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Astrocytes constitute a large portion of the brain’s total cell population and play specialized roles in ion homeostasis, neuronal function, synaptic transmission, blood-brain barrier integrity, and injury response. Astrocytes arise later than neurons during in vivo development as well as during in vitro differentiation protocols and contribute significantly to neural circuit development¹. To model relevant neural cell-cell interactions in vitro, it is often advantageous to co-culture astrocytes with neurons. This protocol describes how complex intercellular interactions can be partially recapitulated in two-dimensional culture by separately deriving forebrain-type neurons and astrocytes from human pluripotent stem cells (hPSCs), then combining them into a co-culture.

Materials

• STEMdiff™ Forebrain Neuron Differentiation Kit (Catalog #08600)
• STEMdiff™ Forebrain Neuron Maturation Kit (Catalog #08605)
• STEMdiff™Astrocyte Differentiation Kit (Catalog #100-0013)
• STEMdiff™Astrocyte Maturation Kit (Catalog #100-0016)
• STEMdiff™ SMADi Neural Induction Kit (Catalog #08581) *✝
• Poly-L-ornithine hydrobromide (PLO) (Sigma P4957) *
• Laminin (Sigma L2020) *
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• Corning® Matrigel® hESC-Qualified Matrix (Corning 354277) ✝
• DMEM/F-12 with 15 mM HEPES (e.g. Catalog #36254) *✝
• Tissue culture-treated cultureware (e.g. Catalog #38015 and/or #38017)
• ACCUTASE™ (Catalog #07920)
• Trypan Blue (e.g. Catalog #07050)
• Hemocytometer
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* Required for forebrain-type neuron differentiation as outlined in the STEMdiff™ Forebrain Neuron Product Information Sheet.

✝ Required for astrocyte differentiation as outlined in the STEMdiff™ Astrocyte Product Information Sheet.

Protocol

Figure 1. Schematic for Co-Culture of hPSC-Derived Forebrain Neurons with Astrocytes

hPSCs are independently differentiated into forebrain-type neurons and astrocytes that are then combined under optimized co-culture conditions.

Part I: Differentiate hPSCs to Astrocytes

1. Follow the protocol outlined in the .
2. Continue the astrocyte maturation phase until astrocytes have been cultured in STEMdiff™ Astrocyte Maturation Medium for at least 3 weeks.
3. Verify successful astrocyte differentiation by performing immunocytochemistry on a subset of cultured cells:
   
   
   Note: With conventional use of the STEMdiff™ astrocyte system, the cell population should be > 70% S100ꞵ⁺, > 60% GFAP⁺, and < 15% positive for ꞵIII-tubulin or doublecortin (DCX).

Part II: Differentiate hPSCs to Forebrain Neurons

1. Follow the protocol outlined in the .
   
   
   Note: When seeding neuronal precursors into STEMdiff™ Forebrain Neuron Maturation Medium (section C, step 1 of the PIS), the suggested density ranges from 1.5 x 10⁴ - 6 x 10⁴ cells/cm², depending on the downstream application and intended culture length. The optimal density should be determined by the user.
2. Continue the forebrain neuron maturation phase until neurons have been cultured in STEMdiff™ Forebrain Neuron Maturation Medium for at least 1 week.
3. Verify successful forebrain neuron differentiation by performing immunocytochemistry on a subset of cultured cells:
   
   
   Note: With conventional use of the STEMdiff™ forebrain neuron system, the cell population should be > 90% positive for ꞵIII-tubulin and FOXG1, and < 10% GFAP⁺.

Part III: Set Up Neuron-Astrocyte Co-Culture

1. Dissociate mature astrocytes from Part I according to steps 1 - 5 of section C (Astrocyte Maturation) of the .
   
   
   Note: Resuspend the cells in an appropriate volume of complete STEMdiff™ Astrocyte Maturation Medium, and perform a cell count using Trypan Blue and a hemocytometer.
2. Dilute the cell suspension in additional complete STEMdiff™ Astrocyte Maturation Medium to obtain the required cell concentration and volume.
   
   
   Note: The concentration and volume of the astrocyte suspension should be optimized by the user and will depend on the desired astrocyte-to-neuron cell-type ratio, the number of culture wells used, and the initial cell density of forebrain neuronal precursors plated in Part II. The recommended astrocyte-to-neuron cell-type ratio ranges from 2:1 to 6:1.
3. Remove and discard the culture medium from the forebrain neurons generated in Part II.
4. Seed the resuspended astrocytes prepared in step 2 onto the forebrain neurons.
5. After 1 day, replace the medium with fresh STEMdiff™ Forebrain Neuron Maturation Medium.
6. Incubate the co-cultures at 37°C and 5% CO₂. Perform a full-medium change with STEMdiff™ Forebrain Neuron Maturation Medium every 2 - 3 days. Co-cultures can be maintained for at least 1 - 2 weeks prior to analysis.