The overarching objective of the Rebuilding a Kidney (RBK) Consortium is the identification and development of strategies to augment or replace renal function in patients with chronic or end- stage kidney disease. A cornerstone of this program is using human pluripotent stem cells (hPSCs) to derive de novo nephrons that may one day have therapeutic potential, which is a worthy but ambitious goal given the structural and physiologic complexity of the kidney. This proposal is based on the hypothesis that a urinary drainage or collecting system is an obligatory component of any approach that hopes to replicate some degree of mature renal function, and in the current state human kidney organoids have no outlet for draining their blind-ended nephrons. To address this problem we are developing methods to culture organoids with hPSC-derived ureteric bud (UB) progenitor cells, and preliminary studies demonstrate that these structures form elongated collecting duct-like tubules interwoven amongst the developing nephrons. The nephrons readily fuse to UB tubules, recapitulating the critical embryonic process that generates epithelial anastomoses required to excrete urine. Importantly, the fusion only occurs at the distal end of the organoid tubules, and it therefore reproduces the normal nephron polarity necessary for kidney function. In Aim 1 of this project, we will further develop these innovative methods into robust protocols and characterize the coordination of the nephrogenic and fusion processes occurring in the organoids. Aim 2 will delineate the impact of the UB-derived collecting system on nephron maturation, both in vitro and in vivo following transplantation into the kidney capsule of immunodeficient mice. These studies will facilitate important advances toward generating a collecting system in kidney organoids and enabling the functional interrogation of hPSC-derived nephrons. We anticipate that this work will synergize with ongoing efforts in the RBK program to move the field closer to resolving the issue of organoid drainage and integration.