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P-001-029
Motor Neurons from Human and Mouse Pluripotent
Stem Cells
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This protocol was generated by an AI system based on available research data and should be carefully reviewed by experienced researchers before implementation. Adjustments may be necessary to optimize results for specific experimental conditions.
Always cross-check with published literature and laboratory best practices.
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This protocol details the step-by-step procedure for the efficient differentiation of motor neurons (MNs) from human induced pluripotent stem cells (iPSCs) and mouse embryonic stem cells (ESCs). It incorporates small-molecule-based neural induction, optimized caudalization and ventralization, co-culture strategies, functional validation assays, and mechanical stress testing.
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Materials & Reagents:
1. Cell Culture & Differentiation Reagents
Human iPSCs (e.g., H9, SB-AD3–1)
Mouse ESCs (e.g., HM1 line)
Matrigel (Corning)
mTeSR1 medium (STEMCELL Technologies)
Neural Induction Medium (NIM):
KnockOut DMEM/F12 (Gibco)
Neurobasal Medium (Gibco)
B-27 Supplement (Gibco)
N-2 Supplement (Gibco)
Non-essential amino acids (NEAA)
GlutaMAX
L-ascorbic acid
Dual-SMAD inhibitors: SB431542 (TGFβ inhibitor), Dorsomorphin (BMP inhibitor)
Wnt activator: CHIR-99021 (GSK-3β inhibitor)
Notch inhibitor: Compound E
2. Differentiation & Patterning Factors
Retinoic Acid (RA) (Sigma)
Smoothened Agonist (SAG) (Cayman Chemicals)
Sonic Hedgehog (SHH) Protein (R&D Systems)
FGF2 (Peprotech)
BDNF, GDNF, CNTF, NT-3 (Peprotech)
3. Co-Culture Reagents
Human astrocytes (ScienCell)
C2C12 mouse myotubes (ATCC)
4. Functional Assay Reagents
Calcium Imaging Dye (Oregon Green 488 BAPTA-2)
Patch-Clamp Electrophysiology Setup
RNA Extraction Kit & Library Prep for RNA-seq
5. Mechanical Stress Testing Setup
Custom-built Uniaxial Stretching Device
Flexible Silicone Culture Substrates
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Step-by-Step Protocol:
Step 1
Maintenance & Expansion of iPSCs / ESCs
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Maintain pluripotent stem cells in feeder-free culture before differentiation.
Coat culture dishes with Matrigel (diluted in KnockOut-DMEM) and incubate at 37°C for 4-12 hours.
Thaw and plate iPSCs/ESCs in mTeSR1 medium, supplementing with 1% Penicillin-Streptomycin (P/S) and RevitaCell for 24 hours.
Maintain cells at 37°C, 5% CO₂, changing the medium daily.
Passage cells at ~80% confluence using Accutase dissociation and replate at a density of 0.5 × 10⁶ cells/well in a 6-well plate.
Step 2
Neural Induction (Days 0-6)
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Convert iPSCs/ESCs into neuromesodermal progenitors (NMPs) using small-molecule inhibitors.
Replace mTeSR1 with Neural Induction Medium (NIM) containing:
KnockOut DMEM/F12 + Neurobasal Medium (1:1)
B-27 + N-2 supplements
Dual-SMAD inhibitors: SB431542 (10 μM) + Dorsomorphin (2 μM)
Wnt activator: CHIR-99021 (3 μM)
Notch inhibitor: Compound E (1 μM)
Change the medium daily for 6 days, monitoring the formation of neural rosettes.
At Day 6, dissociate rosettes using Accutase and plate in fresh Neural Expansion Medium with 10 ng/mL FGF2 + 10 ng/mL EGF.
Step 3
Caudalization & Ventralization (Days 7-14)
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Direct NMPs towards motor neuron fate by modulating signaling pathways.
Replace media with Motor Neuron Induction Medium (MNIM) containing:
Retinoic Acid (RA) (10 μM) (gradual increase over 3 days)
Smoothened Agonist (SAG) (1 μM) instead of high SHH protein exposure
FGF2 (10 ng/mL) to enhance spinal motor neuron identity
Maintain for 7 days, changing the medium every 48 hours.
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Step 4
Motor Neuron Maturation (Days 15-21)
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Mature motor neurons with neurotrophic factors and co-culture approaches.
Replace media with Motor Neuron Maturation Medium, adding:
BDNF (10 ng/mL)
GDNF (10 ng/mL)
CNTF (10 ng/mL)
NT-3 (10 ng/mL)
Co-culture motor neurons with:
Human astrocytes for synaptic support.
C2C12 myotubes to promote neuromuscular junction formation.
Maintain for 7 days, refreshing media every 48 hours.
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Step 5
Functional Assays (Days 22+)
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Validate differentiation with electrophysiology and transcriptomic profiling.
Perform patch-clamp electrophysiology to measure neuronal activity.
Conduct calcium imaging to assess spontaneous network activity.
Extract RNA and perform RNA-seq to confirm MN identity.
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Step 6
Mechanical Stress Testing (Optional for ALS/Injury Models)
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Assess how motor neurons respond to mechanical strain.
Seed motor neurons on flexible silicone substrates.
Apply uniaxial stretch (25%, 45%, 75%) using a custom-built stretching device.
Measure calcium imaging & electrophysiology before and after strain application.
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