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KIDNEY Publication-derived

Gene-Edited Human Kidney Organoids: Podocyte Differentiation and Characterization

Source Kim et al., 2017 · University of Washington School of Medicine · 10.1002/stem.2707

👤 Yong Kyun Kim, Ido Refaeli, Craig R. Brooks, Peifeng Jing, Ramila E. Gulieva, Michael R. Hughes, Nelly M. Cruz, Yannan Liu, Angela J. Churchill, Yuliang Wang, Hongxia Fu, Jeffrey W. Pippin, Lih Y. Lin, Stuart J. Shankland, A. Wayne Vogl, Kelly M. McNagty, Benjamin S. Freedman ⏱ 18 days 📋 7 phases 🧫 Human ESC (WA09), Human iPSC (WTC11, NPHS1-GFP 201B7)

Abstract

This protocol describes the differentiation of human pluripotent stem cells (hPSCs) into kidney organoids containing functional podocytes that phenocopy capillary loop stage podocytes in vivo. The method enables characterization of podocyte ultrastructure, junctional organization, and genetic disease mechanisms through immunofluorescence, electron microscopy, and genome-edited mutant lines, providing a platform for studying kidney development and disease.

Cell source
Human ESC (WA09), Human iPSC (WTC11, NPHS1-GFP 201B7)
Application
Disease modeling

Protocol overview

40 steps across 7 phases

hPSC Maintenance and Plating Day -3 to Day 0
  1. 1 Coat glass plates with GelTrex
  2. 2 Plate hPSCs at specified density
Sequential Medium Changes (Days -2 to 0) Day -2 to Day 0
  1. 1 Change to lower GelTrex concentration
  2. 2 Change to mTeSR1 alone
  3. 3 Change to RPMI with CHIR99021
Kidney Organoid Differentiation (Days 0 to 18) Day 0 to Day 18
  1. 1 Day 1.5 medium change
  2. 2 Routine medium feeding
  3. 3 Harvest organoids on day 18
Fixation and Sample Preparation Day 18 onwards
  1. 1 Fix organoids for immunofluorescence
  2. 2 Wash fixed samples
  3. 3 Prepare samples for cryosectioning
  4. 4 Cryoprotection with sucrose
  5. 5 Mount tissue and freeze
  6. 6 Prepare paraffin-embedded sections
  7. 7 Paraffin embedding
  8. 8 Deparaffinize sections
  9. 9 Antigen retrieval
Immunofluorescence Analysis Post-fixation
  1. 1 Block non-specific binding
  2. 2 Incubate with primary antibodies
  3. 3 Wash samples
  4. 4 Incubate with secondary antibodies
  5. 5 Wash samples again
  6. 6 Mount or stain samples
  7. 7 Image samples
Transmission Electron Microscopy (TEM) Preparation Post-fixation
  1. 1 Initial fixation of organoid cultures
  2. 2 Collect and pellet cells
  3. 3 Resuspend in EM fix
  4. 4 Fix kidney tissue for EM
  5. 5 Post-fixation with osmium tetroxide
  6. 6 Dehydrate in ethanol
  7. 7 Embed in epoxy resin
  8. 8 Section ultramicrotomy
  9. 9 Stain sections
  10. 10 TEM imaging
Image Quantification and Analysis Post-imaging
  1. 1 Draw line scans for fluorescence analysis
  2. 2 Quantify fluorescence intensity
  3. 3 Analyze TEM microvillus density
  4. 4 Measure cell-to-cell separation distance
  5. 5 Count foot processes
  6. 6 Statistical analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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