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BRAIN Publication-derived

Hindbrain Organoids for Disease and Development

Full SOP with Academic or a plan

Source Muguruma et al., 2015 · RIKEN Center for Developmental Biology · 10.1016/j.celrep.2014.12.051

Authors: Keiko Muguruma, Ayaka Nishiyama, Hideshi Kawakami, Kouichi Hashimoto, Yoshiki Sasai Duration: 148 days 4 phases Cell source: Human ESC

Abstract

This protocol generates polarized cerebellar tissue structures from human embryonic stem cells (ESCs) through 3D culture with sequential growth factor addition. The self-organized neuroepithelium differentiates into functional cerebellar cell types including Purkinje cells, granule cells, and interneurons, recapitulating early human cerebellar ontogenesis.

Cell source
Human ESC
Application
Disease modeling and developmental study of cerebellar tissue formation

Protocol overview

21 steps across 4 phases

Phase 1 · Initial hESC Aggregation and Neuroectodermal Induction Days 0-14
  1. a hESC Dissociation and Reaggregation
  2. b FGF2 Addition
  3. c Medium Change at Day 7
  4. d Full Medium Replacement at Day 14
Phase 2 · Cerebellar Plate Specification and Growth (Days 14-35) Days 14-35
  1. a Transfer to Suspension Culture
  2. b Optional: FGF19 Treatment for D-V Polarization
  3. c Optional: SDF1 Treatment for Continuous NE and RL Formation
  4. d Regular Medium Changes
Phase 3 · Purkinje Cell Maturation via Co-culture with Mouse Rhombic Lip Cells Days 35-148
  1. a Isolation of KIRREL2+ Cerebellar Plate Neuroepithelial Progenitors
  2. b Preparation of Mouse Rhombic Lip Cells
  3. c Dissociated Cell Co-culture Setup
  4. d Initial Culture in Serum-Containing Medium
  5. e Transition to Serum-Free, Defined Medium
  6. f Long-term Maintenance with Cytosine Arabinoside Treatment
  7. g Long-term Culture and Maturation (Days 35-148+)
Phase 4 · Granule Cell Differentiation via Reaggregation Culture with Mouse Cerebellar Cells Days 42-53
  1. a Isolation of PSA-NCAM+ Granule Cell Precursors
  2. b GFP Labeling of hESC-Derived Cells
  3. c Preparation of Mouse Cerebellar Cells
  4. d Reaggregate Formation
  5. e Reaggregate Culture Setup
  6. f Observation of Granule Cell Migration and Maturation (Days 5-8+)

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Attribution

This SOP was written by Organthis based on the published method in Muguruma et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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