Nephron Progenitor Cell Engraftment beneath the Murine Kidney Capsule as a Platform for Disease Modeling, Drug Delivery, and Regeneration

Abstract

Approximately 1 in 7 adults globally will be affected by chronic kidney disease, many of whom go undiagnosed. This high prevalence is growing as underlying conditions increase and those who progress to end stage renal disease will require renal replacement therapy in the form of dialysis or a transplant. Human induced pluripotent stem cell (iPSC)-derived kidney organoids could provide a renewable source for implantable kidney tissue. Previous work engrafting terminally differentiated kidney organoids falls short of regenerating functional nephrons and is expected to face several hurdles on its path into the clinic. Wanting to see if these organoids are the ideal starting implant material, we implanted cells from different time points of the differentiation and evaluated their response in vivo. We differentiated NPCs and mature organoids and implanted them beneath the kidney capsule of NOD.SCID mice. Here we show that implanting nephron progenitor cells (NPCs) within the window of opportunity results in improved engraftment reproducibility. NPCs more consistently produced renal structures in grafts compared to organoids and were able to form well vascularized chimeric glomeruli. In contrast, mature organoids dedifferentiated or died rapidly beneath the capsule, and did not become productively vascularized. We also observed human-derived mesangial cells within our grafts, indicating that implanting during this early window of opportunity incorporates stromal progenitor cells and may provide an easier solution to achieve more complex glomeruli.Our goal is to develop a toolbox of resources that enable the use of the kidney capsule implantation model as a platform for studying drug delivery to human cells and modeling genetic diseases in vivo in addition to improving methods of kidney regeneration (as well as other organoid types) with or without the incorporation of biomaterial scaffolds. Because organoids do not reach a level of maturity in vitro that is representative of adult human kidneys, it is possible that engraftment may allow for the creation of a highly complex, human-specific in vivo model. Engraftment of organoids beneath the kidney capsule has been shown to improve overall maturation of nephron segments and promotes interaction between the host vasculature and developing glomeruli that is not observed strictly in vitro. To explore the utility of this platform, three additional directions were investigated. First, to evaluate drug delivery, VHH nanobodies were added to kidney organoid cultures in vitro and systemically perfused in vivo and imaged to measure accumulation. Methods were developed to co-localize signal to specific renal structures, but dosing and delivery will need to be optimized in the future to understand if nanobodies can accumulate in engrafted structures. Second, a novel Ctns-/- NOD.SCID mouse strain was developed to allow for human engraftment in a cystinotic kidney. Cystinosis animal models have been developed previously, but they have all been in immunocompetent strains. With this novel strain, combined with cohorts of patient-derived and CRISPR-edited cystinotic iPSC lines, we can begin to test if the diseased animal's microenvironment exacerbates a disease phenotype in implanted cells and if the implantation of healthy cells can help restore function to kidneys with chronic conditions. Lastly, we wanted to explore the generic utility of kidney capsule implantations. Kidneys are highly vascularized tissues and the capsule helps confine implanted materials to a single location. Over the past several years, we have examined the effects of this environment on implanted biomaterial (hydrogel) scaffolds, tooth organoids, vascular organoids, and salivary gland organoids. Each of these experiments have shed light on the broad usefulness of this technique as well as its limitations. These lessons may be applied to NPC engraftment to help improve our regenerative medicine-based therapeutics in the future.