Enhanced vascularization and longevity of human kidney organoids

Abstract

The global burden of kidney disease affects 10% of the population and is predicted to become the 5th leading cause of death by 2040. Kidneys have very limited regenerative capabilities, so patients with significant kidney damage will need to be treated with dialysis or transplant once they enter end-stage renal disease. Undergoing dialysis, a last resort before transplant, is taxing on a patient's quality of life and can have dangerous side effects. At the same time, a kidney transplants are in short supply with only 28,000 being performed in 2023 while 90,000 patients were on the waiting list. Stem cell derived human kidney organoids have emerged as excellent models to study disease development and test novel therapeutics in vitro while also showing promise as a potential renal replacement therapy. Epithelial organoids are challenging to maintain long-term due to structural degradation and stromal overgrowth, suggesting media limitations. In human kidney organoids, we show that supplementing standard media with tubular-enhancing factors improves yield and extends longevity to six months. Addition of vascular growth factors further increases the number of endothelial cells and their ability to invade podocytes. Generating a transcriptomic single cell RNA-seq atlas of kidney organoid aging reveals that tubular-enhancing factors are necessary to maintain nephron structures, with metabolic activity, signaling, and differentiation pathways all increasing over time. Positive effects of tubular-enhancing and vascular growth factors are protocol agnostic, enabling organoid differentiation with a distinct, suspension-based approach. We have produced a longitudinal atlas of organoid aging using optimized media that improve longevity and vascularization. This platform and dataset offer a robust resource for aging, chronic disease, and tissue engineering applications. Another challenge in the organoid field has been the lack of perfusable vasculature which prevents the development of flow-based functional assays. As a steppingstone toward this objective, we show that kidney organoids can be differentiated from iPSCs entirely within a microfluidic device. This work eliminates a transfer step during or after differentiation which could damage delicate renal structures. Furthermore, organoids grown in the device demonstrate concurrent endothelial cell differentiation, development of concentric layering of nephron components, and a migration of podocytes in longer experiments. These phenotypic changes indicate an increased resemblance to in vivo glomeruli caused by the organoids being differentiated in the confined environment of the microfluidic device. In conclusion, the improvements made in kidney organoid longevity and vascularization demonstrate the potential of human kidney organoids as both a model system and a renal replacement therapy. Together, these applications have the power to help alleviate the global burden of kidney disease and improve quality of life for the individual patients who are affected.