Directed differentiation of ureteric bud and collecting duct organoids from human pluripotent stem cells

Developing models of human kidney tissue in vitro is an important challenge in regenerative nephrology research, given the paucity of novel and effective therapies in kidney disease. However, the de novo generation of kidney tissues from human pluripotent stem cells (hPSCs) is challenging owing to the structural and functional complexity of the organ, as well its developmental origin from two distinct embryologic populations: the metanephric mesenchyme and the ureteric bud (UB). Directed differentiation strategies have been developed to generate kidney organoids containing nephron-like structures; we recently reported an efficient and practical method to generate UB tissues. Here, we describe a detailed step-by-step protocol for differentiation of hPSCs into three-dimensonal UB organoids that exhibit complex morphological development and the capacity to differentiate into functional collecting duct tissues. Over 3 d, hPSCs are induced into PAX2+GATA3+ pronephric (anterior) intermediate mesoderm fates in monolayer cultures at high efficiency. The cells are aggregated into three-dimensional spheroids, which then assemble and organize into nephric duct-like tissue over 4 d. When embedded into an extracellular matrix, the spheroids grow into UB organoids that recapitulate fetal branching morphogenesis for 1 week of culture. When switched to permissive conditions, the UB organoids spontaneously differentiate to form collecting duct principal cells. This approach provides robust and reproducible methods that can be readily adopted by users with basic experience in hPSC and organoid differentiation to generate UB tissues, which may be used to investigate human kidney development, model disease processes and catalyze further efforts in engineering functional kidney tissue.