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BRAIN Publication-derived

DSCAM/PAK1 pathway suppression reverses neurogenesis deficits in iPSC-derived cerebral organoids from patients with Down syndrome

Source Tang et al., 2021 · Department of Stem Cell and Neural Regeneration, State Key Laboratory of Reproductive Medicine, Nanjing Medical University · 10.1172/jci135763

👤 Xiao-Yan Tang, Lei Xu, Jingshen Wang, Yuan Hong, Yuanyuan Wang, Qian Zhu, Da Wang, Xin-Yue Zhang, Chun-Yue Liu, Kai-Heng Fang, Xiao Han, Shihua Wang, Xin Wang, Min Xu, Anita Bhattacharyya, Xing Guo, Mingyan Lin, Yan Liu ⏱ 70 days 📋 11 phases 🧫 Patient-Derived iPSC (Down syndrome trisomy 21)

Abstract

This protocol describes the generation and differentiation of cerebral organoids from Down syndrome patient-derived iPSCs and euploid control iPSCs, along with molecular and genetic interventions to rescue neurogenesis deficits. The protocol involves derivation of 3D cerebral organoids, transcriptomic profiling, DSCAM gene knockdown via CRISPR/Cas9 or CRISPRi, and small-molecule PAK1 inhibition to reverse proliferation and neurogenesis impairments associated with trisomy 21.

Cell source
Patient-Derived iPSC (Down syndrome trisomy 21)
Application
Disease modeling

Protocol overview

68 steps across 11 phases

iPSC Culture and Embryoid Body Formation Days 0–7
  1. 1 Maintain iPSCs under feeder-free conditions
  2. 2 Dissociate iPSCs to single cells
  3. 3 Initiate neural differentiation
  4. 4 Monitor EB formation and morphology
Cerebral Organoid Formation and Maturation Days 7–30
  1. 5 Embed EBs in Matrigel
  2. 6 Transfer Matrigel droplets to culture
  3. 7 Change differentiation medium
  4. 8 Assess organoid growth and morphology
  5. 9 Harvest day-30 organoids for analysis
Organoid Maturation and Late Neurogenesis (Days 30–70) Days 30–70
  1. 10 Continue medium changes
  2. 11 Assess late-stage cortical neurogenesis markers
  3. 12 Harvest day-70 organoids
CRISPR/Cas9-Mediated DSCAM Gene Knockdown Days 0–14 (genomic modification phase)
  1. 13 Design and validate guide RNAs for DSCAM
  2. 14 Prepare and validate CRISPR plasmid
  3. 15 Electroporate DS1 iPSCs
  4. 16 Apply antibiotic selection
  5. 17 Clone and expand single-cell isolates
  6. 18 Verify DSCAM knockdown at mRNA and protein level
CRISPR Interference (CRISPRi)-Mediated DSCAM Suppression Days 0–26 (genomic modification phase)
  1. 19 Design and validate CRISPRi guide RNAs
  2. 20 Clone sgRNAs into lentiviral vector
  3. 21 Prepare lentiviral particles
  4. 22 First lentiviral transduction: dCas9-KRAB expression
  5. 23 Select dCas9-KRAB-expressing cells
  6. 24 Second lentiviral transduction: sgRNA delivery
  7. 25 Select and isolate single clones
  8. 26 Validate CRISPRi-mediated DSCAM knockdown
Differentiation of DSCAM-KD and CRISPRi-Modified iPSCs into Cerebral Organoids Days 0–70 (parallels Phase 1–3; applies modified iPSC lines)
  1. 27 Culture DSCAM-KD or CRISPRi-modified iPSCs
  2. 28 Differentiate modified iPSCs to organoids
  3. 29 Assess rescue of proliferation at day 30
  4. 30 Assess rescue of neurogenesis at days 50–70
  5. 31 Perform scRNA-Seq on DSCAM-KD organoids
Small-Molecule PAK1 Inhibitor Treatment (FRAX486) Days 0–70 (treatment concurrent with organoid differentiation)
  1. 32 Prepare FRAX486 stock solution
  2. 33 Add FRAX486 to wild-type DS1 organoid culture
  3. 34 Assess p-PAK1 reduction at day 30
  4. 35 Assess rescue of proliferation at day 30
  5. 36 Assess neuroepithelial loop architecture at day 30
  6. 37 Assess rescue of neurogenesis at day 50
  7. 38 Assess rescue of upper-layer neurogenesis at day 70
Immunofluorescence Staining and Imaging Days 30, 50, 70 (performed as needed)
  1. 39 Fix organoids with paraformaldehyde
  2. 40 Wash with PBS
  3. 41 Cryoprotect with sucrose
  4. 42 Embed in OCT and cryosection
  5. 43 Permeabilize and block tissue sections
  6. 44 Incubate primary antibodies overnight
  7. 45 Incubate secondary antibodies
  8. 46 Final washes and mounting
  9. 47 Image acquisition
EdU Click-iT Cell Proliferation Assay Day 30 (or as desired)
  1. 48 Pulse organoids with EdU
  2. 49 Fix organoids and remove EdU medium
  3. 50 Wash and permeabilize
  4. 51 Perform Click-iT reaction
Western Blot Analysis Day 30, 50, 70 (as desired)
  1. 52 Lyse organoids
  2. 53 Measure protein concentration
  3. 54 Run SDS-PAGE
  4. 55 Transfer to PVDF membrane
  5. 56 Block membrane
  6. 57 Incubate primary antibodies
  7. 58 Wash membrane
  8. 59 Incubate secondary antibodies
  9. 60 Final washes
  10. 61 Detect protein bands using ECL
  11. 62 Quantify band intensity
Quantitative Real-Time PCR (qPCR) Day 30 (or as desired)
  1. 63 Extract total RNA
  2. 64 Perform reverse transcription
  3. 65 Design or obtain qPCR primers
  4. 66 Set up qPCR reaction
  5. 67 Perform qPCR thermocycling
  6. 68 Analyze qPCR data

Full SOP

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Attribution

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

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