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INTESTINAL Publication-derived

Intestinal Organoids for Developmental Studies (2011)

Full SOP with Academic or a plan

Source Spence et al. · Cincinnati Children's Hospital Medical Center, Cincinnati, USA · 10.1038/nature09691

Authors: Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, Shroyer NF, Wells JM Duration: 140 days 7 phases Cell source: Human iPSC / Human ESC

Abstract

The pioneering paper demonstrating directed differentiation of human pluripotent stem cells into three-dimensional intestinal tissue. Using a temporal series of growth factors mimicking embryonic intestinal development, Spence et al. generated HIOs containing all major epithelial cell types plus mesenchyme.

Cell source
Human iPSC / Human ESC
Application
Developmental Biology

Protocol overview

24 steps across 7 phases

Phase 1 · Maintenance of Pluripotent Stem Cells Ongoing (passage every ~4 days)
  1. a Culture PSCs on Matrigel in mTesR1 medium
  2. b Passage PSCs using dispase
Phase 2 · Differentiation into Definitive Endoderm Days 0-3
  1. a Treat PSCs with Activin A for definitive endoderm induction
  2. b Harvest definitive endoderm
Phase 3 · Hindgut Specification and Spheroid Formation Days 3-7 (post-Activin A treatment)
  1. a Treat definitive endoderm with FGF4 and WNT3A
  2. b Collect hindgut spheroids
Phase 4 · Maturation into Intestinal Organoids (3D Culture in Matrigel) Days 7-140 (up to ~20 weeks of culture)
  1. a Embed spheroids in Matrigel with growth factors
  2. b Add organoid medium overlay
  3. c Change organoid medium every 4 days
  4. d Monitor organoid morphogenesis (Days 0-14)
  5. e Monitor organoid maturation and villus formation (Days 14-28)
  6. f Passage and expand organoids (Optional, after Day 14)
  7. g Monitor intestinal stem cell and progenitor domain formation (Days 28-56+)
Phase 5 · Functional Analysis of Intestinal Cell Types Days 18-28 and beyond
  1. a Assess enterocyte brush border formation
  2. b Test enterocyte peptide transport function
  3. c Quantify differentiated cell types by immunofluorescence
  4. d Confirm expression of intestinal lineage markers by RT-qPCR
Phase 6 · Gain-of-Function Analysis: NEUROG3 Overexpression Day 28
  1. a Prepare adenoviral NEUROG3 stocks
  2. b Transduce day-28 organoids with Ad-NEUROG3
  3. c Analyze enteroendocrine cell fate 7 days post-transduction
Phase 7 · Loss-of-Function Analysis: NEUROG3 Knockdown iPSC line generation and differentiation
  1. a Generate NEUROG3 shRNA lentiviral vectors
  2. b Transduce low-passage H9 ES cells with shRNA lentiviral vectors
  3. c Select and expand puromycin-resistant clones
  4. d Differentiate NEUROG3 shRNA ES cells and analyze knockdown efficiency

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Attribution

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

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