Differentiation of PSCs to Neural Progenitors

Lucia Dutan Polit; Roland Nagy; Deepak P Srivastava

Feb 26, 2025

Differentiation of PSCs to Neural Progenitors

DOI

dx.doi.org/10.17504/protocols.io.j8nlk9z81v5r/v1

Lucia Dutan Polit^1,2,
Roland Nagy^1,2,
Deepak P Srivastava^1,2

¹Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK;
²MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK

Deepak P Srivastava

Department of Basic and Clinical Neuroscience, Institute of ...

DOI: dx.doi.org/10.17504/protocols.io.j8nlk9z81v5r/v1

Protocol Citation: Lucia Dutan Polit, Roland Nagy, Deepak P Srivastava 2025. Differentiation of PSCs to Neural Progenitors. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9z81v5r/v1

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: Working

This protocol is adapted from Shum C, Dutan L, Annuario E, Warre-Cornish K, Taylor SE, Taylor RD, Andreae LC, Buckley NJ, Price J, Bhattacharyya S, Srivastava DP. Δ9-tetrahydrocannabinol and 2-AG decreases neurite outgrowth and differentially affects ERK1/2 and Akt signaling in hiPSC-derived cortical neurons. Mol Cell Neurosci. 2020 Mar;103:103463. doi: 10.1016/j.mcn.2019.103463. Epub 2020 Jan 7. PMID: 31917333.

Created: February 18, 2025

Last Modified: February 26, 2025

Protocol Integer ID: 123449

Keywords: Thawing, Haemocytometer, Neural progenitors

Abstract

This protocol describes the differentiation of pluripotent stem cells (PSCs) to neural progenitors.

Guidelines

Definitions

SMADi- SMAD Inhibitor
Rocki- Rock Inhibitor
DMEM- Dubecco’s Modified Eagle Medium
HBSS- Hank’s Balanced Salt Solution
NP- neural passage
SF- StemFlex
PDL- Poly-D-Lysine

Materials

Reagents:

Geltrex (Gibco; A14133)
ReagentHBSS, no calcium, no magnesiumThermo FisherCatalog #14170070 
ReagentVersene SolutionThermo FisherCatalog #15040066 
ReagentStemFlex MediumThermo Fisher ScientificCatalog #A3349401 
Rocki (LTK; Y1000)
ReagentAccutase Gibco - Thermo FischerCatalog # A1110501 
ReagentN2 supplementGibco - Thermo FisherCatalog #17502048 
ReagentDMEM/F-12Merck MilliporeSigma (Sigma-Aldrich)Catalog #D6421 
ReagentB-27 SupplementGibco - Thermo FischerCatalog #17504044 
ReagentNeurobasal mediumGibco - Thermo FischerCatalog #21103049 
ReagentGlutaMAXGibco - Thermo FisherCatalog #35050038 
SB431542 (Cell guidance systems; SM33)
ReagentLDN193189 hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #SML0559 
ReagentXAV939Merck MilliporeSigma (Sigma-Aldrich)Catalog #X3004 
ReagentRevitaCell Suppplement 100XThermo Fisher ScientificCatalog #A2644501 
ReagentPoly-D-LysineThermo Fisher ScientificCatalog #A3890401 
ReagentL-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4403 
ReagentLamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020 
ReagentTrypan BlueMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8154 
ReagentDAPT Santa Cruz BiotechnologyCatalog #sc-201315A 

Media:

N2 media

AB
DMEM49ml
N2 supplement 0.5ml
Glutamax 0.5ml

B27
 
AB
Neurobasal medium48.5ml
B27 1ml
Glutamax 0.5ml

Geltrex Coating

Slowly thaw a Amount200 µL   aliquot of Geltrex TemperatureOn ice   or in the refrigerator until fully thawed.

Dilute the thawed Geltrex by adding the Amount200 µL   aliquot to Amount20 mL   of cold DMEM/F12. Keep the mixture TemperatureOn ice   during preparation.

Immediately add Amount1 mL  /well of a 6-well plate or Amount100 µL  /well of a 96-well plate. Gently swirl the plate(s) to ensure even coating.

Incubate the plate(s) at Temperature37 °C   for at least Duration02:00:00  . Prevent the plates from drying during incubation.

After incubation, aspirate the unbound Geltrex solution by tilting the plate slightly to avoid disturbing the coated surface. Add the appropriate volume of cell culture media to the coated wells and warm the plate(s) in the incubator prior to adding cells.

Note
Storage of Coated Plates:

If not used immediately, seal the plates with parafilm to prevent dehydration. Store them at 2–8°C for up to 7 days. Before use, let the plate(s) equilibrate to room temperature (15–25°C) for 30 minutes, and then aspirate the Geltrex solution.

Thawing PSCs

1d 0h 2m

Prewarm the PSC defrosting medium by adding SF medium + RevitaCell, 1:100 dilution. Use Amount2 mL   per well for a 6-well plate.

Ensure a Geltrex-coated well is prepared for each vial of PSCs to be thawed.

Place cryovials on dry ice.

Quickly thaw a vial in a 37°C water bath until only a small ice pellet remains.

Gently add Amount1 mL   of TemperatureRoom temperature   defrosting medium to the cryovial. Transfer the diluted cells to a 15 mL tube.

Centrifuge the cell suspension at Centrifigation1250 rpm, 00:02:00 .

Carefully aspirate the supernatant and gently resuspend the cell pellet in the prewarmed defrosting medium.

Add Amount2 mL   per well of the cell suspension for a 6-well plate using a P1000 micropipette.

Ensure the cells are evenly distributed across the well by gently swirling the plate. Place the plate in an incubator (37°C; 5% CO2; 5% O2) and allow the cells to recover for Duration24:00:00  .

After 24 hours, replace the defrosting medium with prewarmed StemCell medium. Continue changing the medium daily until cells reach confluence.

Freezing PSCs

1d 0h 5m

Cool a Mr. Frosty to Temperature4 °C  .

Prepare PSC freezing medium by adding 10% DMSO to StemFlex medium. Use Amount1 mL   per well from a 6-well plate.

Aspirate the medium from one confluent well of the PSC culture. Rinse the well with Amount1 mL   of HBSS (without calcium and magnesium).

Add Amount1 mL   of Versene to the well and swirl the dish to coat the entire surface. Periodically check plates for signs of colonies detaching.

Incubate the dish at Temperature37 °C   for Duration00:02:00  –Duration00:03:00  .

Aspirate the Versene and replace it with Amount1 mL   of StemFlex medium per well.

Gently dislodge the cells using a cell lifter.

Using a P1000 micropipette, transfer the cell suspension to a 15 mL tube containing Amount1 mL   of TemperatureRoom temperature   StemFlex medium.

Centrifuge the cell suspension at Centrifigation1250 rpm, 00:02:00 .

Label cryovials.

Carefully remove the supernatant and resuspend the cells in PSC freezing medium, using Amount1 mL   per well of a 6-well plate.

Transfer Amount1 mL   of the suspension into each cryovial and place in a Temperature4 °C   Mr. Frosty. Transfer to a -80°C freezer and leave for Duration24:00:00  .

After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.

Expansion of PSCs

1d 0h 3m

Check that the cells are approximately 90% confluent before proceeding.

Aspirate the medium from one confluent well of the PSC culture. Rinse the well with Amount1 mL   of HBSS (without calcium and magnesium).

Add Amount1 mL   of Versene to the well and swirl the dish gently to coat the entire surface. Periodically check plates for signs of colonies detaching.

Incubate the dish at Temperature37 °C   for Duration00:02:00  –Duration00:03:00  .

Aspirate the Versene and replace it with Amount1 mL   of StemFlex medium per well.

Gently dislodge the cells using a cell lifter.

Using a P1000 micropipette, transfer the cell suspension to a 15 mL tube containing Amount5 mL   of TemperatureRoom temperature   StemFlex medium (for 2 wells).

Mix the cell suspension by pipetting up and down 3 times with a 10 mL serological pipette.

Add Amount3 mL   of the cell suspension to each of 2 wells in a 6-well plate.

Gently swirl the plate to ensure the cells are evenly distributed across the wells.

Place the plate in an incubator (Temperature37 °C  ; 5% CO2; 5% O2) and allow the cells to recover for Duration24:00:00  .

Differentiating PSCs to Neural Progenitors: Day -1

1d 0h 5m

Note
Plate PSCs in StemFlex medium onto 6-well Geltrex-coated plates, at a density that will ensure cells reach approaching 100% confluence within 24 hours of plating 24 hours prior to desired neuralisation start time. Cells should be ~60% confluent prior to replating.

See Appendix for details regarding timing - If cells do not reach total confluence by D-1 +3d, discard them. Allowing cells to remain at high confluence for longer periods of time diminishes PSC quality and results in variable growth across the well, as well interfering with the timing of subsequent passages. 

PSC passage number must fall between 20 – 30 at the start of neuralisation. 

Passaging: 

Aspirate total volume of media from each well, and rinse with HBSS at TemperatureRoom temperature   (Amount1 mL  /well) to remove calcium.

Add Amount1 mL  /well TemperatureRoom temperature   Versene (EDTA) and incubate (Temperature37 °C  ; 5% CO2; 5% O2) for Duration00:03:00  -Duration00:05:00  . Periodically check plates for signs of colonies detaching.

Aspirate total volume of Versene and replace with Amount1 mL  /well TemperatureRoom temperature   StemFlex media. Working rapidly, use a cell lifter to gently detach colonies from plate.

Using a 1000 μL pipette, carefully pipette up and down once (collect cells, pipette suspension across well, collect) to achieve a homogenous suspension of large PSC clusters. Or use a cell lifter to gently detach the cells from the well and then collect the cells with a P1000 pipette.

Note
Breaking the colonies up too much will delay PSCs in reaching 100% confluence.

Using the same procedure, collect suspension from all wells into a 50 mL tube. With a 10 mL serological pipette add to existing cell suspension the correct volume of TemperatureRoom temperature   StemFlex, such that you have twice the volume of suspension as you do Geltrex coated wells.

Aspirate Geltrex and aliquot StemFlex across plates at Amount1 mL  /well.

Using a 10 mL serological pipette transfer (a well at a time) Amount2 mL   of the cell suspension to each well.

Gently swirl plates to distribute cells evenly across wells and incubate (Temperature37 °C  ; 5% CO2; 5% O2) for Duration24:00:00  .

Following 24-hour incubation, check plates. If cells are not approaching 100% confluent/are loosely packed or appear to have suffered during passage, change media (Amount3 mL  /well TemperatureRoom temperature   StemFlex) and wait an additional day before neuralisation.

Differentiating PSCs to Neural Progenitors: Day 0

Note
Cells must be 100% confluent, or approaching 100% confluence prior to start of neuralisation. Inducing neuralisation while cells are <100% confluent will result in differentiation towards neural crest or nonspecific cells.
Aspirate total volume of StemFlex medium from each well, and replace with Amount2 mL  /well of warm neuralisation medium containing 50% N2/50% B27 media + Concentration0.1 micromolar (µM)   LDN193189 + Concentration10 micromolar (µM)   SB43 (SMAD inhibitors 2i).

Incubate plate for Duration24:00:00   (37°C; 5% CO2; 20% O2).

Following 24-hour incubation, aspirate neuralisation medium and replace with Amount2 mL  /well warm neuralisation medium. Incubate plate for Duration24:00:00   (37°C; 5% CO2; 20% O2). Continue replacing medium every 24 hours until Day 7, or until formation of a uniform neuroepithelial sheet occurs.

Note
A substantial amount of cell death occurs within the first 24 hours of neuralisation; shake plates to resuspend dead cells prior to aspirating medium on Day 1. Cell layer thickens during initial stage of neuralisation – take care when changing media (especially from ~ Day 5 onwards), as forceful pipetting can cause cell layer to detach from plate.

Differentiating PSCs to Neural Progenitors: Day 7 – np1

2d 0h 7m 30s

Note
At this stage cells must be passaged 1:1.
Remove plate(s) from incubator.

Aspirate total volume of medium from each well, and rinse with Amount1 mL  /well TemperatureRoom temperature   HBSS. Let sit for Duration00:00:30  .

30s

Remove total volume of HBSS and add Amount1 mL  /well TemperatureRoom temperature   (Temperature4 °C  ) Accutase (keep protected from light). Immediately transfer plate to incubator and incubate for Duration00:02:00   – Duration00:05:00   (Temperature37 °C  ; 5% CO2; 20% O2), until material can be lifted with gentle pipetting.

Prepare 15 mL tubes containing TemperatureRoom temperature   DMEM at twice the volume of Accutase to be added to the tubes.

Using a P1000 micropipette, attempt to lift all cells by pipetting up and down as gently and as little as possible no more than 5 times.

Note
Any remaining attached cells can be collected by rinsing the plate with 1mL/well RT DMEM.

Collect cells in Accutase, and transfer directly into DMEM.

Note
Do this one well at a time (i.e. collect cells and transfer to DMEM before detaching cells from adjacent well) to minimize exposure of cells to accutase. Avoid exposing cells to air – do not introduce air bubbles; transfer cells in accutase directly into DMEM. Attempt to produce a homogenous suspension of large cell clusters.

Rinse plates with DMEM (Amount1 mL  /well). Add to tube(s) containing Accutase/DMEM/cells.

Centrifuge cell suspension (Centrifigation1250 rpm, 00:02:00 ) and carefully remove supernatant, leaving ~Amount50 µL   atop pelleted cells.

Prepare plates. Aspirate Geltrex and aliquot fresh neuralisation medium + Concentration10 micromolar (µM)   ROCKi across plates at Amount1 mL  /well.

Add TemperatureRoom temperature   neuralisation medium + Concentration10 micromolar (µM)   ROCKi to tube with cell pellet, at a volume equal to the number of wells passaged. Resuspend by gently pipetting up and down using a 10 mL serological pipette.

Note
If pellet failed to resuspend during step 61, pipette up and down with a 10mL serological pipette to break up pellet as much as possible, and allow any cell clusters that fail to resuspend to settle to the bottom of the tube. Try not to carry these over to your new plate – remove any unbroken clusters that are transferred using a p100 pipette.

Aliquot cell suspension across plates (Amount1 mL  /well, one well at a time). Rock plate to distribute cells and incubate for Duration24:00:00   (Temperature37 °C  ; 5% CO2; 20% O2).

Following incubation, remove total volume of medium and replace with Amount2 mL  /well N2:B27 ONLY (i.e. discontinue use of SMADi). Incubate plate (Temperature37 °C  ; 5% CO2; 20% O2) for Duration24:00:00  . Continue replacing medium with fresh N2:B27 every 24 hours until Day 12.

Differentiating PSCs to Neural Progenitors: Day 12 – np2

1d 0h 5m

Passage cells at Day 12, following steps 52 - 63 and replating 1:1 in N2:B27 + Concentration10 micromolar (µM)   ROCKi + Concentration200 micromolar (µM)   AA2P. Continue replacing medium with fresh N2:B27 + Concentration200 micromolar (µM)   AA2P every 24 hours until Day 15/16.

Differentiating PSCs to Neural Progenitors: Day 15/16 – np3

1d 0h 5m

Passage cells at Day 15/16, following steps 52 - 63 and replating 1:1 in N2:B27 + 10 µM ROCKi + 200 µM AA2P.

Note
Passaging cells as clusters at Days 7 and 12 is critical for survival of the culture. Following Day 15/16 cells can and should be passaged as single cells, to promote proliferation and further differentiation. To this end, previous restrictions implemented in order to minimise fragmentation of the cell layer should now be removed. Use RT HBSS and accutase, and use a p1000 micropipette to fully dissociate cells. Cells may be resuspended using a 5mL serological pipette. From this point forward, centrifuge cells at 1250RPM for 2 minutes when passaging.

Continue replacing medium with fresh N2:B27 +AA2P every 24 hours until Day 18/19.

Differentiating PSCs to Neural Progenitors: Day 18/19 – np4

Passage cells at Day 18/19, following steps 52 - 63 and replating 1:1 in N2:B27 + Concentration10 micromolar (µM)   ROCKi + Concentration200 micromolar (µM)   AA2P.

Gently shake flasks to evenly distribute cells and incubate (Temperature37 °C  ; 5% CO2; 20% O2) for Duration24:00:00  .

Differentiating PSCs to Neural Progenitors: Day 19/20

1d 0h 5m

Following 24-hour incubation, remove total volume of media from each flask and replace with Amount15 mL  /flask B27 + Concentration10 micromolar (µM)   DAPT + Concentration200 micromolar (µM)   AA2P.

Freezing NPCs

1d 0h 5m

Cool a Mr. Frosty to Temperature4 °C  .

Prepare freezing medium by adding 10% DMSO to N2:B27 + Concentration200 micromolar (µM)   AA2P medium.

Aspirate the medium from the NPC culture and rinse each well with Amount1 mL   of HBSS (without calcium and magnesium).

Add Amount1 mL   of Accutase to each well containing cells. Swirl the dish gently to coat the entire surface.

Incubate the dish at Temperature37 °C   for Duration00:02:00  –Duration00:03:00  . When the cells begin to round up, remove the dish from the incubator.

Prepare 15 mL tubes containing TemperatureRoom temperature   DMEM/F12 at twice the volume of the Accutase to be added to the tubes.

Using a P1000 micropipette, gently lift the cells by pipetting up and down no more than five times.

Collect the cells in Accutase and transfer them directly into the prepared DMEM/F12.

Rinse the plates with Amount1 mL   of DMEM/F12 per well and add the rinse to the tube containing Accutase, DMEM/F12, and cells.

Centrifuge the cell suspension at Centrifigation1250 rpm, 00:02:00 .

Label cryovials.

Carefully remove the supernatant and resuspend the cells in freezing medium, using Amount1 mL   per well of a 6-well plate.

Transfer Amount1 mL   of the suspension into each cryovial and place in a Temperature4 °C   Mr. Frosty. Transfer the cryovials to a -80°C freezer and leave for Duration24:00:00  .

After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.

Thawing NPCs

1d 0h 2m

Prepare and prewarm the required volume of N2:B27 + Concentration200 micromolar (µM)   AA2P medium containing Concentration10 micromolar (µM)   ROCK inhibitor.

Prepare Geltrex-coated plates for one well of a 6-well plate per vial of cells.

Retrieve the required cryovials of NPCs from storage and place them on dry ice.

Rapidly thaw a vial of NPCs by placing it in a 37°C water bath until a small ice pellet remains.

Add Amount1 mL   of prewarmed NPC medium to the cryovial and transfer the cell mixture to a 15 mL tube.

Centrifuge the cell suspension at Centrifigation1250 rpm, 00:02:00 .

Aspirate the supernatant and gently resuspend the pellet in N2:B27 + Concentration200 micromolar (µM)   AA2P medium containing Concentration10 micromolar (µM)   ROCK inhibitor.

Add Amount2 mL   of the cell suspension per well of a 6-well plate using a P1000 micropipette or Amount100 µL   per well of a 96-well plate using a P200 micropipette.

Ensure the cells are evenly spread in the well and incubate at Temperature37 °C   for Duration24:00:00  .

Terminal Plating of Neural Progenitors: Coating Plates

12h

Note
The following instructions are for plastic tissue culture-treated plates only. Plates must be coated 24 hours prior to terminal plating.
Coat plates with PDL Amount3 mL  /flask (T25 flask) Amount1 mL  /well (6w plate) Amount500 µL  /well (12w plate) Amount300 µL  /well (24w plate) Amount100 µL  /well (96w plate) and incubate (Temperature37 °C  ; 5% CO2; 5% O2) for at least Duration04:00:00  . Wash 3 times with 1 mL/well of DMEM or PBS.

Working TemperatureOn ice  , dilute laminin stock (Amount1 µL  ) to a concentration of Amount20 µL   in cold DMEM.

Following incubation, aspirate total volume of DMEM or PBS solution from plates. Coat plates with laminin solution at the following volumes: Amount3 mL  /flask (T25 flask) Amount1 mL  /well (6w plate) Amount500 µL  /well (12w plate) Amount250 µL  /well (24w plate) Amount100 µL  /well (96w plate) and incubate (37°C; 5% CO2; 5% O2) DurationOvernight  .

Terminal Plating of Neural Progenitors: Terminal Plating

1d 0h 5m

Rinse wells to be passaged with Amount1 mL  /well TemperatureRoom temperature   HBSS.

Add TemperatureRoom temperature   Accutase at Amount1 mL  /well and incubate at Temperature37 °C   for Duration00:03:00  . While plates are incubating, prepare 15 mL tube containing TemperatureRoom temperature   DMEM at twice the volume of the number of wells being passaged.

Check that cells have detached by gently shaking plate. Using a P1000 micropipette, pipette up and across the well several times in order to create a homogenous, single-cell suspension. Transfer cells in Accutase to tube containing DMEM.

Centrifuge cells at Centrifigation1250 rpm, 00:02:00 .

Prepare plates by aspirating 1x DPBS and replacing with Amount1 mL  /well (6w plates) Amount600 µL  /well (12w plates) Amount300 µL  /well (24w plates) Amount70 µL  /well (96w plates) B27 + Concentration10 micromolar (µM)   DAPT + Concentration200 micromolar (µM)   AA2P + Concentration10 micromolar (µM)   ROCKi.

Aspirate supernatant from atop pelleted cells. Resuspend in at least Amount1 mL   B27 + Concentration10 Mass Percent   DAPT + Concentration200 micromolar (µM)   AA2P + Concentration10 micromolar (µM)   ROCKi.

Using a P1000 micropipette, gently pass the cell suspension through a 40 µm cell strainer into a new tube to remove any remaining large colonies.

Collect Amount10 µL   cell suspension and transfer to 0.6 mL Eppendorf tube. Add Amount10 µL   trypan blue and mix by gently pipetting.

Use an automated cell counter or a hemocytometer to calculate the number of viable cells.

Calculate the volume of cell suspension containing the number of cells required to plate  Amount200000 µL   across all coated plates and transfer to a clean 50 mL tube.

Add a sufficient volume of B27 + Concentration10 micromolar (µM)   DAPT + Concentration200 micromolar (µM)   AA2P + Concentration10 micromolar (µM)   ROCKi to cell suspension, such that the total volume will be enough to distribute cell suspension across all plates at the following volumes: Amount6 mL  /flask (T25 flask) Amount1 mL  /well (6w plate) Amount365 µL  /well (12w plate) Amount200 µL  /well (24w plate) Amount30 µL  /well (96w plate).

Aliquot cell suspension across plates according to the volumes given in the previous. Gently shake plates to evenly distribute cells and incubate (37°C; 5% CO2; 20% O2) for Duration24:00:00  .

Following 24-hour incubation, remove total volume of media and replace with Amount2 mL  /well (6w plate) Amount750 µL  /well (12w plate) Amount400 µL  /well (24w plate) Amount100 µL  /well (96w plate) B27 + Concentration10 micromolar (µM)   DAPT + Concentration200 micromolar (µM)   AA2P. Replace medium every 24 hours until D7 following terminal plating.

Note
While cells should be treated with DAPT for 6 days following terminal plating, it is not essential to change medium every day. Specifics are left to the discretion of the operator.

On D7 following terminal plating, remove the total volume of medium from cells and replace with Amount2 mL  /well (6w plate) Amount750 µL  /well (12w plate) Amount400 µL  /well (24w plate) Amount100 µL  /well (96w plate) B27 + Concentration200 micromolar (µM)   AA2P.

Part media change (i.e. remove half the media and replace with an equal volume of fresh B27 + Concentration200 micromolar (µM)   AA2P) every 7 days for the remaining time the cells are in culture.

After day 30 add Amount2 µL   of laminin to the media for feeding cells every 7 days.

Public workspaceDifferentiation of PSCs to Neural Progenitors

Differentiation of PSCs to Neural Progenitors