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BRAIN Publication-derived

Generation of Human Isogenic Huntington Disease Cerebral Organoids and Analysis of Neuroepithelial Development

Source Zhang et al., 2019 · Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research (A*STAR), Singapore · 10.1101/850586

👤 Jinqiu Zhang, Jolene Ooi, Kagistia Hana Utami, Sarah R. Langley, Obed Akwasi Aning, Dong Shin Park, Magdalena Renner, Shiming Ma, Chit Fang Cheok, Juergen A. Knoblich, Florent Ginhoux, Enrico Petretto, Mahmoud A. Pouladi ⏱ 28 days 📋 10 phases 🧫 Human ESC (isogenic Huntington Disease lines: 30Q, 45Q, 65Q, 81Q CAG repeats) and Human iPSC (18Q, 71Q, 109Q CAG repeats)

Abstract

This protocol describes the generation of cerebral organoids from isogenic human embryonic stem cells carrying different huntingtin (HTT) CAG repeat lengths and analyzes the effects of mutant HTT on early neuroepithelial progenitor expansion and neurogenesis. The organoids are analyzed at day 21 and day 28, corresponding to 2–3 months of human in vivo brain development, using immunostaining, RNA-seq, cell cycle analysis, and functional assays to characterize CAG repeat length-dependent neurodevelopmental impairments.

Cell source
Human ESC (isogenic Huntington Disease lines: 30Q, 45Q, 65Q, 81Q CAG repeats) and Human iPSC (18Q, 71Q, 109Q CAG repeats)
Application
Disease modeling (Huntington Disease neurodevelopmental pathology)

Protocol overview

42 steps across 10 phases

Cell Culture and Maintenance Days 0–5
  1. 1 Maintain IsoHD hESC lines
  2. 2 Detach hESCs for organoid initiation
Embryoid Body Formation and Neuroectoderm Induction Days 0–15
  1. 3 Initial embryoid body culture (Days 0–5)
  2. 4 Medium transition to neural induction (Days 5–6)
  3. 5 Neuroectoderm formation and quality check (Day 11)
  4. 6 Forebrain patterning (optional; Day 11–14)
  5. 7 Matrigel embedding and transfer (Day 11–15)
Organoid Expansion and Differentiation Days 15–28
  1. 8 Begin orbital shaking culture (Day 14)
  2. 9 Medium changes during expansion (Days 14–28)
  3. 10 Organoid harvest at day 28
Tissue Preparation and Immunohistochemistry Day 28 onwards
  1. 11 Fixation of organoids
  2. 12 Cryoprotection with sucrose
  3. 13 Embedding and sectioning
  4. 14 Permeabilization and blocking
  5. 15 Primary antibody incubation
  6. 16 Washing and secondary antibody incubation
  7. 17 Final washing and DAPI staining
  8. 18 Image acquisition
Quantitative PCR and Molecular Analysis Day 28 organoids
  1. 19 RNA extraction
  2. 20 Reverse transcription
  3. 21 Quantitative PCR (qPCR)
RNA-Seq and Transcriptional Profiling Day 28 organoids
  1. 22 RNA extraction for sequencing
  2. 23 Library preparation and sequencing
  3. 24 Data quality assessment and alignment
  4. 25 Differential expression analysis
  5. 26 Functional enrichment analysis
Cell Cycle Analysis (FUCCI Assay) Neural stem cell cultures (parallel to organoid development)
  1. 27 FUCCI lentiviral vector production
  2. 28 Neural stem cell differentiation and viral infection
  3. 29 FUCCI live-cell imaging
  4. 30 Cell cycle phase duration analysis
Analysis of Apical Progenitor Division Day 28 organoids
  1. 31 Dual immunostaining for mitosis and apical surface markers
  2. 32 Measurement of cleavage angle
  3. 33 Quantification of apical mitotic cells
Western Blotting for ATM-p53 Pathway Day 28 organoids
  1. 34 Organoid lysis and protein extraction
  2. 35 Protein quantification
  3. 36 SDS-PAGE and protein transfer
  4. 37 Membrane blocking and antibody probing
  5. 38 Chemiluminescent detection
ATM Inhibitor Treatment and Rescue Experiment Days 14–28
  1. 39 ATM inhibitor treatment
  2. 40 Medium changes during ATM inhibitor treatment
  3. 41 Organoid analysis and quantification
  4. 42 Statistical analysis of rescue

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhang et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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