Thursday, May 16, 2024
5/16/2024
SPECIFIC AIMS There are more than 100k American waiting for a kidney transplant, but demand far exceeds supply. Ex vivo organogenesis is a potential source for functional tissue for renal replacement therapy. We and others established techniques to generate cellularly complex kidney organoids from human induced pluripotent cells (iPSCs). Theoretically, iPSC-derived renal organoids would be grafted to host tissue and would integrate with vascular and urinary systems to supplement lost kidney function. However, data indicates that implanted organoids integrate poorly with the host kidney. Based on experimental observations, we identified at least 3 key obstacles that must be overcome to generate integrated renal organoids (Fig. 1): Tissue organization: Organoid tubules, blood vessels, and stroma lack cortico-medullary pattern. Tubular fusion: With current strategies, organoid tubules and vessels do not anastomose with host tissues. Functional assays: We lack assays to identify successful renal replacement by organoid tissue. The goal of our collaborative team is to generate the tools, reagents and protocols to overcome each of these obstacles. In our recent studies, we discovered the following: 1) Distinct stromal cell populations are required to establish correct zonal organization and segment-specific differentiation of tubules and vasculature; 2) Organoids lack a normal distribution and complement of stromal cell populations; 3) Stromal cells and their secreted products can facilitate tubule-tubule interconnection. These findings show that formation of renal organoids that are appropriately patterned for in vivo function depends on creating the correct complement and organization of stromal cells. The concept that anatomically “correct” tissue that is matched to the engraftment site can be generated through manipulation of stromal cell populations is novel and identifies a gap in our understanding of stromal cell biology of developing kidney tissue. To fill this gap and generate resources to determine how this novel patterning mechanism can be exploited to generate kidney tissue for renal replacement, we propose a series of hypothesis-generating investigations that will define the roles of diverse stromal cells in patterning the vasculature and renal epithelium as well as promoting tubular connection to the host. Our longterm goal is to generate functional proximal nephrons with appropriately patterned microvasculature that integrate with the host urinary and circulatory systems for functional renal replacement. To facilitate our efforts to promote tubule and vascular fusion, we will develop a pipeline for in vivo testing of laboratory-derived tissue in animals with impaired kidney function. In preliminary data, we demonstrate novel functional assays including ELISAs and live imaging of tubular fluid flow that can rapidly and easily assess the contribution of implanted organoids to host urine production. We will also generate novel resources, including stromal cell lines, reporter mice, imaging technology, and assays to quantify the extent of renal replacement by organoid tissue. We propose the following Aims: Aim 1: Develop strategies to modulate organoid stroma to promote proximal nephron segments 1A. Develop protocols to promote formation of proximal tubule stroma. 1B. Investigate the role of macrophages in organoid differentiation. Aim 2. Generate robust and functionally regionalized renal vasculature in tissue grafts 2A. Evaluate vascular maintenance, growth, and recruitment in organoids. 2B. Characterize stromal impact on organoid vasculature. 2C. Generate “vascular-primed” organoids. Aim 3. Identify strategies to promote tubule fusion in renal epithelia 3A. Identify the role of stromal interactions on epithelial tubule anastomosis. 3B. Determine the role of HGF and TGFß on tubule anastomosis in organoids. Aim 4. Evaluate function of graft tissue in vivo 4A. Develop a pipeline to test functional replacement potential of graft tissue. 4B. Assess organoid engraftment in regenerative versus fibrotic kidneys. The completion of these aims is expected to greatly advance knowledge of how stromal cell populations provide needed cues for organoid organization, vascularization and connection/engraftment. Successful completion will reveal general concepts and generate much needed resources that will propel research in this field and contribute significantly to the ultimate goal of providing functional renal replacement tissue to the vast number of patients with advanced kidney disease, a goal we believe is achievable in the next 10-20 years. 1
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