Oct 25, 2021
Version 2
Differentiation of hPSCs to hypothalamic neurons V.2
- Cortina Chen1,
- Iman Mali1,
- Florian T Merkle1
- 1Wellcome-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, CB2 0QQ, United Kingdom
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Cortina Chen, Iman Mali, Florian T Merkle 2021. Differentiation of hPSCs to hypothalamic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bzghp3t6Version created by Cortina Chen
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 25, 2021
Last Modified: October 25, 2021
Protocol Integer ID: 54505
Keywords: Differentiation, hPSCs, hypothalamic neurons,
Abstract
This protocol is about Differentiation of hPSCs to hypothalamic neurons.
Attachments
Materials
Materials
1M CaCl2Sigma-aldrichCatalog #21115
L-Ascorbic acidSigma AldrichCatalog #A4403
B-27 SupplementGibco - Thermo FischerCatalog #17504044
Animal-Free Recombinant Human/Murine/Rat BDNFpeprotechCatalog #AF-450-02
CHIR99021Cell Guidance SystemsCatalog #SM13-10
DAPTSigma AldrichCatalog #D5942
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190144
DMEM/F-12, GlutaMAX™ SupplementThermo FisherCatalog #31331028
DMEM/F-12, HEPES, no phenol redThermo FisherCatalog #11039021
DNase Vial (D2)Worthington Biochemical CorporationCatalog #LK003170
Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413202
GABATocrisCatalog #0344
GlutaMAXGibco - Thermo FisherCatalog #35050038
Stemolecule LDN-193189ReprocellCatalog #04-0074
LM 22A4TocrisCatalog #4607
MEM Non-Essential Amino Acids Solution (100X)Thermo Fisher ScientificCatalog #11140035
N2 supplementGibco - Thermo FisherCatalog #17502048
Neurobasal-A MediumThermo Fisher ScientificCatalog #10888022
NKH 477Sigma AldrichCatalog #N3290
PDS Kit Papain VialWorthington Biochemical CorporationCatalog #LK003176
PD 0332991 isethionateSigma AldrichCatalog #PZ0199
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
PurmorphamineMerck MilliporeCatalog #540220
SB 431542 hydrateSigma AldrichCatalog #S4317
MilliporeSigm Calbiochem Smoothened Agonist SAGFisher ScientificCatalog #56-666-01MG
Sodium Bicarbonate 7.5% solutionThermo FisherCatalog #25080060
StemFlex™ MediumThermo Fisher ScientificCatalog #A3349401
Trypan Blue Stain (0.4%) for use with the Countess™ Automated Cell CounterThermo Fisher ScientificCatalog #T10282
Stemolecule XAV939ReprocellCatalog #04-0046
Y-27632 dihydrochloride (Rock Inhibitor)Catalog #DNSK-KI-15-02
Media and Reagents
StemFlex
| Name | Volume | |
| StemFlex basal medium | 450 mL | |
| StemFlex supplement | 50 mL |
N2B27
| Name | Volume | |
| Neurobasal-A | 500 mL | |
| DMEM/F12 with GlutaMAX | 500 mL | |
| Glutamax | 10 mL | |
| Sodium bicarbonate | 10 mL | |
| MEM Nonessential amino acids | 5 mL | |
| Ascorbic acid (200 mM) | 1 mL | |
| Penicillin-streptomycin | 10 mL | |
| Sterile filter, then add the following supplements: | ||
| B27 supplement | 10 mL | |
| N2 supplement | 5 mL |
N2B27 + BDNF (maturation media)
| Name | Volume | |
| N2B27 | 500 mL | |
| BDNF (100 μg/mL stock) | 50 μL |
Trituration medium
| Name | Volume | |
| N2B27 + BDNF | 30 mL | |
| Y-27632 dihydrochloride (Rho kinase inhibitor; 10 mM stock) | 30 μL | |
| Dnase I (2 mg/mL stock; 5990 U/mg) | 500 μL |
Synaptojuice 1 (SJ1; enhanced maturation media)
| Name | Volume | |
| N2B27 + BDNF | 500 mL | |
| DAPT (50 mM stock) | 50 μL | |
| PD0332991 (20 mM stock) | 50 μL | |
| CaCl2 | 185 μL | |
| NKH477 (10 mM stock) | 500 μL | |
| CHIR99021 (20 mM stock) | 50 μL | |
| GABA (300 mM stock) | 500 μL | |
| LM22A4 (10 mM stock) | 50 μL |
Synaptojuice 2 (SJ2; enhanced maturation media)
| Name | Volume | |
| N2B27 + BDNF | 500 mL | |
| PD0332991 (20 mM stock) | 50 μL | |
| CaCl2 | 185 μL | |
| CHIR99021 (20 mM stock) | 50 μL | |
| LM22A4 (10 mM stock) | 50 μL |
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Thawing of human pluripotent stem cell (hPSC) lines:
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 01:00:00 at 37 °C , or Overnight at 4 °C .
Note
Use 3 mL for a 10 cm dish or 1 mL for a well of a 6 well plate.
2h
Aspirate Geltrex
Note
Note: do not let the dish/plate dry out, proceed to the next step immediately.
Add pre-warmed hPSC culture media: StemFlex with 10 micromolar (µM) Rock inhibitor ; 10 mL per 10 cm dish, 2 mL per well of 6 well plate.
Note
Note: the dish/plate is ready to receive cells for plating.
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 00:01:00 -00:03:00 , depending on volume of freeze).
4m
Thoroughly spray vial with 70 % ethanol , 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 1 mL hPSC culture media .
Spin cells at 160 x g, 00:03:00 .
Aspirate media.
Re-suspend the pellet with 10 mL hPSC culture media and mix well by gently pipet it up and down.
Spin at 160 x g, 00:03:00 .
Aspirate media.
Re-suspend pellet with 1 mL hPSC culture media . 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.
Note
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
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
Note
Note: Before induction of differentiation, hPSCs should lack obvious signs of differentiation or contamination, and be in a rapid growth phase.
Aspirate culture medium and briefly and gently wash cells in Room temperature DPBS.
Add 37 °C TrypLE to cell culture, 1 mL per well in 6-well plate, 5 mL per 10 cm plate.
Incubate cell culture for 00:03:00 -00:05:00 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 TrypLE solution 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.
Note
Note: Take care to avoid over-digestion, which can cause cell death.
8m
Gently aspirate TrypLE.
To dissociate cells, add 1 mL /5 mL hPSC culture media 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 = 10 mL )
Note
Note: This wash step dilutes residual TrypLE to slow further digestion.
Spin at 160 x g for 00:03:00 -00:05:00 at Room temperature . Aspirate supernatant, re-suspend cells in 10 mL hPSC culture media .
8m
Spin at 160 x g for 00:03:00 -00:05:00 at Room temperature . Aspirate supernatant, re-suspend cells in hPSC culture media
Note
Note: These wash steps remove any remaining traces of TrypLE.
8m
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 10 µL of this cell suspension with 10 µL Trypan blue , transfer 10 µL 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.
Note
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.
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):
Note
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.
Day 0 (D0): N2B27 + 2 micromolar (µM) XAV939 + 100 millimolar (mM) LDN-193189 + 10 micromolar (µM) SB431542
Day 2 (D2): N2B27 + 2 micromolar (µM) XAV939 + 100 nanomolar (nM) LDN-193189 + 10 micromolar (µM) SB431542 + 1 micromolar (µM) SAG + 1 micromolar (µM) Purmorphamine
Day 4 (D4): N2B27 + 1.5 micromolar (µM) XAV939 + 75 nanomolar (nM) LDN-193189 + 7.5 micromolar (µM) SB431542 + 1 micromolar (µM) SAG + 1 micromolar (µM) Purmorphamine
Day 6 (D6): N2B27 + 1 micromolar (µM) XAV939 + 50 nanomolar (nM) LDN-193189 + 5 micromolar (µM) SB431542 + 1 micromolar (µM) SAG + 1 micromolar (µM) Purmorphamine
Day 8 (D8): N2B27 + 0.5 micromolar (µM) XAV939 + 25 nanomolar (nM) LDN-193189 + 2.5 micromolar (µM) SB431542 + 5 micromolar (µM) DAPT
Day 10 (D10): N2B27 + 5 micromolar (µM) DAPT
Day 12 (D12): N2B27 + 5 micromolar (µM) DAPT
Day 14 (D14): N2B27 + 5 micromolar (µM) DAPT
Neuronal Maturation
Neuronal Maturation
24m
24m
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.
Note
Note: Plate cells based on different experiment requirements/layout.
Wash cells gently with DPBS.
Prepare a mixture of TrypLE and Papain by mixing 10 mL TrypLE 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, 1 mL per well in 6 well plate, 5 mL per 10 cm plate.
Incubate cell culture for 00:03:00 -00:05:00 at 37 °C . After 00:03:00 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 TrypLE and Papain solution 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.
Note
Note: Take care to avoid over-digestion, which can cause cell death and release of genomic DNA.
11m
Gently aspirate TrypLE and Papain solution.
To dissociate cells, add 1 mL /5 mL trituration medium 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 = 10 mL ).
Spin at 160 x g, Room temperature for 00:03:00 -00:05:00 . Aspirate supernatant, re-suspend cells in 10 mL trituration medium .
8m
Spin at 160 x g, Room temperature for 00:03:00 -00:05:00 . Aspirate supernatant, re-suspend cells in desired volume of trituration medium to enable plating at the desired density.
8m
In a 1.5 mL polypropylene tube, mix 10 µL cell suspension with 10 µL Trypan blue , transfer 10 µL of the mixture onto cell counting slide. Count cells with automated cell counter.
Note
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.
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
Note: Plate cells at a density of 3 x 105 cells per cm2 for cells maturing in the enhanced maturation media, Synaptojuice (SJ1/SJ2).
On Day 16, aspirate medium and feed with N2B27 + BDNF or Synaptojuice 1 (SJ1).
Note
Note: laminin (final concentration of 1 µg/mL) may be supplemented in SJ1/SJ2 to provide better cell attachment during the remainder of the maintenance and maturation period.
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).
Change 75 % of media volume every second day.