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LIVER Publication-derived

Biliary Organoids for Disease and Development

Full SOP with Academic or a plan

Source Guan et al., 2017 · Stanford University School of Medicine · 10.1172/jci.insight.94954

Authors: Yuan Guan, Dan Xu, Phillip M. Garfin, Ursula Ehmer, Melissa Hurwitz, Greg Enns, Sara Michie, Manhong Wu, Ming Zheng, Toshihiko Nishimura, Julien Sage, Gary Peltz Duration: 62 days 14 phases Cell source: Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (iPSCs) into three-dimensional hepatic organoids (HOs) that recapitulate hepatocyte and cholangiocyte formation with bile duct-like structures. The HOs model normal liver development and genetic liver diseases such as Alagille syndrome (ALGS) caused by JAG1 mutations.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

78 steps across 14 phases

Phase 1 · iPSC Culture and Maintenance Day 0 - ongoing maintenance
  1. a iPSC Colony Maintenance
Phase 2 · Teratoma Formation Assay Day 0 - Day 21
  1. a Prepare iPSC for Injection
  2. b Subcutaneous Injection into Mice
  3. c Teratoma Monitoring
  4. d Teratoma Harvest and Fixation
  5. e Tissue Processing
  6. f Teratoma Staining for Germ Layer Identification
  7. g Microscopy and Documentation
Phase 3 · CRISPR-Mediated Genome Engineering of iPSC Day 0 - Day 14+ (multi-step selection)
  1. a Design and Clone sgRNA Sequences
  2. b Construct Homology-Directed Repair (HDR) Template with piggyBac
  3. c Prepare Plasmids for Transfection
  4. d Transfect iPSC with CRISPR Components
  5. e First Round Antibiotic Selection (Puromycin)
  6. f Genotyping Clones After First Selection
  7. g Transposon Excision Transfection
  8. h Second Round Antibiotic Selection (Gancyclovir)
  9. i Clone Verification by Sequencing
  10. j Special Handling: Tagged Control Clones
Phase 4 · Hepatic Organoid Differentiation - Phase 1 (Days 0-3) Day 0 - Day 3
  1. a Endoderm Differentiation Initiation
  2. b Monitor Endoderm Formation
Phase 5 · Hepatic Organoid Differentiation - Phase 2 (Days 3-9) Day 3 - Day 9
  1. a Transition to Hepatoblast Medium
  2. b Hepatoblast Development
  3. c Day 9 Characterization
Phase 6 · Hepatic Organoid Formation and Maturation - Phase 1 (HO1; Days 9-50) Day 9 - Day 50
  1. a 3D Culture Initiation
  2. b Organoid Maturation Culture
  3. c Assessment of Organoid and Vesicle Formation (Day 30)
  4. d Characterization at Days 15-20
  5. e Extended Culture to Day 50
Phase 7 · Secondary Organoid Generation (HO2; Days 50-62) Day 50 - Day 62
  1. a HO1 Dissociation and Expansion in Growth Media
  2. b Secondary Organoid Formation in Matrigel with Differentiation Media
  3. c Day 62 HO2 Characterization
Phase 8 · Drug Metabolism Assay (Clemizole) Day 50
  1. a Prepare Control Organoids and Primary Hepatocytes
  2. b Clemizole Incubation
  3. c Metabolite Collection and Analysis
  4. d Normalization to Cellular Protein
Phase 9 · Bile Acid Measurement Day 50
  1. a Supernatant Collection
  2. b Acetonitrile Extraction
  3. c Centrifugation and Drying
  4. d Resuspension in Methanol/Water
  5. e UHPLC-MS Analysis Setup
  6. f UHPLC Gradient Program
  7. g Mass Spectrometry Detection
  8. h Data Analysis and Bile Acid Identification
Phase 10 · Rhodamine 123 MDR1 Transport Assay Day 25
  1. a Prepare Day 25 Organoids
  2. b Optional: Verapamil Pretreatment
  3. c Rhodamine 123 Incubation
  4. d Washing
  5. e Confocal Microscopy Imaging
  6. f Fluorescence Intensity Quantification
  7. g Statistical Analysis
Phase 11 · RNA-Seq and Transcriptome Analysis Day 0 - analysis phase
  1. a cDNA Library Preparation
  2. b cDNA Quality and Quantity Assessment
  3. c Sequencing Library Construction
  4. d Library Cleanup and Pooling
  5. e Illumina Sequencing
  6. f Data Analysis Approach
  7. g Counts Per Million (CPM) Analysis with Morpheus
  8. h Hierarchical Clustering
  9. i Validation by RT-PCR
  10. j Data Deposition
Phase 12 · Reverse Transcription and RT-PCR Quantification Variable - as needed for analysis
  1. a RNA Extraction
  2. b cDNA Synthesis
  3. c TaqMan Gene Expression Analysis
  4. d Data Normalization and Quantification
Phase 13 · Histology and Immunostaining of Liver Tissue (Patient and Control) Variable - processed upon receipt of tissues
  1. a Tissue Collection and Preparation
  2. b Cryosectioning
  3. c Tissue Fixation
  4. d Permeabilization
  5. e Blocking Nonspecific Binding
  6. f Primary Antibody Incubation
  7. g Secondary Antibody Incubation
  8. h Mounting and Imaging
Phase 14 · Preparation of Mouse Embryo Tissue Variable - processed upon tissue collection
  1. a Embryo Tissue Collection
  2. b Frozen Section Preparation
  3. c Tissue Fixation
  4. d Blocking Nonspecific Binding
  5. e Primary and Secondary Antibody Staining
  6. f Imaging and Analysis

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Attribution

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

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