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BRAIN Publication-derived

Generation of Brain Organoids from hiPSCs for Bipolar Disorder Modeling

Source Tran et al., 2021 · University of Michigan, Michigan Medicine · 10.1016/j.neuron.2018.10.007

👤 Henry Tran, Sue O'Shea, Sara Aton, Durga Attili ⏱ 90 days 📋 9 phases 🧫 Patient-Derived iPSC (Bipolar Disorder), Human iPSC (Control)

Abstract

This protocol generates 3-D cerebral organoids from hiPSCs derived from bipolar disorder patients and healthy controls to model brain development and investigate cellular mechanisms underlying bipolar disorder. The protocol includes reprogramming of skin fibroblasts to hiPSCs, neural induction, organoid differentiation, and characterization via qPCR and immunohistochemistry.

Cell source
Patient-Derived iPSC (Bipolar Disorder), Human iPSC (Control)
Application
Disease modeling

Protocol overview

43 steps across 9 phases

hiPSC Culture and Maintenance Ongoing until 80% confluence
  1. 1 Plate hiPSCs on Matrigel-coated dishes
  2. 2 Maintain hiPSCs in E8 medium
  3. 3 Passage hiPSCs at 80% confluence
Embryoid Body Formation Days 0–2
  1. 1 Dissociate hiPSCs with Accutase
  2. 2 Prepare single-cell suspension
  3. 3 Centrifuge and resuspend cells
  4. 4 Add ROCK inhibitor and plate for embryoid body formation
Embryoid Body Expansion and Neural Induction Days 2–25
  1. 1 Transfer embryoid bodies to ultra-low attachment plates
  2. 2 Maintain in hiPSC medium for 4 days (Days 2–6)
  3. 3 Switch to neural induction medium (Days 6–25)
  4. 4 Continue neural induction for 19 days
Neuroectoderm Maturation and Neural Differentiation Days 25 onwards
  1. 1 Transition to neural differentiation medium (NDM) after neuroectoderm formation
  2. 2 Continue long-term culture in NDM
Organoid Imaging and Morphological Analysis Day 6
  1. 1 Image neural spheroids on day 6
  2. 2 Analyze spheroid morphology using ImageJ
  3. 3 Statistical analysis of circularity
RNA Isolation and qPCR Analysis Days 30, 60, 90
  1. 1 Collect organoid samples at days 30, 60, and 90
  2. 2 Isolate RNA using RNeasy Mini Kit
  3. 3 Measure RNA concentration
  4. 4 Perform reverse transcription
  5. 5 Conduct qPCR
  6. 6 Execute qPCR cycling protocol
  7. 7 Normalize and analyze gene expression
Cryosectioning of Organoids Days 30, 60, 90 (preparation)
  1. 1 Wash organoids in DPBS with calcium and magnesium
  2. 2 Fix organoids with 4% PFA
  3. 3 Store fixed organoids at 4°C
  4. 4 Flash freeze organoids in O.C.T. embedding compound
  5. 5 Cut tissue sections using microtome
  6. 6 Store sections at −80°C
Immunohistochemistry Staining Days 30, 60, 90 (staining)
  1. 1 Bring tissue sections to room temperature and remove O.C.T.
  2. 2 Permeabilize tissue sections
  3. 3 Wash sections after permeabilization
  4. 4 Block non-specific antibody binding
  5. 5 Apply primary antibodies overnight
  6. 6 Wash sections after primary antibodies
  7. 7 Apply secondary antibodies
  8. 8 Wash sections after secondary antibodies
  9. 9 Stain nuclei with Hoechst
  10. 10 Final wash and mountslip application
  11. 11 Store slides at room temperature in the dark
Fluorescent Microscopy and Image Analysis After mounting
  1. 1 Image tissue sections using fluorescent microscope
  2. 2 Merge and scale images
  3. 3 Analyze protein expression patterns

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Tran et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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