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Workflow Overview

Figure 1. Schematic of hPSC-Derived Forebrain Neuron and Astrocyte Co-Culture Using STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit

hPSCs are independently differentiated into forebrain-type neurons and astrocytes, which are then combined under optimized co-culture conditions. The schematic above provides an overview of this process and highlights three options for sourcing astrocytes. Astrocytes can be differentiated directly from hPSCs using STEMdiff™ Astrocyte Differentiation Kit.

Protocol

Part I. Obtaining hPSC-Derived Astrocytes

There are multiple options for generating or sourcing mature astrocytes:

Part II. Differentiating hPSCs into Forebrain Neurons

1. Follow the protocol outlined in the STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit Product Information Sheet.
2. Continue with the forebrain neuron maturation phase by culturing the cells in STEMdiff™ Forebrain Neuron Maturation Medium for a minimum of 1 week. To maintain culture purity during this phase, supplement the maturation medium with 2 - 3 µM Uridine and 5-Fluoro-2′-deoxyuridine (FDU/U), starting 48 hours after transitioning to the maturation medium (Day 7), followed by at least two additional feedings every 48 hours (i.e. on Day 9 and Day 11).
   Note: If using Uridine and 5-Fluoro-2′-deoxyuridine (FDU/U) to maintain the purity of the neuronal culture, FDU/U treatment must be terminated prior to introducing astrocytes into the culture. Continuing FDU/U treatment during co-culture may adversely affect astrocyte viability and growth.
3. Verify successful neuronal differentiation by performing immunocytochemistry on a subset of cultured cells.
   Note: When using STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit, the resulting cell population should be more than 90% positive for βIII-tubulin.

Part III. Establishing the Neuron-Astrocyte Co-Culture System

1. Dissociate the mature astrocytes from Part I by following Steps 1 - 5 of Section C (Astrocyte Maturation) in the STEMdiff™ Astrocyte Serum-Free Maturation Kit Product Information Sheet.
   Note: Resuspend the dissociated cells in an appropriate volume of complete STEMdiff™ Astrocyte Serum-Free Maturation Medium. Perform a cell count using Trypan Blue and a hemocytometer.
2. Dilute the cell suspension in additional complete STEMdiff™ Astrocyte Serum-Free Maturation Medium to achieve the desired cell concentration and volume.
   Note: The optimal astrocyte concentration must be empirically determined and depends on the desired astrocyte-to-neuron ratio, number of wells, and neuron density from Part II. A recommended starting ratio is 2:1 to 6:1 (astrocytes:neurons). For example, if forebrain neurons are derived from hPSCs seeded at 75,000 cells/cm², an astrocyte seeding density of 50,000 cells/cm² is recommended to achieve a 5:1 ratio at the time of co-culture setup (i.e. Day 0 in Figure 1). The estimated astrocyte-to-neuron ratio of approximately 5:1 is based on the number of astrocytes seeded and the expected neuronal yield when using STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit.
3. Remove and discard the culture medium from the forebrain neurons generated in Part II.
   Note: If using Uridine and 5-Fluoro-2′-deoxyuridine (FDU/U) to maintain the purity of the neuronal culture, FDU/U treatment must be terminated prior to introducing astrocytes into the culture. Continuing FDU/U treatment during co-culture may adversely affect astrocyte viability and growth.
4. Seed the prepared astrocyte suspension directly onto the neuron culture.
5. After 24 hours, replace complete STEMdiff™ Astrocyte Serum-Free Maturation Medium with fresh STEMdiff™ Forebrain Neuron Maturation Medium.
6. Incubate the co-cultures at 37°C and 5% CO₂, performing a full medium change every 2 - 3 days using STEMdiff™ Forebrain Neuron Maturation Medium.
7. The co-culture can be maintained for 7 - 14+ days prior to analysis.
   Note: The co-culture can be maintained for 7 - 14+ days if the goal is to perform short-term functional assays or assess co-culture quality by immunocytochemistry. For micro-electrode array (MEA) assays or studies requiring more mature networks, cultures can be maintained for 6 weeks. However, cell health and over-confluency should be closely monitored throughout the extended culture period.
8. Verify successful co-culture by performing immunocytochemistry. Stain for MAP2 (neuronal marker) and GFAP (astrocyte marker) to confirm cellular identity. The resulting co-culture should show:
   • MAP2⁺ neurons with well-branched dendrites
   • GFAP⁺ astrocytes displaying typical star-shaped morphology
   • Even distribution of cells with no signs of cell death