Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids

TLDR

Human‑pluripotent‑stem‑cell‑derived kidney cells hold promise for disease modeling and regeneration, yet it remains unclear whether they can recapitulate tissue‑specific phenotypes. In 3‑D cultures, epiblast‑stage hPSCs form spheroids with hollow, amniotic‑like cavities, and GSK3β inhibition drives these spheroids to differentiate into nephron‑like organoids containing proximal tubule, podocyte, and endothelial cell populations. The resulting organoids self‑assemble, recapitulate proximal tubule, podocyte, and endothelial physiology, and exhibit disease phenotypes—including dextran/methotrexate uptake, KIM‑1 expression after injury, podocalyxin‑knockout junctional defects, and PKD1/PKD2‑knockout cyst formation—demonstrating tissue‑specific functional modeling and establishing a versatile 3‑D platform for disease study and regenerative medicine.

Abstract

Abstract Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.