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OTHER Publication-derived

Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening

Source Guo et al., 2024 · Hebei Medical University · 10.1007/s12035-023-03836-4

👤 Ruiyun Guo, Yimeng Chen, Jinyu Zhang, Zijing Zhou, Baofeng Feng, Xiaofeng Du, Xin Liu, Jun Ma, Huixian Cui ⏱ 180 days 📋 7 phases 🧫 Human iPSC

Abstract

This protocol generates wild-type and C9orf72-knockdown human iPSCs, differentiates them into neural stem cells, astrocytes, and motor neurons, and develops three-dimensional spinal cord organoids (SCOs) that recapitulate spinal cord development. The SCOs model amyotrophic lateral sclerosis (ALS) caused by C9orf72 mutation and enable investigation of neuroinflammatory pathways.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

46 steps across 7 phases

Generation of WT-iPSCs from healthy donor fibroblasts Day 0 to Day 27
  1. 1 Fibroblast isolation and culture
  2. 2 Reprogramming fibroblasts to iPSCs using Sendai virus
  3. 3 Selection and expansion of single iPSC colonies
  4. 4 Mycoplasma detection
  5. 5 Karyotype analysis
  6. 6 Immunofluorescence staining for pluripotency markers
  7. 7 Flow cytometry for pluripotency marker validation
  8. 8 Trilineage differentiation to verify pluripotency
C9orf72 knockdown in iPSCs via lentiviral transfection Day 28 to Day 60
  1. 1 Design and construct C9orf72-targeting shRNA lentiviral vectors
  2. 2 Lentiviral transfection of WT-iPSCs
  3. 3 Assess transfection efficiency and select stable clones
  4. 4 Validate C9orf72 knockdown by qRT-PCR
  5. 5 Validate C9orf72 knockdown by Western blot
  6. 6 Confirm pluripotency of C9-knockdown iPSCs
Differentiation of iPSCs into neural stem cells (NSCs) Day 61 to Day 100
  1. 1 Neural induction of iPSCs to NSCs
  2. 2 NSC expansion
  3. 3 Assess NSC proliferation
  4. 4 Validate NSC identity by immunofluorescence
Differentiation of NSCs into astrocytes Day 101 to Day 180
  1. 1 Astrocyte induction from NSCs
  2. 2 Astrocyte maturation
  3. 3 Validate astrocyte differentiation by immunofluorescence
  4. 4 Confirm C9orf72 knockdown in mature astrocytes
  5. 5 Assess astrocyte proliferation
Differentiation of iPSCs into motor neurons Day 0 to Day 28
  1. 1 Preparation of motor neuron differentiation medium
  2. 2 Initial neural differentiation (Day 0–6)
  3. 3 Neuroepithelial progenitor (NEP) differentiation (Day 6–7)
  4. 4 OLIG2+ motor neuron progenitor (MNP) differentiation (Day 7–13)
  5. 5 OLIG2+ MNP expansion (Day 13–19)
  6. 6 MNX1+ motor neuron differentiation (Day 19–25)
  7. 7 Mature CHAT+ motor neuron generation (Day 25–28)
  8. 8 Validate motor neuron differentiation by immunofluorescence
  9. 9 Confirm C9orf72 knockdown in mature motor neurons
Generation and characterization of spinal cord organoids (SCOs) Day 0 to Day 60
  1. 1 Initial aggregation in ultra-low attachment plates (Day 0–2)
  2. 2 Cluster expansion and neural induction (Day 2–10)
  3. 3 Matrigel solidification and initial culture (Day 10–14)
  4. 4 Transfer to spinner flask culture (Day 14–18)
  5. 5 Maturation and neurotropic factor addition (Day 18–60)
  6. 6 Cryosectioning of organoids for immunofluorescence
  7. 7 Immunofluorescence staining of cryosections
  8. 8 Confirm C9orf72 knockdown in mature SCOs
  9. 9 Optional: Assessment of SCO morphology and structure
RNA extraction, qRT-PCR, and inflammatory cytokine analysis Day 30–60 (sampling timepoint)
  1. 1 RNA extraction from cells and organoids
  2. 2 Reverse transcription to cDNA
  3. 3 Quantitative real-time RT-PCR (qRT-PCR)
  4. 4 Analyze qRT-PCR results
  5. 5 Statistical analysis and visualization

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Guo et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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