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INTESTINAL Publication-derived

Neural Crest Cell Implantation to Restore Enteric Nervous System Function in Human Tissue-Engineered Small Intestine

Source Schlieve et al., 2017 · The Saban Research Institute at Children's Hospital Los Angeles · 10.1016/j.stemcr.2017.07.017

👤 Christopher R. Schlieve, Kathryn L. Fowler, Matthew Thornton, Sha Huang, Ibrahim Hajjali, Xiaogang Hou, Brendan Grubbs, Jason R. Spence, Tracy C. Grikscheit ⏱ 105 days 📋 9 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol describes the derivation of human pluripotent stem cell (hPSC)-derived enteric neural crest cells (ENCCs) and their co-implantation with human intestinal organoids (HIOs) to generate tissue-engineered small intestine (ENCC-HIO-TESI) with a functional enteric nervous system. The co-implantation approach establishes mature ganglia, diverse neuronal subtypes, and neuroepithelial connections in vivo, restoring neuron-dependent contractility for potential therapeutic treatment of enteric neuropathies.

Cell source
Human iPSC, Human ESC
Application
Disease modeling; Tissue engineering of functional small intestine with enteric nervous system restoration

Protocol overview

45 steps across 9 phases

hPSC Maintenance and HIO Generation Days 0-35
  1. 1 Maintain hPSCs on Matrigel substrate
  2. 2 Direct differentiate hPSCs into intestinal organoids (HIOs)
ENCC Derivation and Neurosphere Formation Days 0-15
  1. 1 Derive ENCCs from unmodified hPSCs
  2. 2 Plate unsorted ENCCs into ultra-low attachment plates
  3. 3 Culture ENCCs in neurosphere medium for 4 days
Scaffold Seeding and Surgical Implantation Day 15
  1. 1 Prepare biodegradable scaffolds
  2. 2 Seed scaffolds with HIOs and ENCC neurospheres
  3. 3 Implant constructs into NOD/SCID mice omentum
In Vivo Maturation Days 15-105 (3 months)
  1. 1 Allow implants to mature in vivo
  2. 2 Perform surgical explantation
Tissue Fixation and Histological Processing Day 105 (post-explantation)
  1. 1 Fix implants in buffered formalin
  2. 2 Embed fixed tissues in paraffin
  3. 3 Section paraffin blocks
  4. 4 Perform H&E staining
Immunofluorescence Analysis Post-fixation
  1. 1 Deparaffinize and rehydrate tissue sections
  2. 2 Perform antigen retrieval
  3. 3 Block non-specific binding
  4. 4 Apply primary antibodies
  5. 5 Wash and apply secondary antibodies
  6. 6 Mount and image sections
  7. 7 Quantify immunostaining
Light-Sheet and Confocal Microscopy Analysis Post-fixation
  1. 1 Prepare tissue for light-sheet microscopy
  2. 2 Freeze-thaw cycle for permeabilization
  3. 3 Rehydrate and block tissue
  4. 4 Incubate with primary antibodies
  5. 5 Apply secondary antibodies
  6. 6 Clear tissue
  7. 7 Perform light-sheet microscopy
  8. 8 Perform confocal microscopy
Live Video Imaging of Mechanical Contractility Post-explantation (same day preferred)
  1. 1 Equilibrate explants in imaging medium
  2. 2 Baseline contractility imaging
  3. 3 Test ICC-dependent contractility with methylene blue
  4. 4 Test neuron-dependent contractility with tetrodotoxin (TTX)
  5. 5 Image control explants without pharmacological treatment
  6. 6 Process and analyze videos
RNA-Seq and Transcriptome Analysis Post-explantation
  1. 1 Extract total RNA from tissue samples
  2. 2 Quantify RNA concentration
  3. 3 Prepare cDNA libraries for RNA-seq
  4. 4 Perform deep sequencing
  5. 5 Align reads to human genome reference
  6. 6 Normalize data and remove batch effects
  7. 7 Identify differentially expressed genes
  8. 8 Perform gene ontology and pathway analysis
  9. 9 Analyze epithelial cell type genes
  10. 10 Deposit data in public repository

Full SOP

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Attribution

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

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