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PRODUCT INTRODUCTION

Intended Use
This product is for research use only (RUO). Not for use in diagnostic or therapeutic procedures.

Important User Note
These cells exhibit biological characteristics associated with the obese donor phenotype, which may impact their growth, differentiation, and metabolic behavior compared to cells from lean donors. It is the user's responsibility to determine the suitability of this product for their specific application. Proper training in the handling of human-derived materials is required.

PRODUCT DESCRIPTION

Regeneration Biology offers these human spinal motor neurons (MNs) derived from a genetically engineered induced Pluripotent Stem Cell (iPSC) line carrying a homozygous M337V mutation in the TARDBP gene, which encodes the TDP-43 protein. This mutation is strongly associated with familial and sporadic Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). The cells are differentiated using a robust, floor-plate-directed protocol to generate a highly pure population of functional motor neurons.

This isogenic model provides a precise and powerful tool for studying TDP-43 proteinopathy, a pathological hallmark of nearly all ALS cases. These neurons robustly recapitulate key disease features, including cytoplasmic TDP-43 mislocalization, stress granule dynamics, increased sensitivity to proteotoxic stress, and altered mitochondrial function. They are indispensable for dissecting the mechanisms of TDP-43-mediated neurodegeneration and for high-content screening of targeted therapeutics.

KEY FEATURES & BENEFITS

• Precise Genetic Model: Features a homozygous, well-characterized ALS/FTD-associated point mutation (M337V) in the TARDBP gene for consistent, mechanism-driven research.

• Isogenic Control Available: A genetically corrected, wild-type control line (isogenic) is available, enabling definitive attribution of phenotypes directly to the TDP-43 mutation.

• Strong Disease Phenotype: Consistently exhibits cytoplasmic TDP-43 mislocalization, particularly under stress conditions, providing a robust readout for drug screening.

• High Purity & Functionality: Differentiated into a highly pure population of spinal motor neurons expressing key markers and capable of firing action potentials.

• Ready for Functional Assays: Cryopreserved at a post-mitotic stage, ready for recovery and use in high-content imaging, electrophysiology, and biochemical assays.

QUALITY CONTROL SPECIFICATIONS

Each lot is tested to meet the following release criteria:

Viability (post-thaw): ≥ 70% (Test Method: Live/Dead Staining)
Cell Yield per Vial: ≥ 2 million or 5 million viable cells (Test Method: Cell Count)
Purity (MN Marker Expression): ≥ 70% ISL1+/HB9+ (Test Method: Immunocytochemistry (ICC))
Neuronal Marker Expression: Positive: β-III-Tubulin (Tuj1), MAP2 (Test Method: ICC)
Genotype Verification: Confirmed Homozygous TARDBP c.1009A>G (Test Method: Sanger Sequencing)
TDP-43 Expression: Positive for Nuclear TDP-43 (basal state) (Test Method: ICC)
Pluripotency Marker Absence: Negative: Oct3/4 (Test Method: ICC)
Microbiological Sterility: No growth (Test Method: USP <71>)
Mycoplasma: Negative (Test Method: PCR Method)

APPLICATIONS

• TDP-43 Proteinopathy Studies: Investigate mechanisms of nuclear-to-cytoplasmic mislocalization, aggregation, and cleavage of TDP-43.

• High-Content Screening: Screen for compounds that reduce TDP-43 mislocalization, enhance autophagy/proteasomal clearance, or improve neuronal survival.

• RNA Metabolism & Stress Granules: Study altered RNA binding, splicing, and stress granule dynamics in a disease-relevant context.

• Electrophysiology: Characterize functional deficits in neuronal excitability and synaptic transmission using patch-clamp or MEA.

• Biochemical Pathway Analysis: Investigate nucleocytoplasmic transport defects, mitochondrial dysfunction, and ER stress.

CELL CULTURE INFORMATION

1. Materials Supplied

• Product: iMN-TDP43-M337V-HOM-RG66001 (Cryopreserved Vial)

• Components: One vial containing cryopreserved human iPSC-derived motor neurons in suspension.

2. Recommended Media & Reagents

• Complete Motor Neuron Maintenance Medium (Cat. No. M-4530):

  • Basal Medium: BrainPhys Neuronal Medium (recommended for electrophysiology) or Neurobasal Medium.

  • Essential Supplements: B-27 Supplement (2%), N-2 Supplement (1%).

  • Trophic Factors: BDNF (20 ng/mL), GDNF (20 ng/mL), CNTF (10 ng/mL).

  • Signaling Molecules: Ascorbic Acid (200 µM), db-cAMP (1 µM).

• Plating Medium: Complete Maintenance Medium for the first 24-48 hours post-thaw.

• Coating Substrate: Critical for success. iPSC-coated solutions are required.

Thawing and Plating Procedure

Pre-Culture Requirement: Pre-coat culture vessels for at least 2 hours at 37°C. Rinse with sterile PBS before plating.

1. Rapid Thaw: Thaw the vial in a 37°C water bath for 60-90 seconds until it has just thawed.

2. Sterilize and Transfer: Wipe the vial with 70% ethanol and transfer it to the biosafety cabinet.

3. Gentle Dilution: Gently transfer cell suspension to a 15 mL tube. Slowly add 5-9 mL of pre-warmed Plating Medium drop-wise over 2 minutes.

4. Centrifuge: Spin at 200 x g for 5 minutes.

5. Resuspend and Plate: Aspirate supernatant. Gently resuspend the pellet in fresh Plating Medium.

   • Recommended Seeding Density: 70,000 - 150,000 cells/cm². Higher densities may promote network survival and stronger TDP-43 phenotype expression.

6. Incubate: Plate cells onto pre-coated vessels and place in a humidified 37°C incubator with 5% CO₂.

7. Medium Change: After 24-48 hours, carefully perform a 50% medium change with fresh Complete Maintenance Medium. Feed cells every 2-3 days with a 50% medium change.

Maintenance & Phenotype Induction

• Maturation: Allow 10-21 days in culture for full maturation and robust expression of TDP-43-related phenotypes.

• Stress Induction: To consistently induce cytoplasmic TDP-43 mislocalization, treat cells with mild stressors after 14 days in culture (e.g., 50-100 µM Sodium Arsenite for 30-60 min, or 1 µM Staurosporine for 6-24 hours). Optimize conditions for your specific assay.

• Handling: These are delicate neurons. Avoid mechanical disruption. Use pre-warmed media for changes to minimize temperature shock.

GENETIC & DONOR BACKGROUND

• Gene: TARDBP

• Mutation: c.1009A>G (p. Met337Val)

• Zygosity: Homozygous

• Parental Line: The engineered line is based on a well-characterized, karyotypically normal human iPSC background.

• Isogenic Control: The availability of a genetically corrected, otherwise identical control line is critical for validating mutation-specific effects.

ORDERING INFORMATION

Essential Isogenic Control:

Required Reagents:

KEYWORDS

Human Motor Neurons, TDP-43 M337V, Homozygous mutation, TARDBP gene, TDP-43 gene, Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), Neurodegenerative disease, Motor neuron disease, Cytoplasmic accumulation, Aggregation of TDP-43, Nuclear Loss of TDP-43, Disease modeling, Biochemical analysis, Cell-based studies, Neurotoxicity, Oxidative stress, Synaptic function.