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KIDNEY Publication-derived

Organoid Cystogenesis in Human Polycystic Kidney Disease: Microenvironment-Mediated Disease Modeling

Source Cruz et al., 2017 · University of Washington School of Medicine, Division of Nephrology · 10.1038/nmat4994

👤 Nelly M. Cruz, Xuewen Song, Stefan M. Czerniecki, Ramila E. Gulieva, Angela J. Churchill, Yong Kyun Kim, Kosuke Winston, Linh M. Tran, Marco A. Diaz, Hongxia Fu, Laura S. Finn, York Pei, Jonathan Himmelfarb, Benjamin S. Freedman ⏱ 87 days 📋 11 phases 🧫 Human ESC (WA09/H9); Human iPSC (WTC11); CRISPR-targeted PKD1[-/-] or PKD2[-/-] hPSC mutants

Abstract

This protocol describes the generation of kidney organoids from CRISPR-mutant human pluripotent stem cells and their culture under varied physical microenvironments to model polycystic kidney disease (PKD) cystogenesis. By systematic manipulation of adherent cues and stromal components, the protocol achieves high-efficiency cyst formation in PKD organoids, producing phenotypically relevant disease models that recapitulate early-stage human PKD pathology.

Cell source
Human ESC (WA09/H9); Human iPSC (WTC11); CRISPR-targeted PKD1[-/-] or PKD2[-/-] hPSC mutants
Application
Disease modeling of polycystic kidney disease cystogenesis

Protocol overview

71 steps across 11 phases

hPSC Maintenance and Differentiation Initiation Days 0-2
  1. 1 Maintain hPSCs on feeder-free culture
  2. 2 Plate hPSCs for kidney differentiation (Protocol A)
  3. 3 Sequential media changes (Protocol A, hours 16-86)
  4. 4 Continue RB media maintenance (Protocol A)
Alternative Kidney Differentiation Protocol (Protocol B) Days 0-14
  1. 1 Plate undifferentiated hPSCs overnight
  2. 2 Treat with CHIR99021 in APEL
  3. 3 FGF stimulation phase
  4. 4 Long-term APEL culture
Cyst Formation in Adherent Cultures Day 21 (with optional day 21 onward for Forskolin/8-Br-cAMP treatment)
  1. 1 Identify cysts in untreated adherent cultures
  2. 2 cAMP-stimulated cyst formation (optional)
  3. 3 Quantify cysts by light microscopy
High-Efficiency Cyst Formation in Suspension Culture Day 21 (transfer to suspension) through Day 35 (assessment) and beyond for long-term culture
  1. 1 Microdissect and transfer organoids to suspension
  2. 2 Maintain suspension cultures with regular media changes
  3. 3 Assess cystogenesis at two weeks post-transfer
  4. 4 Long-term suspension culture (optional, for massive cyst expansion)
Generation of KTEC Monolayer Cultures from Organoids Day 21 onward (from day of organoid isolation)
  1. 1 Plate organoids on GelTrex for outgrowth
  2. 2 Expand KTEC monolayers (optional further passage)
  3. 3 Alternative: Flow cytometry isolation of LTL+ KTECs
  4. 4 Harvest monolayers for analysis
Collagen Droplet Embedding and Compaction Assay Days 21-35
  1. 1 Prepare Parafilm mold for collagen droplets
  2. 2 Place organoids and prepare collagen droplets
  3. 3 Polymerize collagen droplets
  4. 4 Transfer droplets to suspension in untreated wells
  5. 5 Maintain suspension culture and monitor compaction
  6. 6 Measure droplet diameter and normalize
  7. 7 Fix droplets for histological analysis
  8. 8 Stain for collagen with Picro-sirius red (optional)
siRNA Knockdown and Immunoblotting for PC1/PC2 Analysis Day 0–3 (from siRNA transfection)
  1. 1 Transfect hPSCs with PKD2 siRNA
  2. 2 Change media post-transfection
  3. 3 Harvest cells for lysis
  4. 4 Quantify protein concentration
  5. 5 Prepare protein samples for SDS-PAGE
  6. 6 Perform SDS-PAGE electrophoresis
  7. 7 Transfer proteins to PVDF membrane
  8. 8 Immunoblot with primary antibodies
  9. 9 Secondary antibody incubation and detection
  10. 10 Quantify band intensity and normalize
Immunofluorescence Analysis of Organoids and Tissues Varies by sample (organoid fixation typically days 21-35+)
  1. 1 Fix organoids in suspension culture
  2. 2 Block and permeabilize organoids
  3. 3 Incubate with primary antibodies
  4. 4 Secondary antibody and nuclear staining
  5. 5 Final wash and mounting
  6. 6 Image organoids by fluorescence microscopy
  7. 7 Quantify pH3+ proliferating cells (optional)
  8. 8 Immunofluorescence on frozen tissue sections
Global Gene Expression Analysis (Microarray) Days 71–87 of culture (sample collection and processing)
  1. 1 Manually dissect cysts and tubule remnants
  2. 2 Flash freeze samples
  3. 3 Extract total RNA including small RNA
  4. 4 Assess RNA integrity
  5. 5 Label RNA and prepare cDNA targets
  6. 6 Hybridize samples to microarray chips
  7. 7 Stain and wash hybridized arrays
  8. 8 Scan microarray chips
  9. 9 Process image files and generate CEL files
  10. 10 Quality control and normalization
  11. 11 Identify differentially expressed genes
  12. 12 Gene set enrichment analysis (GSEA)
  13. 13 Analyze GSEA results
RNA-Seq Analysis of hPSCs (Optional, for transcriptomic comparisons) Days 0–3 (from RNA collection to library preparation)
  1. 1 Extract RNA from undifferentiated hPSCs
  2. 2 Check RNA integrity
  3. 3 Prepare sequencing libraries
  4. 4 Sequence libraries
  5. 5 Align reads to reference genome
Transmission Electron Microscopy (TEM) of Collagen Droplets Days 21–35 (sample collection and processing)
  1. 1 Prepare EM fixative
  2. 2 Primary fixation of collagen droplets
  3. 3 Post-fixation with osmium tetroxide
  4. 4 Dehydration in graded ethanol series
  5. 5 Infiltration and embedding in epoxy resin
  6. 6 Ultramicrotomy (sectioning)
  7. 7 Post-staining of TEM sections
  8. 8 Transmission electron microscopy imaging

Full SOP

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Attribution

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

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