SAT-118 COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF CULTURED PODOCYTES AND HUMAN KIDNEY ORGANOIDS AT SINGLE-CELL RESOLUTION

Abstract

Podocytes are terminally differentiated kidney cells that are vital to kidney filter function. Podocyte injury and loss is central to the pathogenesis of proteinuric kidney diseases. Conditionally-immortalized differentiated mouse podocytes and human kidney organoids provide in vitro models to test hypotheses regarding mechanisms of podocyte injury and responses to treatment. To assess the maturity of cultured mouse podocytes and podocyte-like cells in human kidney organoids, we characterized transcriptomic changes of podocyte differentiation at single-cell resolution. Mouse podocyte cultures and day 23 human kidney organoids were disassociated, fixed, suspended (10X Chromium), and sequenced (Illumina HiSeq). To assess transcriptomic changes associated with differentiation, cells were clustered with principal component analysis (PCA) and visualized with Uniform Manifold Approximation and Projection (UMAP). Cell clusters were labeled by inspection of expression of published cell-type specific markers. We leveraged publicly-available scRNAseq data of mouse and human kidney to identify differentially expressed genes associated with podocyte maturation and to infer the trajectory of differentiation along the podocyte lineage. We analyzed 1.8 billion reads from 17467 differentiated mouse podocyte cells and 3.6 billion reads from 36307 cells from human kidney organoids. Differentiated podocytes in culture expressed markers of S-shaped bodies and early podocytes (Col4a1, Col4a2, Col4a5, Pax2) as well as some markers of mature podocytes (Synpo, Podxl, Nupr1, Plce1, Foxd1). We detected robust expression of marker genes (MAFB, NPHS1, NPHS2, PODXL, PTPRO, WT1) at levels observed in podocytes of human fetal kidneys, demonstrating the existence of podocyte-like cells in our differentiated organoids. The transcriptomes of podocyte-like cells from human kidney organoids resemble those of podocytes from fetal human kidneys, and podocyte-like cells are on a trajectory toward fully-differentiated podocytes from adult human kidneys. Because podocytes are critical to kidney function, a robust, reliable model of mature podocytes is highly desirable to be able to assess mechanisms of human kidney disease and responses to treatment. Future work will continue to push the differentiation of human kidney organoids in vitro and in transplanted organoids in vivo to achieve further maturation.