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BRAIN Publication-derived

Generation of Cerebral Organoids from hESCs and iPSCs and Analysis of Cortical Development in Schizophrenia

Source Stachowiak et al., 2017 · State University of New York at Buffalo, Department of Pathology and Anatomical Sciences · 10.1038/s41398-017-0054-x

👤 E. K. Stachowiak, C. A. Benson, S. T. Narla, A. Dimitri, L. E. Bayona Chuye, S. Dhiman, K. Harikrishnan, S. Elahi, D. Freedman, K. J. Brennand, P. Sarder, M. K. Stachowiak ⏱ 35 days 📋 6 phases 🧫 Human ESC (H9, HUES8) and Patient-Derived iPSC (Schizophrenia, Control)

Abstract

This protocol generates 3D cerebral organoids from human embryonic stem cells (hESCs) and patient-derived induced pluripotent stem cells (iPSCs) to model early cortical development and investigate schizophrenia-related developmental abnormalities. Organoids recapitulate the first trimester of human brain development, revealing disrupted neural progenitor cell migration, reduced cortical neuronal maturation, and altered FGFR1 signaling in schizophrenia models, with implications for understanding early pathogenic mechanisms in psychiatric disease.

Cell source
Human ESC (H9, HUES8) and Patient-Derived iPSC (Schizophrenia, Control)
Application
Disease modeling

Protocol overview

30 steps across 6 phases

Embryoid Body (EB) Formation and Neuroectodermal Induction Days 0–8
  1. 1 Aggregate pluripotent stem cells into embryoid bodies
  2. 2 Culture EBs in neuroectodermal media
  3. 3 Embed EBs in matrigel scaffolds
  4. 4 Culture organoids to day 8
Organoid Maturation and Zone Development Days 8–18 (or Days 8–35 for extended cultures)
  1. 1 Continue culture in appropriate differentiation media
  2. 2 Apply FGFR1 inhibitor (if performing inhibition studies)
  3. 3 Harvest organoids at desired timepoint
Pulse-Chase BrdU Labeling (Optional, for cell migration studies) Days 14–18
  1. 1 Pulse label with BrdU
  2. 2 Wash and chase phase
  3. 3 Harvest and immunostain
Organoid Sectioning and Immunocytochemical Staining Concurrent with/after Phase 2 harvest (Day 18 or later)
  1. 1 Fix organoids
  2. 2 Cryoprotection and sectioning
  3. 3 Block and permeabilize sections
  4. 4 Primary antibody incubation
  5. 5 Wash and secondary antibody incubation
  6. 6 Nuclear counterstaining and mounting
  7. 7 Image acquisition
RNAseq Analysis of FGFR1-Regulated Gene Networks (Optional) Parallel with organoid experiments; on 2D NPC/NCC cultures
  1. 1 Differentiate NPCs to neuronal committed cells (NCCs)
  2. 2 Transfect NPCs with FGFR1 expression constructs
  3. 3 Harvest RNA and RNA extraction
  4. 4 RNAseq library preparation and sequencing
  5. 5 Read alignment and differential expression analysis
  6. 6 Gene ontology and pathway analysis
  7. 7 Network correlation analysis
Quantitative Image Analysis and Statistical Evaluation Post-imaging
  1. 1 Define regions of interest (ROIs)
  2. 2 Cell counting and identification
  3. 3 Fluorescence intensity measurements
  4. 4 Computational analysis: Ki67 NPC dispersion
  5. 5 Computational analysis: Calretinin interneuron orientation
  6. 6 Statistical analysis and data presentation

Full SOP

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Attribution

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

This wording is awaiting legal review.

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