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Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development
Yong Kyun Kim1,2,3,4, Ido Refaeli5,6, Craig R Brooks7
1Division of Nephrology, Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
Abstract:
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.
Insights
Human pluripotent stem cell-derived podocytes mimic capillary loop stage cells, aiding kidney disease research. These cells reveal podocalyxin
Area of Science:
- Nephrology
- Developmental Biology
- Stem Cell Biology
Background:
- Podocyte differentiation is crucial for kidney organogenesis.
- Human pluripotent stem cell-derived podocytes (hPSC-podocytes) offer a model for studying kidney development and disease.
- The precise developmental stage and disease modeling capacity of hPSC-podocytes remain to be fully elucidated.
Purpose of the Study:
- To determine the developmental maturity of hPSC-podocytes.
- To assess the utility of hPSC-podocytes in recapitulating kidney disease mechanisms.
- To investigate the role of podocalyxin in podocyte development and function.
Main Methods:
- Generation of kidney organoids and hPSC-podocytes.
- Ultrastructural, biophysical, and transcriptomic analysis.
- CRISPR/Cas9 gene editing to create podocalyxin-knockout models.
- Comparison with in vivo mammalian podocyte development and disease models.
Main Results:
- hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage, exhibiting similar ultrastructure, gene expression, and mutant phenotypes.
- hPSC-podocytes develop junction-rich basal and microvillus-rich apical membranes.
- Podocalyxin deficiency in hPSC-podocytes and mice leads to defects in microvilli assembly, lateral space formation, and junctional migration, impairing urine production.
Conclusions:
- hPSC-podocytes represent a valuable model for studying human kidney development and disease, recapitulating key features of capillary loop stage podocytes.
- Podocalyxin plays a conserved and essential role in mammalian podocyte maturation.
- This study establishes a framework for utilizing hPSC-podocytes to investigate kidney pathophysiology and explore regenerative strategies.
