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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about Differentiation of hPSCs to hypothalamic neurons.</div></div>

[STEPS]
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Thaw an aliquot of 1:10 diluted Geltrex  or in the fridge.
on ice
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Aspirate Geltrex and rinse the dish/plate 1x with an equal volume of DPBS.
Note: do not let the dish/plate dry out.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Add pre-warmed hPSC culture media: StemFlex with ;  per 10 cm dish,  per well of 6 well plate.
[Rock inhibitor]
10 mL
2 mL
Note: the dish/plate is ready to receive cells for plating.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Take vial cells from liquid nitrogen to TC room on dry ice.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Dip bottom half of vial into  water bath and swirl until partially thawed (approximately -, depending on volume of freeze).
37 °C
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Thoroughly spray vial with , and complete thaw by gently transferring pre-warmed hPSC culture media into the partially thawed cells.
[ethanol]
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Transfer cells into a 15 mL V-bottom polypropylene tube with pre-warmed wash media. Wash residual cells out of vial with .
[hPSC culture media]
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Spin cells at .
Centrifuge: 160 34, 3 min
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Aspirate media.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Re-suspend the pellet with  and mix well by gently pipet it up and down.
[hPSC culture media]
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Spin at .
Centrifuge: 160 34, 3 min
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Aspirate media.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Re-suspend pellet with . Dilute into appropriate volume depending on culture dishes/plates used.
[hPSC culture media]
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Add and evenly distribute cells into dish/plate with pre-warmed hPSC culture media (from step 5).
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Transfer to incubator, cells should attach over a few hours.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Change media the following day to StemFlex without Rock inhibitor.
Note: Withdrawal of Rock inhibitor will result in a notable change in morphology, from ‘spikey’ look cells with thin processes, to a smoother appearance. Some cell death may also occur.
?. [Hypothalamic Differentiation]
Coat 6-well or 10 cm plates with Geltrex for differentiation as described above.
?. [Hypothalamic Differentiation]
Nearly confluent hPSCs are dissociated and re-plated for differentiation
Note: Before induction of differentiation, hPSCs should lack obvious signs of differentiation or contamination, and be in a rapid growth phase.
?. [Hypothalamic Differentiation]
Aspirate culture medium and briefly and gently wash cells in  DPBS.
37 Room temperature
?. [Hypothalamic Differentiation]
Add  TrypLE to cell culture,  per well in 6-well plate,  per 10 cm plate.
37 °C
1 mL
5 mL
?. [Hypothalamic Differentiation]
Incubate cell culture for - at . 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 ~ 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.
37 °C
[TrypLE solution]
Note: Take care to avoid over-digestion, which can cause cell death.
?. [Hypothalamic Differentiation]
Gently aspirate TrypLE.
?. [Hypothalamic Differentiation]
To dissociate cells, add / 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.
1 mL
[hPSC culture media]
?. [Hypothalamic Differentiation]
Collect cells in 15 ml V-bottom polypropylene tube, and adjust volume with hPSC culture media (Total volume = )
10 mL
Note: This wash step dilutes residual TrypLE to slow further digestion.
?. [Hypothalamic Differentiation]
Spin at  for - at . Aspirate supernatant, re-suspend cells in .
Centrifuge: 160 34
37 Room temperature
[hPSC culture media]
?. [Hypothalamic Differentiation]
Spin at  for - at . Aspirate supernatant, re-suspend cells in hPSC culture media
Centrifuge: 160 34
37 Room temperature
Note: These wash steps remove any remaining traces of TrypLE.
?. [Hypothalamic Differentiation]
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).
?. [Hypothalamic Differentiation]
In a 1.5 mL polypropylene tube, mix  of this cell suspension with , transfer  of that mixture onto cell counting slide. Count cells with automated cell counter
10 µl
[Trypan blue]
10 µl
?. [Hypothalamic Differentiation]
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.
Note: If cells are sparser, wait until cells reach the desired density before starting the differentiation. Sparse or over-confluent cells will not pattern well.
?. [Hypothalamic Differentiation]
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 (- per 6-well plate, - per 10 cm plate):
5 mL
6 mL
10 mL
15 mL
Note: Observe cells daily for changes in morphology. From Days 0-2, the culture should reach confluence and cells should have a simple and uniform hPSC-like morphology. By Day 4, cultures are highly compacted and cells adopt a more rounded appearance. Between Days 4 and 8, the cultures take on a dense neuro-epithelial morphology with identifiable neural ridge-like structures. A neuro-epithelial morphology is still evident before passaging on Day 14.
?. [Neuronal Maturation]
Coat plates with Geltrex for maturation as described above (Optional: use a  final Geltrex concentration to facilitate neuronal attachment and long term culture).
?. [Neuronal Maturation]
On Day 15, neural progenitors generated above are dissociated and re-plated to encourage neurogenesis and neuronal survival and maturation.
Note: Plate cells based on different experiment requirements/layout.
?. [Neuronal Maturation]
Wash cells gently with DPBS.
?. [Neuronal Maturation]
Prepare a mixture of TrypLE and Papain by mixing  with 1 vial of Papain (140 U/vial). Papain aids in neuronal dissociation and will ensure significantly higher survival upon re-plating.
[TrypLE]
?. [Neuronal Maturation]
Add TrypLE with Papain to cells,  per well in 6 well plate,  per 10 cm plate.
1 mL
5 mL
?. [Neuronal Maturation]
Incubate cell culture for - at . After  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 ~ 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.
37 °C
[TrypLE and Papain solution]
Note: Take care to avoid over-digestion, which can cause cell death and release of genomic DNA.
?. [Neuronal Maturation]
Gently aspirate TrypLE and Papain solution.
?. [Neuronal Maturation]
To dissociate cells, add / 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.
1 mL
[trituration medium]
?. [Neuronal Maturation]
Collect cells in 15 mL V-bottom polypropylene tube, and adjust volume with trituration medium (Total volume = ).
10 mL
?. [Neuronal Maturation]
Spin at  for - . Aspirate supernatant, re-suspend cells in .
Centrifuge: 160 34
[trituration medium]
?. [Neuronal Maturation]
Spin at  for -. Aspirate supernatant, re-suspend cells in desired volume of trituration medium to enable plating at the desired density.
Centrifuge: 160 34
?. [Neuronal Maturation]
In a 1.5 mL polypropylene tube, mix  with , transfer  onto cell counting slide. Count cells with automated cell counter.
[cell suspension]
[Trypan blue]
[of the mixture]
Note: If desired, cultures can be frozen at this point for later thawing as progenitors/immature neurons using the same procedure used for freezing hPSCs.
?. [Neuronal Maturation]
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)
Note: Plate cells at a density of 3 x 105 cells per cm2 for cells maturing in the enhanced maturation media, Synaptojuice (SJ1/SJ2).
?. [Neuronal Maturation]
On Day 16, aspirate medium and feed with N2B27 + BDNF or Synaptojuice 1 (SJ1).
?. [Neuronal Maturation]
On Day 17, aspirate medium and add twice the normal volume of Synaptojuice 1 (e.g.  per well of a 6 well plate,  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).
4 mL
20 mL
?. [Neuronal Maturation]
Change  of media volume every second day.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
Dilute aliquot 1:10 in ice-cold DMEM/F12 to a final concentration of 1:100.
?. [Thawing of human pluripotent stem cell (hPSC) lines:]
To coat plates, add 1:100 diluted Geltrex to TC dish/plate and incubate for  at , or  at .
37 °C
4 °C
Use   for a 10 cm dish or   for a well of a 6 well plate.
?. [Hypothalamic Differentiation]
Day 0 (D0): N2B27 +  +  +
[XAV939]
[LDN-193189]
[SB431542]
?. [Hypothalamic Differentiation]
Day 2 (D2): N2B27 +  +  +  +  +
[XAV939]
[LDN-193189]
[SB431542]
[SAG]
[Purmorphamine]
?. [Hypothalamic Differentiation]
Day 4 (D4): N2B27 +  +  +  +  +
[XAV939]
[LDN-193189]
[SB431542]
[SAG]
[Purmorphamine]
?. [Hypothalamic Differentiation]
Day 6 (D6): N2B27 +  +  +  +  +
[XAV939]
[LDN-193189]
[SB431542]
[SAG ]
[Purmorphamine]
?. [Hypothalamic Differentiation]
Day 8 (D8): N2B27 +  +  +  +
[XAV939]
[LDN-193189]
[SB431542]
[ DAPT]
?. [Hypothalamic Differentiation]
Day 10 (D10): N2B27 +
[ DAPT]
?. [Hypothalamic Differentiation]
Day 12 (D12): N2B27 +
[ DAPT]
?. [Hypothalamic Differentiation]
Day 14 (D14): N2B27 +
[ DAPT]