Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

1. Cell Culture and Reagent Preparation

NOTE: The protocols in this section are written in the order in which they appear in the differentiation protocol (section 2). See the Table of Materials for materials and catalog numbers.

1. Prepare coated plates for cell culture.
   1. Dilute extracellular matrix (ECM) coating solution with DMEM/F12 media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of 3 mL each and immediately store in -20 °C. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringing the total volume to 36 mL for a concentration of 80 µg/mL.
   2. Coat 1 mL per well of a 6-well cell culture plate with ECM working solution. Add an additional 1 mL DMEM/F12 per well (if necessary) to ensure that the surface of the well is fully covered. Allow the coated 6-well cell culture plates to sit at room temperature for at least 1 h before use.
      NOTE: Coated plates may be stored in the incubator or at 4 °C for up to 2 weeks.
2. Prepare media formulations, growth factors, and small molecules.
   1. To prepare 500 mL of human pluripotent stem cell (hPSC) medium²⁷, add 20x and 500x supplements according to manufacturers’ instructions.
   2. To prepare 500 mL of neural medium (NM), add heparin to a final concentration of 2 mg/mL, 1x of antibiotic/antimycotic solution, 1x B27 supplement or 1x N2 supplement to a 500 mL bottle of DMEM/F12 with L-glutamine supplement as previously detailed²⁸.
   3. To prepare a 10 mM (1,000x) stock solution of Rho-Kinase Inhibitor Y27632 (Y), add 10 mg of powder into 3 mL of phosphate buffered saline (PBS). Filter sterilize, aliquot, and store at -20 °C. Use at a 10 µM working concentration in media.
   4. To prepare a 20 mM (10,000x) stock solution of SB431542, add 10 mg of powder into 1.3 mL of dimethyl sulfoxide (DMSO). To prepare a 2 mM (1,000x) stock solution of DMH1, add 10 mg of powder into 13.1 mL of DMSO. Filter sterilize, aliquot, and store each solution at -20 °C. Use each at a 2 µM working concentration in media (NM + SB431542 + DMH1).
      NOTE: This protocol recommends 2 µM working concentrations of both SB431542 and DMH1 based upon our observations and reports from others²⁹ stating that this concentration effectively promotes neural induction from hPSCs. Higher concentrations have also been reported³⁰. Additionally, with alternative media, it has also been shown that small molecules are not necessary for high neural conversion³¹. Thus, working concentrations will vary depending on alternative cell culture environments, and optimal concentrations should be tested by individual researchers.
   5. To prepare individual 100 µg/mL (10,000x) stock solutions of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2), add 1 mg of each into 10 mL of PBS + 0.1% BSA. Aliquot EGF and FGF2 stock solutions into 50 µL aliquots and store at -80 °C. Use each at a 10 ng/mL working concentration in media; e.g., add 5 µL EGF and 5 µL FGF2 to 50 mL of NM (NM + EGF + FGF2).
   6. To prepare a stock solution of doxycycline hydrochloride (Dox), first dissolve powder in DMSO to 100 mg/mL and store at -20 °C. Further dilute it to make a 2 mg/mL (1,000x) stock solution in PBS and store at -20 °C. Use at 2 µg/mL working concentration in media; e.g., add 50 µL Dox to 50 mL of NM (NM + Dox).
      Note: Do not re-freeze solutions after thawing.

2. Generation of Neural Subtypes from Human Induced Pluripotent Cells (hPSCs)

NOTE: All cell cultures should be maintained in an incubator with 5% CO₂ at 37 °C. These cultures are maintained at room oxygen levels, though lower levels may be utilized.

1. Generate and maintain astrocyte progenitors (hAstros) from hPSCs.
   NOTE: This section provides a brief and simplified differentiation protocol. For a detailed description (with embryoid body formation, rosette selection and regional patterning steps) refer to the previously described protocol²⁸ (Figure 1A, dotted green box).
   1. Seed clusters of hPSCs (Figure 1B) onto ECM-coated 6-well plates (see step 1.1) with 2 mL of hPSC medium + Y per well. Feed the stem cells every day until they are about 50% confluent and split as needed.
      1. To split hPSCs, add 1 mL of passaging reagent to wells and aspirate after 1 min. Incubate cells at room temperature for 5 min, add 1 mL of hPSC medium, and split aggregates 1:10 onto new ECM-coated wells in 2 mL of hPSC medium + Y per well.
   2. Maintain the stem cells until they are about 50% confluent, then change media to NM + SB431542 + DMH1 to induce neural differentiation (day 0). When cells are about 95% confluent, split 1:6 into new ECM-coated wells in the same medium.
   3. On day 14, dissociate the cells with detachment solution and transfer to a non-coated flask with Y to promote formation of aggregates.
      NOTE: The addition of Y is utilized to promote cell survival and sphere formation but is not included during every feed.
   4. For the generation of neuronal progenitors and neurons (hNeurons), use these day-14 cells as described below. Alternatively, utilize these cells at later time points from monolayer or sphere cultures before neuronal maturation occurs or gliogenesis commences.
      NOTE: By default, this protocol produces dorsal-cortical astrocytes. However, astrocyte subtypes can be regionally specified by the addition of patterning morphogens if desired^28,32,33. Retinoic acid (RA) may be added to caudalize cells into spinal cord phenotypes, while sonic hedgehog (SHH), smoothened agonist (SAG), or purmorphamine will produce ventral phenotypes.
   5. For the generation of astrocyte progenitors and astrocytes in spontaneously-formed 3D aggregates (hAstrospheres), switch to NM + EGF + FGF2 and feed weekly (or as needed to ensure stable pH) as previously detailed³⁴.
      1. Gently dissociate hAstro aggregates with detachment solution when dark centers appear and remove spheres that spontaneously attach. Break spheres gently once a week to maintain sphere health and to avoid necrotic cores.
         NOTE: Do not exceed 5 min of treatment with detachment solution.
      2. After 4-6 months of expansion, confirm cell identity and either freeze in cryopreservation medium (according to manufacturer’s instructions) or alternative storage conditions for long-term preservation in liquid nitrogen, or use immediately for experimentation.
   6. To thaw a frozen stock of hAstrospheres, quickly thaw a vial at room temperature, transfer the contents to an empty 15 mL conical tube, and centrifuge at 300 x g for 1 min. Carefully aspirate supernatant, wash with DMEM/F12, and repeat for a total of two wash steps. Add to a T25 flask with a total volume of 6 mL of NM + EGF + FGF2 + Y (or scale up as needed).
2. Generate and maintain inducible neurons (iNeurons) from hPSCs.
   1. Seed transgenic hPSCs (with a stable doxycycline-inducible neurogenin 2 transgene³⁵) on ECM-coated 6-well plates with 2 mL of hPSC medium + Y per well (as described above for non-transgenic lines). Feed everyday with 2 mL of media per well until they are ready to lift at 70% confluency. Split cells as described in step 2.1.2.
   2. When cells are about 35% confluent, add NM + Dox to induce differentiation into iNeurons. Maintain as a monolayer culture with NM + Dox for 2 days before proceeding to step 3.2.
      NOTE: Cells will transition into neuronal progenitors but will not yet extend neurites (Figure 1C).

3. Preparation and Maintenance of 3D Sphere Cocultures

1. Dissociate hAstros from 3D aggregates in suspension (as described in step 2.1.5.1).
2. Dissociate hNeurons or iNeurons from a 2D monolayer.
   1. Remove media from 6-well plate and add 500 µL of detachment solution to each well containing monolayer cultures. Incubate at 37 °C for 5 min. Gently add 2-3 mL DMEM/F12 to remove attached cells.
   2. Collect cells and media in a 15 mL conical tube and centrifuge at 300 x g for 1 min. Aspirate the supernatant, add 1 mL of fresh media, and pipette up and down gently with a 1,000 µL micropipette to achieve a single cell suspension.
3. Form 3D spheres using microwell culture plates.
   1. Prepare plate by adding 0.5 mL of anti-adherence rinsing solution to each well of the microwell plate. Centrifuge plate at 2,000 x g for 5 min. Aspirate rinsing solution, add 1 mL of DMEM/F12 to each well to wash, and aspirate again.
      NOTE: Always ensure that the centrifuge is balanced during use.
   2. Count each cell type using a hemocytometer or automated cell counter. Add desired ratio of dissociated hAstros and hNeurons or iNeurons to each well in a total volume of 2 mL of NM + Y (include Dox if iNeurons are utilized). Centrifuge plate at 100 x g for 3 min and return to the incubator.
      NOTE: 24-well microwell plates (see the Table of Materials) contain 300 microwells per well. Add a minimum of 6 x 10⁵ cells (for spheres of 2 x 10³ cells each) and a maximum of 6 x 10⁶ cells (for spheres of 2 x 10⁴ cells each) in 2 mL of media per well. For viable spheres of a specific density within this range, use a defined quantity of cells and divide by 300 to calculate the number of cells per sphere. Alternative sphere forming methods and tools may also be utilized if desired. Follow manufacturer’s instructions for acceptable ranges of cell densities.
   3. Allow cells to self-assemble into densely packed spheres within microwell plates over the next 2 days (Figure 2A). Replace 50% media if the culture is yellowing.
      NOTE: Exchange only half media and pipette gently when removing and adding media in order not to disturb spheres. Spheres may lift up from microwells and fuse together with higher force.
   4. After 2 days, gently remove spheres from microwells with a 1,000 µL micropipette (Figure 1D). Lightly apply force to the bottom of microwells with media to remove any additional adhered spheres. Let spheres settle in a 15 mL conical tube, aspirate the old media, and add fresh media.
4. Set up the spinner flask bioreactor system for forming 3D spheres.
   1. Clean the surface of a magnetic stir plate with 70% ethanol, place it into a cell culture incubator. Autoclave individual spinner flasks to sterilize.
   2. Add spheres with a minimum of 50–60 mL media to each spinner flask (Figure 1D, inset). Place the flask on the magnetic stir plate set at 60 rpm. Culture spheres in spinner flasks until they are ready for data collection.
      NOTE: A minimum of 3 weeks in culture is needed for synapse formation. If the spinner flask bioreactor system is not desired, spheres may be cultured in stationary conditions in cell culture flasks or dishes. Spheres may also be embedded in ECM or hydrogel.

4. Measurement of Live Synaptic Physiology with Multielectrode Arrays (MEAs)

1. Prepare MEAs for cell culture.
   1. Clean surface of each MEA with 1 mL of detergent for 1 h. Rinse with sterile deionized (DI) water, rinse with 70% ethanol to sterilize, and then air dry in biosafety cabinet under UV light.
      NOTE: MEAs can be stored in DI water at 4 °C under sterile conditions until use.
   2. Prepare a 0.5 mg/mL solution of poly-ornithine (PLO) by dissolving 50 mg of PLO into 100 mL of boric acid buffer. Coat the surface of each MEA with 1 mL of PLO to render the surface hydrophilic and incubate at 37 °C for a minimum of 4 h. Remove the PLO, wash the surface with DI water, add 1 mL of ECM (see step 1.1.1), and incubate at 37 °C for a minimum of 4 h.
   3. Remove ECM and place spheres in 1.5 mL of media on a MEA surface, ensuring that the spheres are positioned on top of the electrode array (Figures 3A, 3A’). Allow to adhere for 2 days. Change half of the media every 2–3 days (or more often if needed), ensuring that the spheres are not disturbed.
      NOTE: Addition of growth factors may improve culture survival and maturation.
2. Measure electrical activity of neural spheres.
   1. Set up a multielectrode array (MEA) system with a temperature-controlled headstage. Create a software program with a 5 Hz high-pass filter, and a 200 Hz low-pass filter, and a spike threshold of 5x standard deviation (SD).
   2. Place the MEA on headstage and record spontaneous electrical activity (Figure 3B, B’). Save raw data including spike frequency and amplitude for statistical analysis.
      NOTE: A perfusion system may be utilized with the MEA to add pharmacological agents or to increase flow of fresh media for long-term recording. Additional post-processing of spikes may be performed as desired^36,37.

5. Measurement of Synaptic Density with Immunocytochemistry

1. Prepare the reagents.
   1. To make a 500 mL stock solution of 4% paraformaldehyde (PFA), add 400 mL of 1x PBS to a glass beaker or a stir plate in a ventilated hood. Add stir bar and heat to 60 °C while stirring; do not boil. Add 20 g of PFA powder to the heated PBS solution and raise the pH to 6.9 by slowly adding 1 N NaOH dropwise.
      NOTE: 4% PFA solution can be aliquoted and stored at 4 °C for up to one month.
   2. Add 20 g or 30 g of sucrose to 100 mL of PBS to make 20% and 30% solutions of sucrose, respectively.
   3. Add 1 mL of detergent to 10 mL of PBS to prepare a 10% stock solution. Invert several times to homogenize.
   4. Add 250 µL of 10% detergent stock solution (final concentration of 0.25%) and 500 µL each of goat and donkey serums (final concentration of 5% each) to 10 mL PBS to prepare the primary blocking buffer.
   5. Add 100 µL each of goat and donkey serums (final concentration of 1% each) to 10 mL PBS to prepare the secondary blocking buffer.
2. Prepare the samples.
   1. Transfer the spheres into a 15 mL conical tube, allow them to settle at the bottom of the tube, and aspirate the old media. Rinse spheres with 500 µL of PBS, let settle, and aspirate PBS. Add 500 µL of 4% PFA (or enough to cover spheres) and incubate at 4 °C for 30 min.
   2. Aspirate the PFA and gently wash twice with PBS. Add 20% sucrose solution and incubate at 4 °C for several hours or overnight. Carefully aspirate, add 30% sucrose solution, and incubate at 4 °C for several hours or overnight.
      NOTE: Samples may be stored at 4 °C in either PBS or sucrose for up to 1 week before proceeding to the next step. If not slicing, maintain as 3D spheres (Figure 4A-C) in a 1.5 mL tube and proceed to step 5.3.
   3. If slicing the spheres, carefully transfer spheres to an embedding cryogenic mold on top of dry ice. Aspirate the 30% sucrose solution and slowly pour tissue embedding solution into the mold until it fully covers the sample, avoiding air bubbles. Wait until solution freezes and store at -80 °C until cryostat slicing.
   4. Using a cryostat at -20 °C, slice the embedded blocks into 30 µm sections (or as desired) and transfer to glass slides. Store at -80 °C until ready to stain.
3. Immunostain the spheres.
   1. Outline the edges of the slides with a hydrophobic PAP pen to prevent liquid spillage. Prepare primary and secondary blocking solutions with antibodies (see the Table of Materials). Add 500 µL of primary blocking buffer to each slide or tube and incubate at room temperature for 30 min.
   2. Remove the liquid, add 500 µL of fresh primary blocking buffer with diluted primary antibodies to each slide or tube, and incubate overnight at 4 °C. Remove primary antibody solution and wash 3x with PBS for 10 min each.
   3. Add 500 µL of secondary blocking buffer with diluted secondary antibodies to each slide or tube and incubate at 4 °C for 1 h. Remove secondary antibody solution and wash 3x with PBS for 10 min each.
      NOTE: A list of recommended antibodies is provided in the Table of Materials. Other antibodies or dyes may be used as desired. Do not expose fluorescent samples to light (step 5.3.3 onward).
   4. Add 50 µL of mounting solution dropwise to cover the surface and carefully place a coverslip over the slide, avoiding bubbles. Allow the slides to dry at room temperature for 24 h and store at 4 °C.
4. Image the slides.
   1. Use a confocal or two-photon fluorescent microscope with a 63X oil immersion objective to image pre- and post-synaptic protein abundance and colocalization within sphere slices (Figure 4D). Use a 20X or 40X objective to visualize morphological features of the hAstros.
   2. Open fluorescence image files with ImageJ software. Count pre- and post- synaptic puncta manually using the Cell Counter plug-in, using an unbiased counting method as previously detailed³⁸, or with alternative methods.