Differentiation of hPSCs to hypothalamic neurons
Cortina Chen, Iman Mali, Florian T Merkle
Abstract
This protocol is about Differentiation of hPSCs to hypothalamic neurons.
Before start
Prepare Media and Reagents as described in section 'Materials'.
Attachments
Steps
Thawing of human pluripotent stem cell (hPSC) lines:
Thaw an aliquot of 1:10 diluted Geltrex On ice or in the fridge.
Dilute aliquot 1:10 in ice-cold DMEM/F12 to a final concentration of 1:100.
To coat plates, add 1:100 diluted Geltrex to TC dish/plate and incubate for 1h 0m 0s at 37°C, or 1h 0m 0s at 4°C.
Aspirate Geltrex
Add pre-warmed hPSC culture media: StemFlex with 10micromolar (µM); 10mL per 10 cm dish, 2mL per well of 6 well plate.
Take vial cells from liquid nitrogen to TC room on dry ice.
Dip bottom half of vial into 37°C water bath and swirl until partially thawed (approximately 0h 1m 0s-0h 3m 0s, depending on volume of freeze).
Thoroughly spray vial with 70%, and complete thaw by gently transferring pre-warmed hPSC culture media into the partially thawed cells.
Transfer cells into a 15 mL V-bottom polypropylene tube with pre-warmed wash media. Wash residual cells out of vial with 1mL.
Spin cells at 160x g.
Aspirate media.
Re-suspend the pellet with 10mL and mix well by gently pipet it up and down.
Spin at 160x g.
Aspirate media.
Re-suspend pellet with 1mL. Dilute into appropriate volume depending on culture dishes/plates used.
Add and evenly distribute cells into dish/plate with pre-warmed hPSC culture media (from step 5).
Transfer to incubator, cells should attach over a few hours.
Change media the following day to StemFlex without Rock inhibitor.
Hypothalamic Differentiation
Coat 6-well or 10 cm plates with Geltrex for differentiation as described above.
Nearly confluent hPSCs are dissociated and re-plated for differentiation
Aspirate culture medium and briefly and gently wash cells in 37Room temperature DPBS.
Add 37°C TrypLE to cell culture, 1mL per well in 6-well plate, 5mL per 10 cm plate.
Incubate cell culture for 0h 3m 0s-0h 5m 0s 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 ~100µL 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.
Gently aspirate TrypLE.
To dissociate cells, add 1mL/5mL 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.
Collect cells in 15 ml V-bottom polypropylene tube, and adjust volume with hPSC culture media (Total volume = 10mL)
Spin at 160x g,0h 0m 0s for 0h 3m 0s-0h 5m 0s at 37Room temperature. Aspirate supernatant, re-suspend cells in 10mL.
Spin at 160x g,0h 0m 0s for 0h 3m 0s-0h 5m 0s at 37Room temperature. Aspirate supernatant, re-suspend cells in hPSC culture media
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).
In a 1.5 mL polypropylene tube, mix 10µL of this cell suspension with 10µL, transfer 10µL of that mixture onto cell counting slide. Count cells with automated cell counter
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.
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 (8mL-10mL per 6-well plate, 20mL-25mL per 10 cm plate):
Day 0 (D0): N2B27 + 2micromolar (µM) + 100millimolar (mM) + 10micromolar (µM)
Day 2 (D2): N2B27 + 2micromolar (µM) + 100nanomolar (nM) + 10micromolar (µM) + 1micromolar (µM) + 1micromolar (µM)
Day 4 (D4): N2B27 + 1.5micromolar (µM) + 75nanomolar (nM) + 7.5micromolar (µM) + 1micromolar (µM) + 1micromolar (µM)
Day 6 (D6): N2B27 + 1micromolar (µM) + 50nanomolar (nM) + 5micromolar (µM) + 1micromolar (µM) + 1micromolar (µM)
Day 8 (D8): N2B27 + 0.5micromolar (µM) + 25nanomolar (nM) + 2.5micromolar (µM) + 5micromolar (µM)
Day 10 (D10): N2B27 + 5micromolar (µM)
Day 12 (D12): N2B27 + 5micromolar (µM)
Day 14 (D14): N2B27 + 5micromolar (µM)
Neuronal Maturation
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).
On Day 15 , neural progenitors generated above are dissociated and re-plated to encourage neurogenesis and neuronal survival and maturation.
Wash cells gently with DPBS.
Prepare a mixture of TrypLE and Papain by mixing 10mL with 1 vial of Papain (140 U/vial). Papain aids in neuronal dissociation and will ensure significantly higher survival upon re-plating.
Add TrypLE with Papain to cells, 1mL per well in 6 well plate, 5mL per 10 cm plate.
Incubate cell culture for 0h 3m 0s-0h 5m 0s at 37°C. After 0h 3m 0s 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 ~100µL 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.
Gently aspirate TrypLE and Papain solution.
To dissociate cells, add 1mL/5mL 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.
Collect cells in 15 mL V-bottom polypropylene tube, and adjust volume with trituration medium (Total volume = 10mL).
Spin at 160x g,0h 0m 0s for 0h 3m 0s-0h 5m 0s . Aspirate supernatant, re-suspend cells in 10mL.
Spin at 160x g,0h 0m 0s for 0h 3m 0s-0h 5m 0s. Aspirate supernatant, re-suspend cells in desired volume of trituration medium to enable plating at the desired density.
In a 1.5 mL polypropylene tube, mix 10µL with 10µL, transfer 10µL onto cell counting slide. Count cells with automated cell counter.
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)
On Day 16 , aspirate medium and feed with N2B27 + BDNF or Synaptojuice 1 (SJ1).
On Day 17 , aspirate medium and add twice the normal volume of Synaptojuice 1 (e.g. 4mL per well of a 6 well plate, 20mL 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).
Change 75% of media volume every second day.
