Junhyong Kim (PI)
University of Pennsylvania
James Eberwine (PI)
University of Pennsylvania
The goal of the Penn project is to develop molecular biology and informatics tools to identify key regulatory factors that determine kidney cell phenotypes. Single cell resolution transcriptome data can reveal cryptic cell types as well as help identify regulatory systems related to cell phenotypes. We hypothesize that cell phenotype determining genes are often at low to moderate abundance in a cell, making them difficult to approach using current methods. The Penn group will develop novel molecular biology techniques to enrich single cell transcriptomes for low abundance genes. We will also develop informatics tools that will help identify targets for trans-differentiation experiments, leveraging single cell level measurements.
Homan, Kimberly A.; Gupta, Navin; Kroll, Katharina T.; Kolesky, David B.; Skylar-Scott, Mark; Miyoshi, Tomoya; Mau, Donald; Valerius, M. Todd; Ferrante, Thomas; Bonventre, Joseph V.; Lewis, Jennifer A.; Morizane, Ryuji. Nature Methods. February 2019.
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.
Lindström, NO; McMahon, JA; Guo, J; Tran, T; Guo, Q; Rutledge, E; Parvez, RK; Saribekyan, G; Schuler, RE; Liao, C; Kim, AD; Abdelhalim, A; Ruffins, SW; Thornton, ME; Basking, L; Grubbs, B; Kesselman, C; McMahon, AP. J Am Soc Nephrol. February 2018.
Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.
Kim, YK; Refaeli, I; Brooks, CR; Jing, P; Gulieva, RE; Hughes, MR; Cruz, NM; Liu, Y; Churchill, AJ; Wang, Y; Fu, H; Pippin, JW; Lin, LY; Shankland, SJ; Vogl, AW; McNagny, KM; Freedman, BS. Stem Cells. vol. 35(12), 2366–2378. December 2017.
A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378