Application of Progenitor Niche Signals to Ex Vivo Nephrogenesis

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Summary Approximately 750,000 Americans have end stage renal disease, in which kidney function is insufficient to sustain life. Organ function can be supplemented by dialysis in these individuals, however the 10 year survival rate for individuals on dialysis is just over 10%. Survival rates are much better for patients receiving a kidney transplant, but organ supply does not match demand. Ex vivo organogenesis has the potential to provide functional tissue for renal replacement therapy. Furthermore, defining signals that functionally direct nephrogenesis may identify pathways that can be manipulated to augment the regenerative response of the injured kidney in vivo. Several groups, including our own, have established techniques that allow us to generate cellularly complex kidney organoids derived from human and mouse induced pluripotent cells. These tissues seem an ideal source for generating renal replacement tissue. Theoretically, one would take patient-derived renal organoids and transplant them onto a diseased kidney, where they would integrate with the host urinary system and improve renal function. Although several groups have attempted to perform these types of transplantations, there is no evidence to date that they functionally integrate with the host kidney. In our preliminary studies, we have identified three key obstacles that must be overcome in order to generate ex vivo renal organoids that integrate with the host. First, organoid structure is relatively disorganized, which is in contrast to the precise arrangement of cell types along the cortical-medullary axis of healthy, native kidneys. Second, with current strategies, organoid- derived tubules do not connect with host-derived tubules and the organoid-derived tubules involute over time. Third, we lack robust functional assays to identify experimental modifications that improve organoid function. Each of these barriers must be eliminated to generate functional organoids that can be clinically beneficial to patients. We hypothesize that the best approach to achieve integrated organoid tissue is to selectively generate cell types that match the anatomic site of engraftment. Specifically, we will identify conditions that will allow us to generate proximal nephrons, including glomeruli and proximal tubules with their associated interstitium and vasculature, (herein referred to as cortical organoids) for the purposes of engraftment. To this end, our strategy for ex vivo nephron generation is unique in its emphasis on promoting the anatomically “correct” epithelia and its microenvironment for the site of engraftment. Concurrent to this, we will identify factors and techniques that promote tubule-tubule fusion. Thus, once we have generated cortical organoids, we will utilize this technology to stimulate the tubules of the graft to anastomose with the tubules of the host. Finally, we will use live imaging and well-defined functional assays as a readout of tubular function to continually optimize our strategy. The long-term goal of this proposal is to engineer organized, ex vivo renal tissue that can be induced to form functional nephrons in animal hosts through novel grafting strategies.