TITLE: Cortical spheroid differentiation
AUTHORS: Annika Martin, Hanqin Li, Dirk Hockemeyer

[DESCRIPTION]
This protocol describes the procedure of differentiating hPSCs into early cortical spheroids following an adaptation of a published protocol (Yoon et al 2019, Paşca et al. 2015).

Protocol overview
A. Media Formulations
B. Growth and Maintenance of Human Cortical Spheroids 

Initial notes
A list of reagents and relevant vendor information can be found in the table listed under the materials tab.

[STEPS]
SECTION: Media Formulations
1. hESC KSR Media (500 ml)
     400 ml DMEM/F12
     100 ml KSR
     5 ml Pen-strep
     5 ml NEAA
     5 ml Glutamax
     5 mL HEPES buffer

hESC Wash Media (500 ml)
     500 ml DMEM
     25mL ml Calf Serum
     5 ml Pen-strep

hCS media (500 ml)
     500 ml Neurobasal media
     10 ml B27 (minus vitamin A)
     5 ml pen-strep
     5 ml Glutamax
     5 mL HEPES buffer

Neural precursor expansion media
     50 ml hCS media
     40 µl 25 ug/ml FGF (final concentration, 20 ng/ml)
     25 µl 40 µg/ml EGF (final concentration, 20 ng/ml)

Neural induction media
     50 ml hESC KSR Media 
     50 µl 10 mM SB431542 (final concentration, 10 µM)
     25 µl 10 mM dorsomorphin (final concentration, 5 µM)

Cortical differentiation media 
     50 ml hCS media 
     50 µl 20 µg/ml BDNF (final concentration, 20 ng/ml)
     50 µl 20 µg/ml NT-3 (final concentration, 20 ng/ml)

Resuspend Dorsomorphin in DMSO at a concentration of 10 mM (1:2000)
Resuspend SB431542 in DMSO at a concentration of 10 mM (1:1000)
Resuspend Rock Inhibitor in DMSO at a concentration of 10 mM (1:1000)
Resuspend BDNF in water at a concentration of 20 µg/ml (1:1000)
Resuspend NT3 in water a concentration of 20 µg/ml (1:1000)
Resuspend FGF at a concentration of 25 µg/ml (1:1250)
Resuspend EGF at a concentration of 40 µg/ml  (1:2000)

SECTION: Growth and Maintenance of Human Cortical Spheroids
2. Grow stem cells feeder-free on 6-well plates as described in (dx.doi.org/10.17504/protocols.io.b4mcqu2w).
SECTION: Growth and Maintenance of Human Cortical Spheroids
3. On day -1 of differentiation remove hESC Media and feed with hESC KSR Media + 10uM Rock Inhibitor (1:1000).
SECTION: Growth and Maintenance of Human Cortical Spheroids
4. Pre-coat an aggrewell plate according to manufacturer instructions.
SECTION: Growth and Maintenance of Human Cortical Spheroids
5. On day 0 of differentiation, aspirate media and add 1 mL of PBS- onto each well.
SECTION: Growth and Maintenance of Human Cortical Spheroids
6. Immediately Remove PBS- and add 1 mLof Accutase (1:3 Diluted with PBS-) onto each well of the 6 well plate.

SECTION: Growth and Maintenance of Human Cortical Spheroids
7. Return plate to incubator for ~30 min or until cells are dissociated into single cell suspension.

SECTION: Growth and Maintenance of Human Cortical Spheroids
8. Remove plate from incubator. Pool the cells into a 15mL conical tube per plate (6mL total volume).
SECTION: Growth and Maintenance of Human Cortical Spheroids
9. Using 6 mL hESC Wash media, rinse the wells and dilute the cell-Accutase suspension.
SECTION: Growth and Maintenance of Human Cortical Spheroids
10. Spin down cells for 5 min at 200 x g to 300 x g.

SECTION: Growth and Maintenance of Human Cortical Spheroids
11. Remove the supernatant from the falcon tube using a sterile glass pipette. Be careful not to aspirate any of the cells.
SECTION: Growth and Maintenance of Human Cortical Spheroids
12. Resuspend the cells in 5 mL of hESC KSR Media + 10uM Rock Inhibitor (1:1000).
SECTION: Growth and Maintenance of Human Cortical Spheroids
13. Using a 5mL serological pipette, filter cells through a 40 µm cell strainer into a new 50 ml conical tube.
SECTION: Growth and Maintenance of Human Cortical Spheroids
14. Take two sets of 10 µL of cell suspension. Mix each set with 10 μl trypan blue dye, which comes with the Countess Cell Counting Chamber Slides.

SECTION: Growth and Maintenance of Human Cortical Spheroids
15. Count cells with Countess automated cell counter or hemocytometer, averaging the counts from the two sets.
SECTION: Growth and Maintenance of Human Cortical Spheroids
16. Resuspend the cells to a concentration of 18 million cells per 5 mL of media (3.6 million cells per mL) in hESC KSR Media + 10µM Rock Inhibitor (1:1000).
SECTION: Growth and Maintenance of Human Cortical Spheroids
16.1. If needed, combine tubes from multiple plates and re-concentrate by spinning down at 200 x g to 300 x g and resuspending in an appropriate volume of hESC Media + Rock Inhibitor 1:1000.

SECTION: Growth and Maintenance of Human Cortical Spheroids
17. Transfer 5 mLof suspension to one well of a pre-coated 6-well Aggrewell 800 plate. Return plate to the incubator.

SECTION: Growth and Maintenance of Human Cortical Spheroids
18. The next day (Day 1), prepare neural induction media.
SECTION: Growth and Maintenance of Human Cortical Spheroids
19. Remove the aggrewell plate from the incubator and use a serological pipette to transfer the newly formed Embryoid Bodies (EBs) to a 15 ml conical tube.
SECTION: Growth and Maintenance of Human Cortical Spheroids
20. Allow EBs to settle (1-2 mins, but check before aspirating to ensure EB retention) and aspirate off media, taking special care to not suck up the EBs.
SECTION: Growth and Maintenance of Human Cortical Spheroids
21. Resuspend in 5 ml of neural induction media and deposit into one well of a 6-well ultra-low attachment plate.
SECTION: Growth and Maintenance of Human Cortical Spheroids
22. Repeat steps 17-20 to change media each day until Day 6.
SECTION: Growth and Maintenance of Human Cortical Spheroids
23. On Day 6, remove cell suspension from the plate and deposit into a 15 ml falcon tube
SECTION: Growth and Maintenance of Human Cortical Spheroids
24. Allow spheroids to settle to the bottom of the tube (~1-2 mins).
SECTION: Growth and Maintenance of Human Cortical Spheroids
25. While settling, make neural precursor expansion media.
SECTION: Growth and Maintenance of Human Cortical Spheroids
26. Remove the supernatant from the settled spheroid suspension.
SECTION: Growth and Maintenance of Human Cortical Spheroids
27. Resuspend spheroids in neural precursor expansion media.
SECTION: Growth and Maintenance of Human Cortical Spheroids
28. Return to the Ultra-Low Attachment plate and return the plate to the incubator.
SECTION: Growth and Maintenance of Human Cortical Spheroids
29. Repeat steps 22-27 to feed cells every day until Day 16 and then every other day until Day 25
SECTION: Growth and Maintenance of Human Cortical Spheroids
30. On day 25, prepare Cortical differentiation media.
SECTION: Growth and Maintenance of Human Cortical Spheroids
31. Remove cell suspension from the plate and deposit into a 15 ml falcon tube.
SECTION: Growth and Maintenance of Human Cortical Spheroids
32. Allow spheroids to settle to the bottom of the tube (~1-2 mins).
SECTION: Growth and Maintenance of Human Cortical Spheroids
33. Remove the supernatant from the settled cell suspension.
SECTION: Growth and Maintenance of Human Cortical Spheroids
34. Resuspend cells in Cortical differentiation media.
SECTION: Growth and Maintenance of Human Cortical Spheroids
35. Return to the Ultra-Low Attachment plate and return the plate to the incubator.
SECTION: Growth and Maintenance of Human Cortical Spheroids
36. Repeat steps 30-34 every four days (d29, 33, 37, 41) day until Day 43
SECTION: Growth and Maintenance of Human Cortical Spheroids
37. On day 43, change the media as above, but replace with only hCS media with no additives.
SECTION: Growth and Maintenance of Human Cortical Spheroids
38. Change media by sedimenting the spheroids, removing the supernatant, resuspending in hCS Media, and returning to the Ultra-Low Attachment plate every four days until the termination of the experiment.