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KIDNEY Publication-derived

Kidney Organoids for Disease Modelling with Gene Editing

Full SOP with Academic or a plan

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

Authors: 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 Duration: 18 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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