TITLE: Differentiation of hPSCs to hypothalamic neurons
AUTHORS: Cortina  Chen, Iman Mali, Florian T Merkle

[DESCRIPTION]
This protocol is about Differentiation of hPSCs to hypothalamic neurons.

[BEFORE_START]
Prepare Media and Reagents as described in section 'Materials'.

[STEPS]
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
1. Thaw an aliquot of 1:10 diluted Geltrex on ice or in the fridge. 
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
2. Dilute aliquot 1:10 in ice-cold DMEM/F12 to a final concentration of 1:100. 
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
3. To coat plates, add 1:100 diluted Geltrex to TC dish/plate and incubate for 60 min at 37 °C, or 60 min at 4 °C. 
 

SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
4. Aspirate Geltrex
 

SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
5. Add pre-warmed hPSC culture media: StemFlex with 10 micromolar (µM); 10 mL per 10 cm dish, 2 mL per well of 6 well plate.
 

SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
6. Take vial cells from liquid nitrogen to TC room on dry ice.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
7. Dip bottom half of vial into 37 °C water bath and swirl until partially thawed (approximately 1 min-3 min, depending on volume of freeze).
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
8. Thoroughly spray vial with 70 %, and complete thaw by gently transferring pre-warmed hPSC culture media into the partially thawed cells.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
9. Transfer cells into a 15 mL V-bottom polypropylene tube with pre-warmed wash media. Wash residual cells out of vial with 1 mL.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
10. Spin cells at 160 x g, 3 min.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
11. Aspirate media.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
12. Re-suspend the pellet with 10 mL and mix well by gently pipet it up and down.

SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
13. Spin at 160 x g, 3 min.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
14. Aspirate media.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
15. Re-suspend pellet with 1 mL. Dilute into appropriate volume depending on culture dishes/plates used.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
16. Add and evenly distribute cells into dish/plate with pre-warmed hPSC culture media (from step 5).
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
17. Transfer to incubator, cells should attach over a few hours.
SECTION: Thawing of human pluripotent stem cell (hPSC) lines:
18. Change media the following day to StemFlex without Rock inhibitor.
 

SECTION: Hypothalamic Differentiation
19. Coat 6-well or 10 cm plates with Geltrex for differentiation as described above.
SECTION: Hypothalamic Differentiation
20. Nearly confluent hPSCs are dissociated and re-plated for differentiation
 

SECTION: Hypothalamic Differentiation
21. Aspirate culture medium and briefly and gently wash cells in 37 Room temperature DPBS.
SECTION: Hypothalamic Differentiation
22. Add 37 °C TrypLE to cell culture, 1 mL per well in 6-well plate, 5 mL per 10 cm plate.
SECTION: Hypothalamic Differentiation
23. Incubate cell culture for 3 min-5 min at 37 °C. After a 3 minute incubation, check to see if cells are detaching. Under a phase contrast microscope, the cells should start to round up and take on a phase-bright appearance, but not spontaneously detach from the plate. Once cultures adopt this appearance, gently suck up and dispel ~100 µL with a P1000 pipette against the cells. They should easily dislodge and leave a small area devoid of cells. If cells do not dissociate easily, extend TrypLE digestion for another minute and repeat this test.
 

SECTION: Hypothalamic Differentiation
24. Gently aspirate TrypLE.
SECTION: Hypothalamic Differentiation
25. To dissociate cells, add 1 mL/5 mL for a well of a 6 well/10 cm plate, and gently pipette this medium over the plate to detach cells and dissociate them to a single-cell suspension.
SECTION: Hypothalamic Differentiation
26. Collect cells in 15 ml V-bottom polypropylene tube, and adjust volume with hPSC culture media (Total volume = 10 mL)
 

SECTION: Hypothalamic Differentiation
27. Spin at 160 x g for 3 min-5 min at 37 Room temperature. Aspirate supernatant, re-suspend cells in 10 mL.
SECTION: Hypothalamic Differentiation
28. Spin at 160 x g for 3 min-5 min at 37 Room temperature. Aspirate supernatant, re-suspend cells in hPSC culture media
 

SECTION: Hypothalamic Differentiation
29. After re-suspending the cell pellet, adjust volume so that the suspension is visibly turbid, but not milky (approximately 1-5 x 106 cells/mL).
SECTION: Hypothalamic Differentiation
30. In a 1.5 mL polypropylene tube, mix 10 µL of this cell suspension with 10 µL, transfer 10 µL of that mixture onto cell counting slide. Count cells with automated cell counter

SECTION: Hypothalamic Differentiation
31. Plate cells onto Geltrex-coated plates in hPSC culture media at a concentration of 1 x 105 cells per cm2 (corresponding to 9.5 x 105 cells per well of a 6-well plate, or 5.5 x 106 cells per 10 cm plate). This density corresponds to approximately 80% confluence the following day. Ensure that cells are evenly distributed across the plate by gently shaking the plate left to right, then top to bottom before and after transferring it to the incubator.
 

SECTION: Hypothalamic Differentiation
32. If cells plated for differentiation are evenly distributed over the plate and at a density of approximately 75%, start differentiation by washing cultures once with DPBS and adding Day 0 (D0) medium (see below). Every second day, make full medium changes as follows (8 mL-10 mL per 6-well plate, 20 mL-25 mL per 10 cm plate):
 

SECTION: Hypothalamic Differentiation
32.1. Day 0 (D0): N2B27 + 2 micromolar (µM) + 100 milimolar (mM) + 10 micromolar (µM)

SECTION: Hypothalamic Differentiation
32.2. Day 2 (D2): N2B27 + 2 micromolar (µM) + 100 nanomolar (nM) + 10 micromolar (µM) + 1 micromolar (µM) + 1 micromolar (µM)

SECTION: Hypothalamic Differentiation
32.3. Day 4 (D4): N2B27 + 1.5 micromolar (µM) + 75 nanomolar (nM) + 7.5 micromolar (µM) + 1 micromolar (µM) + 1 micromolar (µM)

SECTION: Hypothalamic Differentiation
32.4. Day 6 (D6): N2B27 + 1 micromolar (µM) + 50 nanomolar (nM) + 5 micromolar (µM) + 1 micromolar (µM) + 1 micromolar (µM) 

SECTION: Hypothalamic Differentiation
32.5. Day 8 (D8): N2B27 + 0.5 micromolar (µM) + 25 nanomolar (nM) + 2.5 micromolar (µM) + 5 micromolar (µM)

SECTION: Hypothalamic Differentiation
32.6. Day 10 (D10): N2B27 + 5 micromolar (µM)

SECTION: Hypothalamic Differentiation
32.7. Day 12 (D12): N2B27 + 5 micromolar (µM)

SECTION: Hypothalamic Differentiation
32.8. Day 14 (D14): N2B27 + 5 micromolar (µM)
SECTION: Neuronal Maturation
33. Coat plates with Geltrex for maturation as described above (Note: use a 0.02 % final Geltrex concentration to facilitate neuronal attachment and long term culture).
SECTION: Neuronal Maturation
34. On Day 15, neural progenitors generated above are dissociated and re-plated to encourage neurogenesis and neuronal survival and maturation. 
 

SECTION: Neuronal Maturation
35. Wash cells gently with DPBS.
SECTION: Neuronal Maturation
36. Prepare a mixture of TrypLE and Papain by mixing 10 mL with 1 vial of Papain (140 U/vial). Papain aids in neuronal dissociation and will ensure significantly higher survival upon re-plating.
SECTION: Neuronal Maturation
37. Add TrypLE with Papain to cells, 1 mL per well in 6 well plate, 5 mL per 10 cm plate.
SECTION: Neuronal Maturation
38. Incubate cell culture for 3 min-5 min at 37 °C. After 3 min of incubation, check to see if cells are detaching. Under a phase contrast microscope, the cells should start to round up and take on a phase-bright appearance, but not spontaneously detach from the plate. Once cultures adopt this appearance, gently suck up and dispel ~100 µL with a P1000 pipette against the cells. They should easily dislodge and leave a small area devoid of cells. If cells do not dissociate easily, extend TrypLE digestion for another minute and repeat this test.
 


SECTION: Neuronal Maturation
39. Gently aspirate TrypLE and Papain solution.
SECTION: Neuronal Maturation
40. To dissociate cells, add 1 mL/5 mL for a well of a 6 well /10 cm plate, and gently pipette this medium over the plate to detach cells and dissociate them to a single-cell suspension.
SECTION: Neuronal Maturation
41. Collect cells in 15 mL V-bottom polypropylene tube, and adjust volume with trituration medium (Total volume = 10 mL).
SECTION: Neuronal Maturation
42. Spin at 160 x g for 3 min-5 min . Aspirate supernatant, re-suspend cells in 10 mL.

SECTION: Neuronal Maturation
43. Spin at 160 x g for 3 min-5 min. Aspirate supernatant, re-suspend cells in desired volume of trituration medium to enable plating at the desired density.
SECTION: Neuronal Maturation
44. In a 1.5 mL polypropylene tube, mix 10 µL with 10 µL, transfer 10 µL onto cell counting slide. Count cells with automated cell counter.
 

SECTION: Neuronal Maturation
45. 

Plate cells onto Geltrex-coated plates in maturation media at a concentration of 1 x 105 cells per cm2 for cells maturing in N2B27 + BDNF (corresponding to 9.5 x 105 cells per well of a 6 well plate, or 5.5 x 106 cells per 10 cm plate)
 

SECTION: Neuronal Maturation
46. On Day 16, aspirate medium and feed with N2B27 + BDNF or Synaptojuice 1 (SJ1).
 

SECTION: Neuronal Maturation
47. On Day 17, aspirate medium and add twice the normal volume of Synaptojuice 1 (e.g. 4 mL per well of a 6 well plate, 20 mL per 10 cm plate) for neuronal maintenance for 1 week. This larger volume helps ensure that neurons are exposed to a relatively constant supply of nutrients. After 1 week, maintain the mature neurons on N2B27 + BDNF or Synptojuice 2 (SJ2).
SECTION: Neuronal Maturation
48. Change 75 % of media volume every second day.