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BRAIN Publication-derived

Brain Assembloids

Full SOP with Academic or a plan

Source Workman et al. · Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA · 10.1038/nm.4233

Authors: Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N, Chang CF, Schiesser J, Aubert P, Bhatt SM, Stanley EG, Elefanty AG, Miyaoka Y, Mandegar MA, Conklin BR, Neunlist M, Brber SA, Wells JM, Bhatt AH, Helmrath MA Duration: 70 days 5 phases Cell source: Human iPSC

Abstract

Workman et al. generated intestinal organoids with integrated enteric nervous systems by co-differentiating intestinal and neural crest cells from human iPSCs, demonstrating neurally-mediated peristaltic contractions. This Nature Medicine 2017 study established the first functional gut-brain axis model for studying enteric neuropathies including Hirschsprung disease.

Cell source
Human iPSC
Application
Gut-Brain Axis Research

Protocol overview

29 steps across 5 phases

Phase 1 · Neural Crest Cell (NCC) Generation Days 0-10
  1. a Detach and prepare pluripotent stem cells (PSCs)
  2. b Initiate neural induction
  3. c Collect neurospheres (Day 6)
  4. d Isolate and passage migrated NCCs
Phase 2 · Human Intestinal Organoid (HIO) Generation and NCC Integration Days 0-28 (in vitro)
  1. a Initiate intestinal differentiation from PSCs
  2. b Aggregate HIO spheroids with NCCs
  3. c Culture HIO+ENS in vitro
  4. d Assessment of in vitro ENS formation
Phase 3 · In Vivo Maturation (Kidney Capsule Transplantation) Days 0-70 (in vivo, 6-10 weeks post-transplant)
  1. a Prepare HIO+ENS for transplantation
  2. b Transplant into NSG mice kidney capsule
  3. c Allow organoid maturation and vascularization
  4. d Harvest transplanted organoids
  5. e Morphological and immunological characterization
Phase 4 · Functional Assays: Neuronal Activity and Contractility Variable (performed on in vitro 8-week and in vivo 6-10 week organoids)
  1. a Live-cell calcium imaging (in vitro)
  2. b KCl-induced calcium wave (in vitro and in vivo)
  3. c Electrical field stimulation (EFS) of contractility
  4. d TTX-mediated blockade of neuronal activity
  5. e Isometric-force organ-bath measurements
  6. f Methylene blue ICC inhibition
  7. g ENS activation with DMPP (nicotinic agonist)
  8. h TTX blockade during DMPP stimulation
  9. i NOS inhibition with L-NAME
  10. j Sodium nitroprusside (SNP) NO-donor validation
Phase 5 · PHOX2B Mutation Modeling (Optional Disease Context) Days 0-70 (parallel to standard HIO+ENS protocol)
  1. a Generate PHOX2B mutant iPSCs
  2. b Differentiate PHOX2B mutant lines into vagal NCCs
  3. c Recombine PHOX2B mutant NCCs with wild-type HIOs
  4. d RNA-seq analysis of PHOX2B genotype effects (in vitro)
  5. e In vivo transplantation of PHOX2B mutant organoids
  6. f Quantitative assessment of PHOX2B mutant phenotype

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Attribution

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

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