Myeloid progenitor cell differentiation and microglial cell maturation

iPSC cell culture and microglia differentiation

iPSC cell maintenance (6-well plate)

1. A human iPSC cell line from ATCC (KYOU-DXR0109B human iPSC cells, Cat#ACS-1023) (Takahashi K, et.al., 2007) was received and stored in a liquid N2 tank. 
2. Thaw Geltrex™ in the refrigerator (4°C) overnight or in the ice and aliquot out to 100ul/tube and saved in -20^oC. 
3. Dilute the thawed Geltrex™ to 150 μg/mL in cold DMEM: F12 (roughly 1:100) with ice-cold tips, tubes, and pipette, and mix well, immediately coat each well with 1.5 mL diluted Geltrex™. Swirl the plate gently to ensure that the entire well is evenly covered. Leave the coated plate at 37°C for one hour. The coated plates can be stored at 4^oC for 1 week. 
4. 30 Minutes Prior to handling Cells, prewarm Pluripotent Stem Cell medium mTeSR™1 (StemCell Technologies, # 85850) at RT for at least 30 minutes before adding to cells. 5uM ROCK inhibitor was added to the medium for the new thawed cells. You don’t need to add a ROCK inhibitor following the medium change.
5. Rapidly thaw the cells by placing the cryovial in a 37°C water bath and swirling gently. Remove the cryovial from the water bath when only a few ice crystals remain. Sterilize the cryovial by rinsing it with 70% ethanol. From this point on, all operations should be carried out under germ-free conditions.
6. Using a 1mL pipette, gently transfer the cell suspension to a 15mL conical tube. Slowly add 4 mL stem cell culture medium dropwise, to the conical tube. Use an additional 1 mL of media to rinse the cryovial and transfer the liquid to the 15mL conical tube. Shake the conical tube gently to mix the cells while adding media.
7. Gently pipette the cells up and down several times to mix thoroughly. Avoid breaking apart the aggregates into a single-cell suspension. Centrifuge the cells at 200 x g for 5 minutes at 4^oC.
8. Aspirate the supernatant and discard it. Gently tap on the bottom of the tube to loosen the cell pellet. Add 1.5 mL of stem cell culture medium supplemented with ROCK Inhibitor Y27632 to a final concentration of 5 μM to the tube. Gently resuspend the pellet by pipetting up and down 5-6 times with a 1 mL tip, maintaining the cell aggregates.
9. Aspirate the coating solution and discard; add 1.5 mL of iPS Cell medium, also with ROCK Inhibitor Y27632 at 5 μM, to 3 wells of a 6-well plate.
10. Seed 0.5 mL of cell aggregates into each well prepared in step 9. Incubate the culture at 37°C, 5% CO2, in an air-atmosphere incubator.
11. Change medium 100% every day, pass the cells every 4-5 days (80% confluent) at a split ratio of 1:4, or cryopreservation. 

iPSC passage (6-well plate)

1. Thaw 2 Geltrex-treated 6-well plates and incubate in the 37^oC incubator for 1 hour, then remove them and leave them in the hood for another 1 hour. 
2. Warm an aliquot of Stem Cell Dissociation Reagent working solution (ReleSR, StemCells) and DMEM/F12 medium to room temperature, then warm up the stem cell culture medium at RT and thaw the ROCK inhibitor.
3. Aspirate and discard the stem cell culture medium. Rinse the cells twice by adding and discarding 2 mL of DMEM: F12.
4. Add 1 mL of Stem Cell Dissociation Reagent working solution to the well. Incubate at 37°C for 5 minutes.
5. Aspirate the Stem Cell Dissociation Reagent, taking care not to dislodge the cells during manipulation. 
6. Add 2 mL of stem cell culture medium to each well and detach the cells by pipetting up and down 2 to 5 times with a 1 ml tip; take care not to over pipette the culture into a single-cell suspension, as single cells will not establish colonies after seeding.
7. Transfer the cell aggregates to a 15 mL conical tube. Add an additional 2 mL of stem cell culture medium to the well to collect any remaining cells. Transfer this rinse to the 15 mL conical tube containing the cell aggregates.
8. Centrifuge the cell aggregates at 200 x g for 5 minutes at 4^oC. Aspirate the supernatant and discard. 
9. Add 6 mL of stem cell culture medium with rock inhibitor. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 10 mL pipped, maintaining the small cell aggregates. Take care not to over pipette the culture into a single-cell suspension, as single cells will not establish colonies after seeding.
10. Add 1.5ml of stem cell culture medium with 5uM ROCK inhibitor to 2 Geltrex-coated plates, and transfer 0.5ml of the resuspended iPS cells to each well. 
11. Incubate the plates at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day. Passage the cells every 4 to 5 days (80% confluent).

iPSC Cryopreservation (for 6-well plate)

1. Thaw the Stem Cell Freezing Media (FreSR-S, StemCells) at 4^oC overnight. 
2. Put the Stem Cell Freezing Media (FreSR-S, StemCells) on ice. Decontaminate by spraying with 70% alcohol before using.
3. Cryopreserve stem cell colonies when the cell cultures are 60-80% confluent. Collect 1 6-well plate of the cells in the tube as described in the passage culture protocol (steps 2-8).
4. Add 6 mL of cold Stem Cell Freezing Media to the tube. Resuspend the pellet by pipetting up and down 5-6 times with a 10mL pipette, maintaining the cell aggregates.
5. Immediately transfer 1 mL of the cell suspensions into labeled cryovials and store them on the ice. 
6. Freeze the cells gradually at -20°C 2hrs, then at -80°C overnight. The cells should not be left at 80°C for more than 24 to 48 hours. Once at 80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.

Embryoid body (EB) Formation by Spinning

1. Embryoid body (EB) formation employed the Spin-EBs formation method performed in AggreWellsTM800 microwell culture plates (StemCell Technologies, Catalog # 34825) (Haenseler W. et al., 2017). 
2. Cultured hiPSC cells in a 6-well plate were washed with warm PBS once. After incubating the cells with 1 mL of TrypLE Express (Gibco by Life Technologies) for 5 min at 37^oC, the cells and TrypLE mix were harvested into a 15 ml tube (1 well of the cells) after being pipetted 2-5 times. 
3. The cells and TrypLE were mixed well into a single-cell suspension by pipetting up and down with a 5ml pipette, then diluted 1:10 with PBS. 
4. Cells were counted and spun down for 5 minutes with 200g at 4^oC.
5. Meanwhile, AggreWellsTM800 plates were first prepared by rinsing each AggreWell with PBS once and aspirating the PBS. 1 mL of mTeSRTM-1 spin-EB medium was added to the AggreWell (which did not yet contain cells). The AggreWell plate with medium but no cells was centrifuged at 3000 g for 2 min (ensuring the centrifuge was balanced) to remove microscopic air bubbles. 
6.  After hiPSC centrifugation, PBS was aspirated, and the cell pellet was tapped loose and resuspended in mTeSRTM-1 spin-EB medium following the cell count as 4x10⁶ /ml.
7. Transfer 1 mL of 4x10⁶ iPSC to each well of the prepared AggreWellsTM800 plate. 
8. The plate containing iPSC and 2 mL of spin-EB medium per well was centrifuged at 800 rpm for 3 minutes. The plate was examined under the microscope to verify cells were evenly distributed among the micro-wells. The plate was very gently put into the incubator and left for 4 days.
9. From day 5 to day 8, the EBs were fed daily with spin-EB medium (warm up at room temperature) by gently aspirating 1 mL medium out using a p1000 Gilson and very gently adding in1 mL fresh spin-EB medium in a drop-wise manner down the side of the well so the EBs were not washed out of the microwells. 
10. On day 8, check the EB formation under the microscope. When the EBs are generated evenly in each microwell, it is the time to harvest. The EBs of the wells were pipetted up and down 5-10 times using a 5 mL serological pipette to dislodge the EBs from the micro-wells. 
11. Prepare the 40um cell strainer. Open a 40um cell strainer and insert it over a 50ml centrifuge tube. Wash the strainer once with 2 ml of PBS. 
12. The contents (EBs + medium) in the AggreWellsTM800 plate were transferred onto a 40um cell strainer over a 50 mL centrifuge tube. The cells in the wells were washed out 2-3 times with PBS and transferred onto the same strainer to collect all EBs and wash the EBs on the strainer. This way, the EBs were on top of the strainer, and the media/PBS passed through the strainer into the 50 mL collection tube. 
13. The strainer with the EBs balanced on top was carefully inverted over onto a new 50 mL centrifuge tube so that the EBs were now at the bottom of the strainer and could be collected into the new 50 mL tube by passing through differentiation media (contents of media described in next section). The strainer was held at an angle to facilitate the collection of EBs. EBs could be counted by transferring 50 uL EB mix to a well of a 96-well plate and using a light microscope.

spin-EB medium

1x mTeSRTM-1 medium

5 uM Rock-inhibitor

50 ng/ml BMP4

20 ng/ml SCF

50 ng/ml VEGF

Microglia cell progenitor cells differentiation from EBs

1. For differentiation, 60-100 EBs were transferred to a 75-cm² flask, 8-10 EBs/well were transferred into one well of a 6-well tissue culture plate in 3 mL medium, or 1 EB was transferred into one well of a 24-well tissue culture plate. Two-thirds of the medium was replaced every five days. 
2. Once microglia progenitor cells were visible in the supernatant of the cultures (from 2–4 weeks onwards), non-adherent progenitor cells were harvested by collecting the medium and floating cells another day or every 3 days. 
3. The collected floating cells and medium were transferred to the wells of a 6-well plate or another new 75-cm2 flask for continued culture for microglia cell differentiation. 12 ml of microglia progenitor culture medium was added to the EB culture flask. 
4. The floating microglia progenitor cells can be reattached to the well or flask. If the cell density is low, then continue to add the collected floating microglia progenitor cells to the same well or flask next time collection until it is around 60-70% confluent. 

Microglia progenitor culture medium

X-VIVOTM15 (Lonza, # BEBP04-744Q)

100 ng/mL M-CSF (Invitrogen)

25 ng/mL IL-3 (R&D)

2 mM glutamax (Invitrogen) or glutamine

Optional:

100 U/mL penicillin(option)

100 mg/mL streptomycin (Invitrogen) (option)

0.055 mM b-mercaptoethanol (Invitrogen). (option)

Microglia cell differentiation 

1. When the density of harvested floating cells in the 6-well plate or flask is enough, change the medium every 2 days and let the cells differentiate for 2 weeks. The cells will differentiate into completed differentiated microglia cells.  
2. The floating cells can also be cultured in slide chambers for differentiation and staining.
3. Some floating cells in the new flasks can divide and produce new floating microglial progenitor cells as the floating microglial progenitor cells' source. 

Microglia cell differentiation media 

50% conditional medium: The medium from EB culture flasks after microglia progenitor cell generation (filtered through 0.2um filter)

50% DMEM/F12 (gibco, #11330)

Plus:

2 mM Glutamax, 

50 ng/mL M-CSF (Invitrogen)

100 ng/mL IL-34 (Peprotech)

10 ng/mL TGFb1

2-5ng/ml TGFb2

20ng/ml CX3CL1

References

Takahashi K, et.al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861-872, 2007. PubMed: 18035408.

Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, Chintawar S, Schnell C, Antel JP, Allen ND, Cader MZ, Wade-Martins R, James WS, Cowley SA. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response. Stem Cell Reports. 2017 Jun 6;8(6):1727-1742. doi: 10.1016/j.stemcr.2017.05.017. PMID: 28591653; PMCID: PMC5470330.