Stepwise developmental mimicry generates proximal-biased kidney organoids.

The kidney maintains body fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for renal reabsorption of a range of sugars, ions, and amino acids, are highly susceptible to damage, leading to pathologies necessitating dialysis and kidney transplants. While human pluripotent stem cell-derived kidney organoids are used for modeling renal development, disease, and injury, the formation of proximal nephron cells in these 3D structures is incomplete. Here, we describe how to drive the development of proximal tubule precursors in kidney organoids by following a blueprint of in vivo human nephrogenesis. Transient manipulation of the PI3K signaling pathway activates Notch signaling in the early nephron and drives nephrons toward a proximal precursor state. These "proximal-biased" (PB) organoid nephrons proceed to generate proximal nephron precursor cells. Single-cell transcriptional analyses across the organoid nephron differentiation, comparing control and PB types, confirm the requirement of transient Notch signaling for proximal development. Indicative of functional maturity, PB organoids demonstrate dextran and albumin uptake, akin to in vivo proximal tubules. Moreover, PB organoids are highly sensitive to nephrotoxic agents, display an injury response, and drive expression of HAVCR1 / KIM1 , an early proximal-specific marker of kidney injury. Injured PB organoids show evidence of collapsed tubules, DNA damage, and upregulate the injury-response marker SOX9 . The PB organoid model therefore has functional relevance and potential for modeling mechanisms underpinning nephron injury. These advances improve the use of iPSC-derived kidney organoids as tools to understand developmental nephrology, model disease, test novel therapeutics, and for understanding human renal physiology.

Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

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.