Background

Human pluripotent stem cells (hPSCs), including embryonic stem (ES) and induced pluripotent stem (iPS) cells, can undergo neural induction to generate neural progenitor cells (NPCs). Highly enriched cultures of CNS-type NPCs may be obtained through the use of standard monolayer culture or embryoid body (EB) protocols. Further differentiation of NPCs into neurons and glia provides a physiologically relevant model for neurological disease research, drug discovery and cell therapy validation. Many published protocols for neuronal differentiation use Brewer’s B27¹ supplement and/or Bottenstein’s N2² supplement with a basal medium of choice. While traditional neuronal basal media support cell survival, they impair neurological activities, including action potential generation and synaptic activity. BrainPhys™ was designed by Dr. Cedric Bardy in Dr.Fred H. Gage’s laboratory to better support in vitro neuronal function.³ In this technical bulletin, we describe a method (based on Yuan et al.⁴) for neuronal differentiation (Figure 1), using BrainPhys™ Neuronal Medium as a basal medium, supplemented with NeuroCult™ SM1, N2 Supplement-A and other factors. Using this protocol, forebrain-type neurons can be generated from NPCs in 2 - 4 weeks. Further culture in BrainPhys™ Neuronal Medium results in cultures that are phenotypically mature and, after 65 days in culture, synaptically active.

Results

When NPCs are cultured in BrainPhys™ Neuronal Medium with NeuroCult™ SM1 and N2 Supplement-A, a mixed population of forebrain-type neurons is generated. A 2 - 4 week culture period is required for the cells to differentiate and for neuronal morphology to become apparent (Figure 2A). Further maturation under the same conditions results in neurons showing high expression of the pre-synaptic marker Synapsin 1 (Figure 2B). After 65 days of maturation, neurons show increased frequency and amplitude of spontaneous excitatory and inhibitory channel currents, compared to cultures differentiated using a traditional basal medium (see data in Document #27010). To evaluate neuronal differentiation, the markers Class III β-Tubulin, Microtubule-Associated Protein2 (MAP2), Gamma Aminobutyric Acid (GABA), etc. can be used.

Protocol: Neuronal Differentiation of hPSC-Derived Neural Progenitor Cells

This protocol describes a method for neuronal differentiation of NPCs derived from H9 hPSCs.⁴ Some protocol optimization may be required for cells derived from other cell lines.

Generation of Neural Progenitor Cells

Generate NPCs using the EB protocol. For complete instructions, refer to the Technical Manual: Generation and Culture of Neural Progenitor Cells using the STEMdiff™ Neural System (Document #28782). After the first single-cell passage onto poly-L-ornithine/laminin-coated dishes (15 μg/mL and 10 μg/mL, respectively), culture NPCs in STEMdiff™ Neural Progenitor Medium for 1-3 days, until 25 - 50% confluent, replenishing the medium daily.

Procedure for Neuronal Differentiation

1. Prepare complete differentiation medium:
   • Basal medium: BrainPhys™ Neuronal Medium
   • Supplements (working concentrations)
   • 2% NeuroCult™ SM1
   • 1% N2 Supplement-A
   • 20 ng/mL BDNF
   • 20 ng/mL GDNF
   • 1 mM dibutyryl cAMP
   • 200 nM Ascorbic Acid
2. When cells have reached 25 - 50% confluency, add an equal volume of complete differentiation medium to the existing medium in each well.
3. Perform a half-medium change every 2 - 3 days by removing ~50% of the culture medium from each well and replacing with an equal volume of fresh complete differentiation medium. A 2 - 4 week culture period will be required for cells to differentiate and for mature neuronal morphology to become apparent.
4. Analyze for neuronal differentiation using markers such as Class III b-Tubulin, MAP2, GABA.

Additional Reagents

anti-Class III b-Tubulin Mouse IgG2a (Catalog #01409)

anti-MAP2 Rabbit IgG (Catalog #01410)

anti-GABA Rabbit IgG (Sigma-Aldrich; Catalog #A2052)

Important Notes

• If using the monolayer protocol for neural induction, cells should be moved into STEMdiff™ Neural Progenitor Medium at passage 3. Then, culture cells in complete STEMdiff™ Neural Progenitor Medium for 1 - 3 days at 37°C, until cells are 50% confluent, replenishing medium daily, and proceed with the neuronal differentiation protocol.
• The key optimization step is the seeding density of NPCs.

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