCs. By following this protocol, iNPC lines can be derived from any hPSC lines, exhibiting a stable cell proliferation rate and retaining NPC marker gene expression over long-term culture. We also describe a protocol to generate nephron organoids from the iNPC lines. These iNPC-derived nephron organoids show minimal off-target cell types compared to hPSC-derived kidney organoids, with enhanced podocyte maturity. This protocol consists of a modified 10-d protocol to generate iNPCs from hPSCs, an iNPC expansion phase with a unique chemically defined iNPC expansion medium called 'hNPSR-v2' and a stepwise 21-d differentiation protocol to generate nephron organoids from iNPCs on an air-liquid interface. Experience in culturing and differentiating hPSCs is required to conduct this protocol, which can be executed within 1.5-2 months.
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Background: Epithelial-mesenchymal transition (EMT) is prevalent in human cancer and facilitates tumor metastasis and therapy resistance by enhancing cancer cell motility, invasiveness, survival, and immune evasion. However, the molecular mechanisms underlying the cellular changes during EMT remain largely elusive, making it challenging to simultaneously target these diverse malignant phenotypes. Results: Here, we show that the EMT-inducing ZEB transcription factors directly repressed WWC1 (also known as KIBRA), a key upstream activating component of the Hippo signaling pathway. The EMT program thus inherently downregulated WWC1, leading to impaired Hippo signaling and constitutive activation of the downstream effector and transcriptional coactivator YAP. The YAP-dependent transcriptional program promotes manifold cellular phenotypes that resemble those induced during EMT. Indeed, pharmacological inhibition of YAP suppressed EMT-stimulated cell migration and invasion, apoptosis resistance, and cell size growth, identifying active YAP as a common essential mediator of multiple EMT-associated phenotypes. Moreover, YAP activation directly induced transcription of B7 family immune checkpoint proteins VSIR (VISTA) and PD-L2, and rendered cancer cells resistant to effector CD8 T cells. Conclusions: Collectively, the results suggest that EMT intrinsically activates YAP by repressing WWC1, providing a non-genetic mechanism for pervasive YAP activation in cancer. Activated YAP, in turn, critically contributes to diverse EMT-enhanced malignant phenotypes and immune evasion. Therefore, pharmacological targeting of YAP may suppress various EMT-associated malignant properties and improve the efficacy of anti-PD-1 immunotherapy, offering a promising therapeutic strategy against cancer cells exhibiting EMT characteristics.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.
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