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INTESTINAL Publication-derived

Directed Differentiation of Human PSCs into Intestinal Tissue In

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

👤 Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, Shroyer NF, Wells JM ⏱ 140 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

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

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