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BRAIN Publication-derived

Generation and Neuronal Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) into Forebrain 3D Multilayered Structures

Source Mariani et al., 2012 · <UNKNOWN> · 10.1073/pnas.1202944109

👤 Mariani, et al. ⏱ 70 days 📋 7 phases 🧫 Patient-Derived iPSC (Autism/Schizophrenia risk variant carriers)

Abstract

This protocol describes the reprogramming of human skin fibroblasts into induced pluripotent stem cells (hiPSCs) via retroviral delivery of Yamanaka factors, and subsequent directed neuronal differentiation into 3D forebrain multilayered structures resembling cortical architecture. The 3D organoids develop region-specific neuronal and glial cell populations and were used to model early forebrain development and identify differentially expressed genes implicated in neuropsychiatric disease.

Cell source
Patient-Derived iPSC (Autism/Schizophrenia risk variant carriers)
Application
Disease modeling and developmental study of forebrain neurogenesis

Protocol overview

26 steps across 7 phases

Generation of hiPSCs from Patient-Derived Fibroblasts Day -30 to Day 0 (approximately 1 month post-infection)
  1. 1 Obtain skin biopsy and establish primary fibroblast culture
  2. 2 Infect fibroblasts at passage 3 with Yamanaka factor retroviral vectors
  3. 3 Culture cells in hypoxic conditions post-infection
  4. 4 Pick and expand hiPSC colonies on Matrigel
  5. 5 Prepare hiPSC maintenance medium
Neuronal Induction and Aggregate Formation (Days 0–18) Day 0 to Day 18
  1. 1 Pre-treat colonies with Y27632 and dissociate into single cells
  2. 2 Plate single cells in low-attachment 96-well plates to form embryoid bodies
  3. 3 Culture aggregates in suspension with forebrain induction factors (Days 0–18)
Neuroepithelial Maturation (Days 18–24) Day 18 to Day 24
  1. 1 Transfer aggregates to ultralow-attachment six-well plates
  2. 2 Continue culture in DMEM/F12/GLUTAMAX with reduced factor concentration
Neuronal and Glial Differentiation in Adherent Culture (Days 25–70) Day 25 to Day 70
  1. 1 Replate floating aggregates onto coated culture slides
  2. 2 Culture in Neurobasal medium with B27 supplement
  3. 3 Change medium every other day until endpoint
  4. 4 Fix structures at endpoint for cryosectioning
Immunostaining and Fluorescence Microscopy Performed on fixed samples from Days 25, 45–50, and 70
  1. 1 Prepare cryosections
  2. 2 Block nonspecific antibody binding
  3. 3 Incubate with primary antibodies
  4. 4 Wash sections in PBS
  5. 5 Incubate with secondary antibodies
  6. 6 Mount sections and image
Quantification and Data Analysis Post-imaging analysis
  1. 1 Perform stereological cell counting
  2. 2 Calculate percentage of labeled cells
RNA Extraction and Microarray Analysis Samples from Days 0 (undifferentiated hiPSCs), 25, 45–50, and 70
  1. 1 Extract total RNA from hiPSCs and differentiated aggregates
  2. 2 Hybridize RNA to Illumina HumanHT-12 v4 BEADCHIP
  3. 3 Perform data normalization and quality control
  4. 4 Identify differentially expressed genes

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Mariani et al., 2012. 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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