Home › Cell Biology › Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Cell Biology JoVE (Open Access) Citable · DOI

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

DOI: 10.3791/55702-v
What you'll learn
  • ✓ Differentiate hiPSCs into mixed neuron and glial cultures using embryoid body methods
  • ✓ Generate neuropithelial aggregates and dissociate them for neuronal maturation
  • ✓ Apply hiPSC-derived neurons/glia for neurotoxicity screening and drug testing
Protocol

Biopharma Insights Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Difficulty
advanced
Total time
~3–4 weeks (differentiation protocol from hiPSC expansion through mature mixed culture)
Model organism
Human induced pluripotent stem cells (hiPSCs)
Biosafety
BSL-1

Steps

1
Expand human induced pluripotent stem cells

Culture and passage hiPSCs to generate sufficient cell numbers for downstream differentiation. Maintain pluripotency and viability under standard stem cell conditions.

▶ 01:10
2
Generate embryoid bodies from hiPSCs

Initiate differentiation by forming embryoid bodies, the first step toward neural lineage commitment. Aggregate hiPSCs in suspension to induce spontaneous neural differentiation.

▶ 02:20
3
Form neuropithelial aggregates from embryoid bodies

Culture embryoid bodies under conditions that promote neural tissue organization. Generate neuropithelial aggregates that will serve as precursors for neuron and glia differentiation.

▶ 03:41
4
Dissociate aggregates and differentiate into neurons

Mechanically or enzymatically dissociate neuropithelial aggregates into single cells. Culture dissociated cells under conditions promoting neuronal and glial differentiation and maturation.

▶ 04:53
5
Validate differentiation into neurons and glia

Assess the resulting mixed culture for neuronal and glial markers using immunofluorescence or flow cytometry. Confirm successful differentiation and characterize cell composition.

▶ 06:36
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