Home › Cell Biology › Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Cell Biology JoVE (Open Access) Citable · DOI

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

DOI: 10.3791/3273-v
What you'll learn
  • ✓ Derive human neuronal progenitors from pluripotent embryonic stem cells using small molecule induction
  • ✓ Apply retinoic acid-based neural differentiation in both adhesive and suspension culture systems
  • ✓ Generate mature neuronal phenotypes suitable for CNS repair applications
Protocol

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Difficulty
advanced
Total time
~7-10 days per differentiation protocol
Model organism
Human embryonic stem cells (hESCs)
Biosafety
BSL-1

Steps

1
Prepare media and coat culture plates

Prepare defined culture media and coat plates with appropriate adhesion factors to establish optimal conditions for hES cell maintenance and subsequent neural differentiation.

▶ 01:27
2
Passage and seed undifferentiated hES cells

Maintain pluripotent human embryonic stem cells under defined culture conditions and seed at appropriate density for subsequent neural induction.

▶ 02:33
3
Induce neural differentiation with retinoic acid

Apply retinoic acid treatment to hES cells in adhesive culture to initiate neural induction and promote neuroblast formation from pluripotent precursors.

▶ 04:31
4
Differentiate in suspension and mature neurons

Transfer retinoic acid-induced neural progenitors to suspension culture for continued differentiation, then reattach cultures to promote maturation into neuronal phenotypes.

▶ 05:42
5
Generate feeder-free neuronal cultures with RA

Perform direct neuronal differentiation of serum- and feeder-free pluripotent hES cells using retinoic acid induction to establish efficient derivation protocols.

▶ 08:08
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