Apr 28, 2026

hiPSC derived Neural Stem Cells (NSCs) generation, banking and quality control

DOI
https://dx.doi.org/10.17504/protocols.io.x54v9pxoqg3e/v1
hiPSC derived Neural Stem Cells (NSCs) generation, banking and quality control
  • 1Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany
Valeria Fernandez Vallone
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v9pxoqg3e/v1
Protocol Citation: Valeria Fernandez Vallone, Judit Kuechler, Harald Stachelscheid 2026. hiPSC derived Neural Stem Cells (NSCs) generation, banking and quality control. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pxoqg3e/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
We use this protocol and it's working
Created: February 07, 2024
Last Modified: April 28, 2026
Protocol  Integer ID: 94846
Keywords: NSC, hPSC differentiation, neural stem cells, pluripotent stem cells, neural stem cell, pluripotent stem cell, induced pluripotent stem cell, procedures for nsc cryobanking, nsc, hipsc, flow cytometry, cell, nsc cryobanking
Funders Acknowledgements:
European Union’s Horizon 2020 research and innovation program
Grant ID: 825161
Bundesministerium für Bildung und Forschung, Germany
Grant ID: 16GW0191
Abstract
This protocol describes the differentiation of human induced pluripotent stem cells (hiPSCs) into neural stem cells (NSCs). It includes flow cytometry-based analysis on day 7 of differentiation as a quality control measure, as well as procedures for NSC cryobanking and batch control following thawing and culture.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES

Use sterile material
  • 1.5/2 mL Cryo tubes
  • 1/5/10 mL pipettes
  • 15/50 mL conical tubes
  • 10/200/1000µL tips and micropipettes
  • Culture treated 6-well plates
  • 40/100 µm Cell strainers
  • 5 mL Round-bottom FACS tubes

  • Microscope
  • Centrifuge
  • Vortex
  • Class II Biosafety Cabinet
  • Cell counting equipment
  • Flow cytometer e.g. MACS Quant VYB
  • Aspirator pump with disposable pipette
  • Freezing container (Mr. Frosty) filled with 100% 2-propanol (pre-chilled), alternatively equipment for automated controlled freezing.


MEDIA AND REAGENT

  • Essential 8Life TechnologiesCatalog #A1517001
  • KnockOut™ DMEM/F-12Thermo FisherCatalog #12660012
  • DorsomorphinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5499-5MG or (Biovision, 1686-5) (see appendix)
  • SB 431542 hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4317 (see appendix)
  • B27 supplement without retinoic acid (50x)Gibco - Thermo Fisher ScientificCatalog #17504044 (see appendix)
  • N-2 Supplement (100X)Thermo FisherCatalog #17502048 (see appendix)
  • DPBS, calcium, magnesiumThermo FisherCatalog #14040133
  • DPBS, no calcium, no magnesiumThermo FisherCatalog #14190144
  • Dimethylsulfoxid (DMSO)Carl RothCatalog #A994.2
  • ACCUTASE™STEMCELL Technologies Inc.Catalog #07920
  • TrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029
  • Advanced DMEM/F-12Thermo Fisher ScientificCatalog #12634-010
  • Penicillin/StreptomycinInvitrogen - Thermo FisherCatalog #15140-122
  • Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413302
  • Neurobasal™ MediumThermo Fisher ScientificCatalog #21103049
  • PSC Neural Induction MediumThermo FisherCatalog #A1647801 includes Neural Induction Supplement (Thermo Fisher, A16477-01) (see appendix) and Neurobasal Medium
  • ROCK inhibitor (Ri) Y-27632STEMCELL Technologies Inc.Catalog #72308
  • DMEM/F-12, HEPESThermo FisherCatalog #11330057
  • MACS BSA Stock SolutionMiltenyi BiotecCatalog # 130-091-376
  • FoxP3 Staining Buffer SetMiltenyi BiotecCatalog #130-093-142
  • Corning® 1L Cell Culture Grade Water Tested to USP Sterile Water for Injection SpecificationsCorningCatalog #25-055-CM
  • MACSQuant® Running BuffersMiltenyi BiotecCatalog #130-092-747
  • ULTRAPURE O.5M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
  • Trypan blueThermo Fisher ScientificCatalog #T10282

NSC marker antibodies

  • Nestin-PEMiltenyi BiotecCatalog #130-119-799
  • Pax6-APCMiltenyi BiotecCatalog #130-123-328
  • Sox1-FITCMiltenyi BiotecCatalog #130-111-042
  • Sox2-FITCMiltenyi BiotecCatalog #130-120-721


  • BAMBANKERBioCat GmbHCatalog #BB03-NP 20 mL or 100 mL


SUPPORTING PROTOCOLS

Coating of tissue Culture Vessels for hPSC
Survival factors for hPSC growth
Single cell passaging of hPSC



Protocol materials
FoxP3 Staining Buffer SetMiltenyi BiotecCatalog #130-093-142
DMEM/F-12, HEPESThermo FisherCatalog #11330057
DPBS, calcium, magnesiumThermo FisherCatalog #14040133
TrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029
Penicillin/StreptomycinInvitrogen - Thermo FisherCatalog #15140-122
Pax6-APCMiltenyi BiotecCatalog #130-123-328
Sox2-FITCMiltenyi BiotecCatalog #130-120-721
BAMBANKERBioCat GmbHCatalog #BB03-NP
B27 supplement without retinoic acid (50x)Gibco - Thermo Fisher ScientificCatalog #17504044
DorsomorphinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5499-5MG
Advanced DMEM/F-12Thermo Fisher ScientificCatalog #12634-010
Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413302
PSC Neural Induction MediumThermo FisherCatalog #A1647801
ACCUTASE™STEMCELL Technologies Inc.Catalog #07920
Trypan blueThermo Fisher ScientificCatalog #T10282
Nestin-PEMiltenyi BiotecCatalog #130-119-799
Sox1-FITCMiltenyi BiotecCatalog #130-111-042
SB 431542 hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4317
Essential 8Life TechnologiesCatalog #A1517001
N-2 Supplement (100X)Thermo FisherCatalog #17502048
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190144
Neurobasal™ MediumThermo Fisher ScientificCatalog #21103049
Corning® 1L Cell Culture Grade Water Tested to USP Sterile Water for Injection SpecificationsCorningCatalog #25-055-CM
MACSQuant® Running BuffersMiltenyi BiotecCatalog #130-092-747
ULTRAPURE O.5M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
Dimethylsulfoxid (DMSO)Carl RothCatalog #A994.2
MACS BSA Stock SolutionMiltenyi BiotecCatalog # 130-091-376
KnockOut™ DMEM/F-12Thermo FisherCatalog #12660012
ROCK inhibitor (Ri) Y-27632STEMCELL Technologies Inc.Catalog #72308
Before start
hPSC culture conditions and survival factors choice depend on hPSC line and individual lab practices.
For options refer to protocol collection:
Good practices in hPSC handling

Coating of tissue Culture Vessels for hPSC

Survival factors for hPSC growth

Single cell passaging of hPSC
Differentiation of hiPSC into NSCs
1w 1d 1h 15m
Day -2 or -3
  1. Monitor morphology and confluence (~70%) of human induced pluripotent stem cells (hiPSCs) prior starting.
  2. Perform a single cell passage using TrypLE Select or Accutase.
  3. Seed hiPSCs at a density of 0.5 x 105/cm2 onto Geltrex coated wells of a 6 well-plate in E8 medium containing ROCK inhibitor (10 micromolar (µM) ).
30m
Day -2 to -1
Perform daily medium changes with 2-3 mL E8 medium without ROCK inhibitor and culture for 48:00:00 .
2d
Day 0
When cultures reach 60-80% confluence, replace medium with Neural Induction Medium (NIM) (Table 1). Use 2 mL for one well of a 6 well-plate.
15m

Table 1. Preparation of Neural Induction Medium (NIM)
ABCDE
ComponentFinal concentration500 mL125 mL50 mL
DMEM/F12 + 2.5 mM Glutamine + 15 mM HEPES 480 mL 120 mL 48 mL
B27 Supplement (50X) 1X 10 mL 2.5 mL 1 mL
N-2 Supplement (100X) 1X 5 mL 1.25 ml 0.5 mL
Dorsomorphin (Stock sol. 10 mM) 2 µM 100 µL 25 µL 10 µL
SB431542 (Stock sol. 50 mM) 10 µM 100 µL 25 µL 10 µL
Pen/Strep 1X 5 mL 1.25 mL 0.5 mL
Refer to appendix for stock solution preparation.
Store at +4ºC until media is used up. Use within 2 weeks from the date of preparation.
30m
Day 1-3
Perform daily medium change (2.5 – 3.5 mL / well) with NIM.
3d
Day 4-6
On day 4 of neural induction, cells will be reaching confluency. Increase volume of pre-warmed complete NIM per well to 5 mL .
3d

Note
  • It is possible to perform one double feed (8 mL/ well), with the next medium change 48 h later. However, it is not recommended either to go longer than 1 day without a medium change, or to feed the cultures every other day continuously.

  • Due to high cell density in the culture from day 4 onwards, doubling the volume of NIM is very crucial for cell nutrition. Also, minimal cell death should be observed from day 4 to 7 after neural induction. If the color of cells turns brownish with many floating cells during day 4 to 7 of neural induction, it indicates that the starting density of iPSCs was too high and should be reduced next time.


It is recommended to monitor cell morphology by taking microscopic pictures. See Figure 1 and 2 for exemplary cell morphology during the differentiation.

Expected result

Figure 1. Representative pictures showing morphology change of hiPSC line BIHi250-A (day -1, ROCKi morphology) until day 1 of differentiation. On day 0 (day of induction) the cells should be about 50-80% dense. During the next days, the cells become 100% dense.


Figure 2. Representative images showing the morphology change of hiPSC line BIHi250-A between day 5 and 7 (endpoint) during neural induction and differentiation.



Quality Control of NSCs by Marker Expression Prior to Banking
3h 45m
Before starting prepare solutions needed for FACS staining as follows:

A. Prepare a buffer containing PBS (pH 7.5), 0.5% bovine serum albumin (BSA) and 2 mM EDTA according to Table 2.

Table 2. Preparation of FACS buffer
AB
ReagentVolume [mL]
PBS without Ca2+ and Mg2+ 95
BSA stock solution 10% 5
UltraPure 0.5 M EDTA 0.4
Store at 4°C.
B. Fixation and permeabilization buffer

Note
Prepared according to manufacturer´s instructions. For reference see Tables 3 and 4.FoxP3 Staining Buffer SetMiltenyi BiotecCatalog #130-093-142
It is recommended to freshly prepare the buffers and keep them at 4°C during the whole process.


Table 3. Preparation of fixation buffer
ABCD
Sample NumberVolume to be prepared [mL] Fix/Perm Solution 1 [mL]Fix/Perm solution 2 [mL]
1 0.5 0.125 0.375
6 3 0.75 2.25
12 6 1.5 4.5
Option: cells can be fixed using a 4% PFA solution as an alternative to fixative provided in the kit
Table 4. Preparation of permeabilization buffer
ABCD
Sample NumberVolume to be prepared [mL]Permeabilisation Buffer [mL]Distilled water [mL]
1 1.20.121.08
6 7.20.726.48
12 14.41.4412.96
Option: cells can be permeablized using a 0.1% saponin / 1% BSA solution as an alternative to permeabilization solution provided in the kit

30m
Day 7
Prepare a single cell suspension from one well NSCs using TrypLE Select or Accutase.
20m
Centrifuge NSC suspension at 300 x g, Room temperature for 00:05:00

5m
Resuspend the NSCs in 1 mL Neurobasal media supplemented with ROCK inhibitor
(10 micromolar (µM) ) and perform cell count.
5m
Prepare three FACS tubes with 2 - 3 x 105 cells per tube:
Tube 1: non-stained sample (auto-fluorescence)
Tube 2: SOX2-FITC/PAX6-APC
Tube 3: SOX1-FITC/NESTIN-PE


Note
  • It is recommended to add to the FACS panel respective tubes for isotype control according to antibodies host, concentration and fluorophore.
  • It is recommended for FACS panel establishment to prepare single stainings to perform compensation during or post-acquisition of fluorophores with overlapping emission spectra.

5m
Add 1 mL of FACS buffer (Table 2) to the cell suspension in the FACS tubes.
1m
Centrifuge cells at 300 x g, 00:03:00 .
3m
Decant the supernatant into a waste bottle and carefully blot the tube on a cloth.

Note
Do not tip the tube to avoid losing cells.

3m
Resuspend sample pellets in 0.5 mL of cold, freshly prepared fixation solution (Table 3).
3m
Mix well and incubate for 00:30:00 in the dark at 4 °C .
30m
Wash cells by adding 1 mL of cold permeabilization buffer (Table 4).
3m
Centrifuge at 300 x g, 00:03:00 .
3m
Wash cells by adding 0.5 mL of cold permeabilisation buffer (Table 4)
3m
Centrifuge at 300 x g, 00:03:00 .
3m
Decant the supernatant into a waste bottle and carefully blot the tube on a cloth.
3m
Resuspend each pellet in 100 µL of cold permeabilisation buffer.

3m
Add antibodies to tubes 2 and 3 according to Table 5.

Table 5. Antibody staining
AB
Antibody Volume per tube [µL]
SOX2-FITC / PAX6-APC 2
SOX1-FITC / NESTIN-PE 2

3m
Mix well and incubate for01:00:00 in the dark at 4 °C .
1h
Wash cells by adding 0.5 mL of cold permeabilisation buffer.
3m
Centrifuge at300 x g, 00:03:00 .
3m
Decant the supernatant into a waste bottle and carefully blot the tube on a cloth.
3m
Resuspend each pellet in300 µL of FACS buffer and proceed to FACS measurement.

Note
  • It is recommended to mix stained samples gently.
  • If cell clumps are still visible, pipet the cell suspensions through a 40 µm cell strainer into 5 mL FACS tubes.
  • Perform FACS analysis immediately or store the samples closed and dark at 4°C up to 1 week.


Expected result
Double positive SOX2-FITC/PAX6-APC above 85-90%
Double positive SOX1-FITC/NESTIN-PE above 85-90%

30m
NSC bank cryopreservation
1d 0h 49m
When NSCs reach confluency (usually day 7), wash cells with 1 mL of PBS without Ca2+ and Mg2+
per well of 6 well-plate.

Note
  • From one 100% confluent well of a 6-well plate, up to 1.0 – 1.5 x 107 cells can be harvested.
  • It is recommended to freeze 1.5 – 3.0 x 106 NSCs per cryotube.
  • Based on these estimations, prepare up to 10 cryotubes for one 6 well-plate. Make sure to have enough labelled tubes ready in advance, or use barcoded tubes.

5m
Aspirate PBS and add 1 mL of TrypLE Select or Accutase to each well and incubate for00:10:00 at 37 °C until most cells detach. After incubation, tap the culture vessel to detach all cells.

Note
Incubation time for enzymatic dissociation can be extended additional 00:05:00 to 00:10:00 when NSCs are tighly attached.
Do not pipette the cells. Mechanical dissociation at this point will result in cell death.
It requieres experience to be able to balance incubation times to accelerate the dissociation process without causing excesively cell death.

10m
To stop the enzymatic reaction add 2 mL of pre-warmed NIM containing10 micromolar (µM) ROCK inhibitor.
3m
Gently pipet the cell suspension up and down for 3-4 times using a 1000 µL pipette to break up any remaining cell clumps and detach residual cells from plate surface.

Note
If clumps are still seen, use a 1000 µL microliter pipette to pass the cell suspension through a 100 μm cell strainer and transfer them to a 50 mL conical tube.

3m
Perform cell count.
5m
Centrifuge the cells at 300 x g, 00:05:00 at Room temperature .
5m
Remove supernatant and resuspend pellet in an adequate volume of Bambanker containing10 micromolar (µM) ROCK inhibitor. The cell concentration should be in the range of 1.5 – 3.0 x 106 NSCs per mL of Bambanker.
3m
Transfer 1 mL of cell suspension (containing 1.5 – 3.0 x 106 cells) per cryotube. Place the cryotubes in a freezing container and store at -80°C freezer Overnight .

Note
When available, the use of an automated controlled freezing device is recommended.

15m
After 24:00:00 place NSC bank in a liquid nitrogen tank for long-term storage.

1d
Quality control of NSCs bank by survival upon thawing
2d 1h 3m
Before starting prepare:

1. Geltrex-coated vessels before performing the next steps.

2. 10 mL Neurobasal Medium containing 10 micromolar (µM) ROCK inhibitor and keep it at 37 °C until use.

3. 50 mL Neural Expansion Media (NEM) according to table 6.

Table 6. Preparation of Neural Expansion Medium (NEM)
ABCDE
ComponentFinal concentration500 mL100 mL50 mL
Advanced DMEM/F12 0.5X 245 mL 49 mL 24.5 mL
Neurobasal Medium 0.5X 245 mL 49 mL 24.5 mL
Neural Induction Supplement 50X10 mL 2 mL 1 mL
Pen/Strep 1X5 mL 1 mL 0.5 mL
It is recommended to thaw and prepare aliquots of Neural Induction Supplement in advance (see Appendix).
Store medium at +4ºC. Use within one week from the date of preparation.

40m
Remove cryotube containing NSCs from liquid nitrogen and perform a quick thaw in a 37 °C water bath. Carefully swirl the cryotube in the water, avoid immersing it above the level of the cap.

Note
Remove the cryotube from the warm bath when still contains a remnant piece of ice.

5m
Disinfect the tube by wiping with 80% Ethanol using a tissue.
1m
Using a 5 mL pipette remove cells from cryotube and transfer them to a 15 mL falcon tube.
2m
Slowly (drop wise) add about 9 mL of Neurobasal Medium containing 10 micromolar (µM) ROCK inhibitor to the cells, while gently swirling the tube.

Note
This step helps reducing osmotic shock.

3m
Gently pipet up and down to break up any clumps.
1m
Centrifuge at 300 x g, 00:05:00 .
5m
Remove the supernatant and resuspend pellet in 2 mL of NEM containing 10 micromolar (µM) ROCK inhibitor.
1m
Perform cell count using Trypan Blue and document cell viability.


Expected result
Viability at thawing above 70-80% is recommended as QC upon thawing.


5m
Seed 1 x 105 – 1.5 x 105 NSCs/cm2 in Geltrex coated vessels and incubate at 37 °C , 5% CO2 incubator for 24:00:00 .

Note
Optimal NSC seeding density may vary between hiPSC lines.
It is recommended to test 3-4 seeding densities close to the range suggested in this step.
It is crucial to seed NSCs densely as they require good cell-to-cell contact.

2d
The day after, change media to NEM without ROCK inhibitor and monitor cell growth over the next days.

Note
As reference, use 2 mL media per well of a 6 well-plate. Volume should be adapted to culture density avoiding high metabolic environment.


Optional QC on NSC culture upon expansion include marker staining (immunofluorescence or FACS) for SOX2, PAX6, NESTIN, SOX1. For FACS refer to steps 9-30.
Appendix
1h 30m
REAGENTS PREPARATION

Dorsomorphin (BMP inhibitor)
Prepare a 10 millimolar (mM) stock solution in DMSO, therefore dissolve 5 mg Dorsomorphin in 1.25 mL of DMSO.
Make aliquots á 50 µL and store at -20 °C . Once thawed, keep at 4 °C .

SB431542 (TGF-β inhibitor)
Prepare a 50 millimolar (mM) stock solution in DMSO, therefore dissolve5 mg SB431542 in 260.5 µL DMSO.
Make aliquots á 50 µL and store at -20 °C . Once thawed, keep at 4 °C .

B27 supplement (50X)
Thaw B27 supplement Overnight at 4 °C .
Make aliquots in working volumes (e.g. 1 mL ) and store at -20 °C . Do not freeze-thaw more than twice.

N-2 supplement (100X)
Thaw N-2 supplement Overnight at 4 °C .
Aliquot in working volumes (e.g. 0.5 mL) and store at -20 °C . Do not freeze-thaw more than twice.

Neural Induction Supplement (10 mL )
Thaw Neural Induction Supplement overnight at 4 °C .
Aliquot in working volumes (e.g. 1 mL ) and store at -20 °C . Do not freeze-thaw more than once.