PROJECT SUMMARY The goal of this proposal is to study and control nephron induction and assembly towards the formation of replacement renal tissue. Kidney organoids re-create an astonishing cellular diversity comparable to the early fetal kidney. However, structural connectivity of urine-producing nephrons and their drainage network formed by ureteric epithelium (UE) is required to avoid rapid pathology, yet has not been achieved. Accordingly, there is an urgent need to achieve connectivity between nephrons and ureteric epithelium before kidney organoids can achieve their potential in regenerative medicine. Our long-term goal is to construct ‘higher-order’ synthetic kidney tissues using human autologous stem cell lineages and assembly technologies that mimic the outcomes of morphogenesis. Our overall objectives at this stage are firstly to gain spatial control over nephron formation by determining how the mechanical microenvironment contributes to their induction sites and maturation. Its second objective is to direct nephron fusion with UE at many spatial sites through a controlled invasive process. Achieving these objectives will mark a transformative advance towards creating replacement kidney tissue. Our central hypothesis is that mechanical compaction of mesenchymal cells during kidney morphogenesis permits nephron induction, and subsequently that tight spatiotemporal control over WNT signaling events is necessary for their efficient fusion with UE. We plan to achieve the objectives through two specific aims. Firstly, we will determine the mechanical basis of nephrogenesis and use it to specify nephron positions. We will study mechanical compaction of the nephrogenic mesenchyme, assess biophysical properties of early nephron cells, and optimize nephrogenesis at specific locations using micropatterning technology. Secondly, we will program WNT-induced fusion of nephrons with ureteric epithelium. We will optimize fusion in nephron-ureteric epithelial co-cultures using optogenetic control over WNT signaling, and then trigger nephron assembly with UE spheroids after transferring them from micropatterned surfaces. The proposed research is innovative because we create fundamental knowledge while creating tissues that are biomaterial- free, human-derived (compatible with patient-derived autologous cell strategies), and therefore open to future development for transplantation. The proposed research is significant because higher-order assembly of human kidney tissue will create a step-change in renal replacement technology beyond dialysis, transplant, and “abiotic” filtration. We expect these efforts to have significant positive impact in the areas of fundamental biological discovery, drug target screening, and regenerative medicine.