SUN-130 Kidney organoids and the transplantation of human induced pluripotent stem cell-derived podocytes into newborn mouse kidneys

Abstract

Podocyte depletion is a hallmark of kidney injury that results in progression to fibrosis. Overall, podocyte research has been hampered by the lack of suitable models that permit the direct study of factors regulating podocyte survival and function. Although primary culture of human podocytes is possible, these cells only replicate over a short term and cannot be maintained over long periods. In contrast, podocytes derived from pluripotent stem cells (iPSC) that maintain characteristic podocyte features can undergo long-term proliferation. Reprogramming of adult cells to generate iPSCs will enable organ repair, regeneration and disease pathogenesis leading to therapeutic options for cell replacement. This study differentiated kidney podocytes from human iPSC, using monolayer cultures and a 3D organoid approach, prior to transplantation into newborn mouse kidneys. Differentiation of iPSCs to podocytes (iPSC-POD) was performed in Nphs1 (Nephrin)-green fluorescent protein (GFP) iPSCs by the addition of activin A, bone morphogenetic protein 7 (BMP7) and retinoic acid. GFP+ expression corresponding with nephrin expression of differentiated cells was examined in monolayer culture by genotyping, fluorescence-activated cell sorting (FACs) analysis and quantitative PCR (qPCR). As a comparison, kidney organoids were generated in vitro by the induction of iPSCs into epiblast spheroids and the addition of the Wnt signal agonist CHIR99021. To assess in vivo integration, either CFSE+ labeled iPSC-PODs or iPSC-POD labeled with a cell tracker [endocytosed qdot crystals (LifeTechnologies)], were differentiated over 10 days of culture. Thereafter, 1 x 105 differentiated iPSC-POD were directly injected into the kidneys of mouse pups at postnatal day one (P1) using a novel intrarenal administration technique to achieve cell transplantation. A timecourse analysis of cell integration was assessed in differentiated iPSC-POD, compared to undifferentiated cells, using confocal fluorescence microscopy using the co-expression of podocyte-specific antibodies. Using Nphs1-GFP+ iPSCs a variable protein expression was found following differentiation to kidney podocytes using fluorescent microscopy and qPCR. In contrast to monolayer culture, kidney organoids were generated using a Geltrex matrix that were found to positively recapitulate cell organisation in vivo. The advantages provided using kidney organoid culture were the robust expression of podocyte markers, namely; podocin, Wilms tumour factor (WT)-1 and podocalyxin; and the proximal tubular marker Lotus Tetragonolous Lectin (LTL). The transplantation of differentiated iPSC-POD into newborn mouse kidneys were detected in the renal cortex and interstitium as early as postnatal day 3. The integrated-labeled iPSC-POD were shown to integrate adjacent to developing glomeruli using co-localisation of glomerular and tubular-specific markers. This study provides proof-of-principle that differentiated iPSC–POD are able to survive and integrate into recipient newborn mouse kidneys, that provides an ideal in vivo environment due to the immature and immunoprivileged nature of the developing postnatal kidneys. We also report that iPSC-PODs within 3D kidney organoids may provide a superior in vitro system for cell and disease modeling and ultimately isolation of kidney cells for replacement therapy.