How to Tri-Culture Astrocytes, Microglia, and NGN2 mRNA-LNP-Induced Forebrain Neurons Derived from Human Pluripotent Stem Cells
The human brain is composed of neurons and glial cells, including astrocytes and microglia, that work together to support neural function and maintain homeostasis. In vitro tri-culture systems derived from human pluripotent stem cells (hPSCs) offer a powerful approach to model these interactions in a controlled and physiologically relevant context. Unlike primary tissue, hPSC-derived models enable the generation of pure populations of these various cell types from the same genetic background, allowing for more precise recombination than the mixed populations typically obtained from primary sources.
This step-by-step protocol outlines a method for generating and combining hPSC-derived forebrain neurons, astrocytes, and microglia, then establishing a two-dimensional tri-culture system. Forebrain neurons are generated using the STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit—which utilizes non-integrating NGN2 mRNA delivered via lipid nanoparticles (LNPs) for rapid and transcription factor (TF)-based neuronal induction. This model enables the study of assay development, disease mechanisms, neurotoxicity, and more, in a more physiologically relevant context, by supporting the study of neuronal–glial and neuroimmune interactions.
Materials and Reagents
- STEMdiffâ„¢-TF Forebrain Induced Neuron Differentiation Kit (Catalog #100-1678)
- STEMdiffâ„¢ Forebrain Neuron Maturation Kit (Catalog #08605)
- STEMdiffâ„¢ Astrocyte Differentiation Kit* (Catalog #100-0013)
- STEMdiffâ„¢ Astrocyte Serum-Free Maturation Kit (Catalog #100-1666)
- STEMdiffâ„¢ Hematopoietic Kit** (Catalog #05310)
- STEMdiffâ„¢ Microglia Differentiation Kit (Catalog #100-0019)
- BrainPhysâ„¢ hPSC Neuron Kit (Catalog #05795)
- mTeSRâ„¢ Plus (Catalog #100-0276)
* For researchers prioritizing efficiency, cryopreserved cells differentiated from the human iPSC control line, SCTi003-A (Catalog #200-0511), using STEMdiff™ kits can be used for significant time savings, as outlined below (see Figure 1). The neural induction of hPSCs to generate astrocytes can be skipped by using Human iPSC-Derived Neural Progenitor Cells (Catalog #200-0620). Alternatively, Human iPSC-Derived Astrocytes (Catalog #200-0980) can be directly thawed and incorporated into co-cultures. When paired with the STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit, these approaches allow for the rapid generation of high-purity, reproducible neuron–astrocyte co-cultures.
Alternatively, the generation of astrocytes from hPSCs using the standardized STEMdiffâ„¢ Astrocyte Differentiation Kit enables greater experimental flexibility, allowing customization based on specific genotypes of interest.
** Used to generate hematopoietic progenitor cells for downstream microglia differentiation as outlined in the STEMdiffâ„¢ Hematopoietic Kit Product Information Sheet and the STEMdiffâ„¢ Microglia Differentiation Kit Product Information Sheet.
Important Notes
- This protocol is optimized for use with hPSC maintenance reagents and has been validated across multiple hPSC lines. For upstream protocols and source materials, refer to the mTeSRâ„¢ Plus and the Product Information Sheet for the Healthy Control Human iPSC Line, Female, SCTi003-A, a well-characterized, high-quality human (induced pluripotent stem cell (iPSC) line available from º£½ÇÆƽâ°æ Technologies.
- The kits listed may require additional materials not included in this protocol. Please refer to the relevant Product Information Sheets for a complete list of required materials.
- To tri-culture hPSC-derived forebrain neurons, microglia, and astrocytes generated via directed differentiation, refer to this optimized protocol.
Workflow Overview
Figure 1. Schematic of hPSC-Derived Forebrain Neuron, Astrocyte, and Microglia Tri-Culture Using the STEMdiffâ„¢-TF Forebrain Induced Neuron Differentiation Kit
hPSCs are independently differentiated into forebrain-type neurons, astrocytes, and microglia, which are then combined under optimized tri-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 the STEMdiffâ„¢ Astrocyte Differentiation Kit, derived from cryopreserved neural progenitor cells (NPCs), or obtained as cryopreserved, ready-to-use astrocytes. Microglia are generated using the STEMdiffâ„¢ Hematopoietic Kit followed by the STEMdiffâ„¢ Microglia Differentiation Kit. To establish the tri-culture, matured astrocytes are first added to the forebrain neurons, followed by the addition of microglia. Proceed to the next section for the detailed protocol.
- Notes:
Before combining astrocytes and forebrain neurons (as shown on Day 0 in Figure 1), differentiated astrocytes should be maintained in STEMdiffâ„¢ Astrocyte Serum-Free Maturation Medium for a minimum of 3 weeks, while differentiated forebrain neurons should be cultured in STEMdiffâ„¢ Forebrain Neuron Maturation Medium for at least 1 week. The timeline in Figure 1 serves as a general guideline based on these recommended maturation periods. Depending on experimental goals, the duration of maturation for one or both cell types may be extended. Please consult the relevant Product Information Sheets for detailed protocol timelines prior to starting your experiment. Once established, co-cultures can be maintained for 7 - 14+ days before analysis. - Tri-cultures are typically maintained for 3 - 30 days, depending on downstream applications. To ensure optimal microglia viability, microglia should be used after the 24-day differentiation period (steps 1 - 8 in Section A of the STEMdiffâ„¢ Microglia Differentiation Kit Product Information Sheet).
Protocol
Part I. Obtaining hPSC-Derived Astrocytes
There are multiple options for generating or sourcing astrocytes:
A. Starting from hPSCs
B. Starting from Neural Progenitor Cells (NPCs)
C. Starting from Mature Astrocytes
Part II. Differentiating hPSCs into Forebrain Neurons
- Follow the protocol outlined in the STEMdiffâ„¢-TF Forebrain Induced Neuron Differentiation Kit Product Information Sheet.
- 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).
- Verify successful neuronal differentiation by performing immunocytochemistry on a subset of cultured cells.
Note: When using the STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit, the resulting cell population should be more than 90% positive for βIII-tubulin.
Part III. Differentiating hPSCs into Microglia
- Start with a high-quality hPSC line such as Healthy Control Human iPSC Line, Female, SCTi003-A (Catalog #200-0511) or Healthy Control Human iPSC Line, Male, SCTi004-A (Catalog #200-0769). Refer to this webpage for a range of other genotypes to start from.
- Generate hematopoietic progenitor cells (HPCs) using the complete protocol provided in the STEMdiffâ„¢ Hematopoietic Kit Product Information Sheet.
- Assess the efficiency of HPC differentiation using flow cytometry.
Note: When using the STEMdiffâ„¢ Hematopoietic Kit, the resulting cell population should yield more than 90% CD43+ cells and more than 20% co-expressing CD34 and CD45 cells.
- Continue with microglia differentiation by following steps 1 -8 in Section A of the STEMdiffâ„¢ Microglia Differentiation & Maturation Kit Product Information Sheet.
- Verify successful microglia differentiation by flow cytometry.
Note: When using STEMdiffâ„¢ Microglia Differentiation & Maturation Kits, the resulting cell population should rank for more than 80% positive for co-expression of CD45 and CD11b.
Part IV. Establishing the Initial Neuron-Astrocyte Co-Culture System
- Dissociate the mature astrocytes from Part I by following Steps 1 - 5 of Section C (Astrocyte Maturation) in the STEMdiffâ„¢ Astrocyte Product Information Sheet.
Note: Resuspend the dissociated cells in an appropriate volume of complete STEMdiffâ„¢ Astrocyte Serum-Free Maturation Medium. Perform a cell count using Trypan Blue and a hemocytometer.
- Dilute the cell suspension in additional complete STEMdiffâ„¢ 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 initially seeded at 75,000 cells/cm² the expected final density of forebrain neurons will be ~10,000 cells/cm². Therefore, an astrocyte seeding density of 50,000 cells/cm² is recommended to achieve a 5:1 ratio (astrocytes:neurons).
- Remove and discard the culture medium from the forebrain neurons generated in Part II.
- Seed the resuspended astrocytes onto the forebrain neurons and maintain overnight in the STEMdiffâ„¢ Astrocyte Serum-Free Maturation Kit. Proceed to Part V.
Part V. Establishing the Microglia-Forebrain Neuron-Astrocyte Tri-Culture System
- Prepare the required volume of BrainPhys™ Neuronal Medium with supplements by following the instructions in Section B, “Preparation of Complete Differentiation Medium,†of the BrainPhys™ Product information sheet.
Note: The volume needed will vary depending on the culture plate format and the intended duration of the tri-culture.
- Thaw STEMdiff™ Microglia Supplement 2 (Component #100-0023 from the STEMdiff™ Microglia Differentiation Kit) at room temperature (15 - 25°C) until just thawed, or alternatively overnight at 2 - 8°C. Mix thoroughly before use.
Notes:
- If not used immediately, aliquot and store the supplement at –20°C. Do not use past the expiration date indicated on the label.
- If an insufficient volume of STEMdiffâ„¢ Microglia Supplement 2 is available, contact techsupport@stemcell.com to purchase additional quantities.
- To prepare the optimized tri-culture medium, add STEMdiff™ Microglia Supplement 2 to BrainPhys™ Neuronal Medium + supplements at a final concentration of 1X (e.g. 40 µL supplement per 10 mL medium).
- Mix thoroughly.
Note: Prepared tri-culture medium may be stored at 2 - 8°C for up to 2 weeks if not used immediately.
- Collect microglia from Part III:
- Transfer the entire cell suspension into a 15 mL conical tube. For maximum recovery, perform additional washes with warm DMEM/F-12 containing 15 mM HEPES.
- Centrifuge at 300 × g for 5 minutes.
- Carefully aspirate the supernatant and resuspend the pellet in an appropriate volume of Tri-Culture Medium.
- Count cells using Trypan Blue and a hemocytometer.
- Dilute the microglia cell suspension in additional Tri-Culture Medium to obtain the required final cell concentration and volume.
Notes:
- As a general guideline, microglia should be seeded at approximately half the density of astrocytes. However, this ratio can be further adjusted depending on experimental objectives and the desired cellular composition of the tri-culture.
- Microglia are semi-adherent and may be inadvertently removed during medium changes. To minimize cell loss, centrifuge the culture plate in a swinging-bucket centrifuge equipped with plate adapters at 100 × g for 2 minutes prior to each half-medium change to promote cell settling.
- Carefully remove and discard the culture medium from the forebrain neuron-astrocyte co-cultures prepared in Part IV.
- Gently seed the resuspended microglia onto the existing co-culture of neurons and astrocytes.
- Place the plate in a 37°C, 5% CO2 incubator, and ensure even distribution of the cells by moving the plate in several quick, short motions—first back and forth, then side to side.
- Maintain the tri-culture by performing a half-medium change every 2 - 3 days using freshly prepared tri-culture medium.
Note: The tri-culture can be maintained for 3 - 10 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 4 weeks. However, cell health and over-confluency should be closely monitored throughout the extended culture period.
- Verify successful tri-culture by performing immunocytochemistry. Stain for MAP2 (neuronal marker), IBA1 (microglia marker), and GFAP (astrocyte marker) to confirm cellular identity.
- Neuronal marker: Anti-Human MAP2 Antibody, Polyclonal (Catalog #100-1342 or abcam, Catalog #ab5392)
- Microglia marker: Rabbit polyclonal anti-IBA1 (Synaptic Systems Catalog #234 003)
- Astrocyte marker: Chicken polyclonal anti-GFAP (Aves Labs Catalog #GFAP)
- Total cell density may be assessed by DAPI staining.
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