Investigating nephron-vascular crosstalk in tissue patterning and maturation ABSTRACT Type II diabetes, the number one cause of chronic kidney disease (CKD), is reaching pandemic levels.1 The need for kidney transplants is thus at an all-time high, with over 100,000 patients on the waiting list.1 This stark shortage of organs has led scientists to look to culturing kidney tissue in vitro as an alternative source of replacement tissue. One promising approach has been self-organizing organoids which mimic developmental processes and yield multicellular organ-specific tissues. However, organoid cell types often fail to acquire full maturity and function. While often overlooked, blood vessels are essential to kidney function, and proper integration and patterning of vasculature will be crucial for transplant success. We suggest that developing methods for building functional blood vessels in engineered tissues will transform the field and advance our ability to generate functional, transplantable organs. Accordingly, we must first establish a comprehensive assessment of organoid vasculature. Therefore, we propose to investigate how blood vessels co-develop with nephrons and to map their transcriptional heterogeneity. Using human kidney organoids as a model system will help us define the complexities of vascular-nephron interactions during development and disease. We hypothesize that proper EC patterning is essential to nephron differentiation and/or maintenance. Using RNA-sequencing data from embryonic kidney ECs, we identified candidate signaling molecules by ECs that are expressed in a position to impact nephron progenitor cells (NPCs). In this proposal, we will pursue three aims: Aim 1) We will characterize the kidney organoid vasculature. And Aim 2) We will develop methods to promote and support organoid vascularization. Characterizing the vasculature of kidney organoids and defining the role of signaling molecules during their development will fill a large gap in knowledge about nephron formation and greatly advance the progress we have made toward building a kidney in vitro as well as treatments for CKD. Ultimately, this research will guide in vitro nephron engineering to produce functional tissue for therapeutic replacement in patients with CKD.