Mouse Embryonic Stem Cells Culture and Differentiation to Neural Stem Cells

Abhinav Anand; Francisco Bustos

Feb 20, 2025

Mouse Embryonic Stem Cells Culture and Differentiation to Neural Stem Cells

DOI

dx.doi.org/10.17504/protocols.io.q26g7mdp1gwz/v1

Abhinav Anand^1,2,
Francisco Bustos^1,2,3

¹Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD 57104, USA;
²Department of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA;
³Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD 57105, USA

Abhinav Anand

Sanford Research

DOI: dx.doi.org/10.17504/protocols.io.q26g7mdp1gwz/v1

Protocol Citation: Abhinav Anand, Francisco Bustos 2025. Mouse Embryonic Stem Cells Culture and Differentiation to Neural Stem Cells . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mdp1gwz/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: January 03, 2025

Last Modified: February 20, 2025

Protocol Integer ID: 117655

Keywords: Mouse embryonic stem cells, Neural stem cells, Neural differentiation, Pluripotency, naïve pluripotency, N2B27

Abstract

The property of stem cell pluripotency observed in the inner cell mass of the developing mouse embryo at the blastocyst stage can be modeled in vitro via mouse embryonic stem cells (mESCs). mESCs can be cultured in gelatinized plates with media containing Leukemia inhibitory factor (LIF) and fetal bovine serum (LIF and serum), which maintains a dynamic and heterogeneous pluripotency state. These cells can also be cultured in media with two kinase inhibitors (2i) + LIF, which maintains a homogeneous naive pluripotent state that is germline competent. These cells can be differentiated to the neural lineage by culturing them in neural stem cell (NSC) media, and NSCs can be isolated and cultured from this process. Here we describe protocols for culturing mESCs in LIF and serum or 2i + LIF conditions, and induction of neural differentiation to obtain and culture mESC-derived mouse NSCs.

Image Attribution

Images were obtained by Francisco Bustos, Sanford Research, Sioux Falls, South Dakota.

Protocol materials

ReagentDMEM/F-12, no glutamineThermo FisherCatalog #21331020Step 31.1
 
ReagentGlucose SolutionThermo FisherCatalog #A2494001Step 36.1
 
ReagentFetal Bovine Serum, Regular, USDA Safety TestedCorningCatalog #35-010-CVIn 2 steps
 
ReagentPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122In 3 steps
 
ReagentESGRO® Recombinant Mouse LIF ProteinMerck MilliporeSigma (Sigma-Aldrich)Catalog #ESG1107Step 5.7
 
ReagentNeurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049Step 31.2
 
ReagentGibco™ Bovine Albumin Fraction V (7.5% solution)Thermo Fisher ScientificCatalog #15260037Step 36.3
 
ReagentCHIR99021Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1046Step 30
 
ReagentGST-LIF recombinant proteinMRC-PPU Reagents and ServicesCatalog #DU1715In 2 steps
 
ReagentB-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #17504044In 2 steps
 
ReagentPD 0325901Merck MilliporeSigma (Sigma-Aldrich)Catalog #PZ0162Step 30
 
ReagentGibco™ Mouse EGF Recombinant Protein, PeproTech®Fisher ScientificCatalog #31509100UGStep 37.1
 
ReagentGibco™ DMEM/F-12 HEPESFisher ScientificCatalog #11-330-032Step 36
 
ReagentDMEM, high glucose, no glutamineThermo FisherCatalog #11960069Step 5
 
ReagentHuman LIF Recombinant Protein, PeproTech®Thermo Fisher ScientificCatalog #300-05-25UGStep 5.7
 
ReagentDMSO ≥ 99.9%VWR International (Avantor)Catalog #MK494802In 2 steps
 
ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CVIn 4 steps
 
ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050In 2 steps
 
Reagent2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500In 3 steps
 
ReagentStemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo Fisher ScientificCatalog #A1110501In 2 steps
 
ReagentGibco™ Mouse FGF-basic (FGF-2/bFGF) Recombinant Protein, PeproTech®Fisher ScientificCatalog #4503310UGStep 37.2
 
ReagentGibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055In 2 steps
 
ReagentMouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020In 3 steps
 
ReagentPBS (10X), pH 7.4Thermo FisherCatalog #70011069Step 7.2
 
ReagentExternally and Internally Threaded Cryogenic Storage VialsThermo FisherCatalog #12567501In 2 steps
 
ReagentN2 supplement (100x supplement)Gibco - Thermo Fisher ScientificCatalog #17502048In 2 steps
 
ReagentKnockOut&trade; Serum ReplacementThermo FisherCatalog #10828028Step 5.2
 
ReagentL-Glutamine (200 mM)Thermo FisherCatalog #25030149In 2 steps
 
ReagentGelatin from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890Step 6
 
Reagent0.05% Trypsin-EDTA, phenol redInvitrogen - Thermo FisherCatalog #25300054Step 10

Mouse Embryonic Stem Cells Culture in LIF and Serum conditions

1w 5d

This protocol can be used to culture mouse embryonic stem cell lines including CCE, J1, E14TG2a, and others.
CITATION
Bustos F, Segarra-Fas A, Chaugule VK, Brandenburg L, Branigan E, Toth R, Macartney T, Knebel A, Hay RT, Walden H, Findlay GM (2018). RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation..LINK
https://doi.org/10.1016/j.celrep.2018.04.022

CITATION
Villa F, Fujisawa R, Ainsworth J, Nishimura K, Lie-A-Ling M, Lacaud G, Labib KP (2021). CUL2(LRR1) , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle..LINK
https://doi.org/10.15252/embr.202052164
 

ESCs must be passed every Duration48:00:00   and media changed every Duration24:00:00  . However, occasionally cells can be grown for Duration48:00:00   without a media change if the plate is in low confluency.

ESCs can be grown in Amount10 mL   of ES-DMEM + LIF in a 10 cm plate.

Amount1000000 cells   typically give a confluent plate after Duration48:00:00  , after which a 1/10 or 1/20 splitting is normally sufficient for keeping stocks.

Preparation of ES-DMEM + LIF

For preparation of Amount600 mL   ES-DMEM +LIF: DMEM containing Concentration10 % (v/v)    fetal bovine serum (FBS), Concentration5 % (v/v)   Knock-Out serum replacement, Concentration50 ng/mL   GST-tagged leukemia inhibitory factor (LIF), Concentration100 U/mL   penicillin/streptomycin, Concentration2 millimolar (mM)   glutamine, Concentration0.1 millimolar (mM)   minimum essential media (MEM) Non-essential amino acids, Concentration1 millimolar (mM)    sodium pyruvate and Concentration0.1 millimolar (mM)   β-mercaptoethanol (BME). 

To a Amount500 mL     ReagentDMEM, high glucose, no glutamineThermo FisherCatalog #11960069  bottle add:

ReagentFetal Bovine Serum, Regular, USDA Safety TestedCorningCatalog #35-010-CV  : Amount60 mL  .

ReagentKnockOut&trade; Serum ReplacementThermo FisherCatalog #10828028 : Amount30 mL  .

ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050 : Amount6 mL  .

ReagentL-Glutamine (200 mM)Thermo FisherCatalog #25030149 : Amount6 mL  .

ReagentPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122 :Amount6 mL  .

Reagent2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500 : Amount4 µL  .

ReagentGST-LIF recombinant proteinMRC-PPU Reagents and ServicesCatalog #DU1715  to Concentration50 ng/mL final concentration : Amount10 µL   of Concentration3.12 mg/mL   stock.

Note
Alternative LIF sources:

ReagentESGRO® Recombinant Mouse LIF ProteinMerck MilliporeSigma (Sigma-Aldrich)Catalog #ESG1107   use Concentration500 U/mL final .

ReagentHuman LIF Recombinant Protein, PeproTech®Thermo Fisher ScientificCatalog #300-05-25UG    use  Concentration50 ng/mL final .

Filter in Stericup in biosafety cabinet to sterilize.
Equipment
Millipore® Stericup® Quick Release Vacuum Filtration System
NAME
0.22 um polyethersulfone membrane filtration system
TYPE
Millipore
BRAND
All Photos(3) Key Documents COA/COQ View All Docum
SKU
https://www.sigmaaldrich.com/US/en/product/mm/s2gpu05re
LINK
0.22 um filter
SPECIFICATIONS

Preparation of 0.1% Gelatin in PBS

Prepare 1% Gelatin.
 Dissolve Amount10 g  ofReagentGelatin from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890   powder in Amount1000 mL    ultrapure water.

Autoclave.

Prepare 0.1% Gelatin in PBS.

To a Amount500 mL   bottle add:

Amount45 mL   ReagentPBS (10X), pH 7.4Thermo FisherCatalog #70011069  .

Amount425 mL    ultrapure water.

Amount50 mL   of 1% Gelatin from step 1.

Filter in Stericup in biosafety cabinet to sterilize.
Equipment
Millipore® Stericup® Quick Release Vacuum Filtration System
NAME
0.22 um polyethersulfone membrane filtration system
TYPE
Millipore
BRAND
All Photos(3) Key Documents COA/COQ View All Docum
SKU
https://www.sigmaaldrich.com/US/en/product/mm/s2gpu05re
LINK
0.22 um filter
SPECIFICATIONS

Mouse Embryonic Stem Cells Passaging (10cm plate)

15m

Aspirate the media.

Wash cells in Amount5 mL   ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV  
twice.

Aspirate and add Amount2 mL   Reagent0.05% Trypsin-EDTA, phenol redInvitrogen - Thermo FisherCatalog #25300054 .

Incubate Duration00:05:00   at Temperature37 °C  .

Add Amount4 mL   of ES-DMEM +LIF  to the trypsinized cells and gently pipette 10 times to break up any remaining cell clumps.

Transfer to a Amount15 mL  conical tube.

Centrifuge Centrifigation1000 rpm, 25°C, 00:05:00 , aspirate, and resuspend in Amount1 mL   of media.

Gelatinize plates: add Amount5 mL   per 10 cm plate of Concentration0.1 % (v/v)   gelatin in PBS, spread and incubate for Duration00:05:00  , and aspirate.

Count cells and plate desired seeding density in Amount7 mL   ES-DMEM +LIF per 10 cm plate.

mESCs in ES-DMEM + LIF Media

Mouse Embryonic Stem Cells Freezing

Prepare Freezing media (80% ES-DMEM, 10% DMSO, 10% FBS).

For Amount10 mL   of freezing media add:

Amount8 mL   ES-DMEM + LIF.

Amount1 mL   ReagentDMSO ≥ 99.9%VWR International (Avantor)Catalog #MK494802 .

Amount1 mL   ReagentFetal Bovine Serum, Regular, USDA Safety TestedCorningCatalog #35-010-CV .

Split cells normally (steps 7-12), centrifuge Centrifigation1000 rpm, 00:05:00 , and gently resuspend in freezing media.

Transfer to ReagentExternally and Internally Threaded Cryogenic Storage VialsThermo FisherCatalog #12567501   and incubate overnight in a: 
Equipment
Freezing container, Nalgene® Mr. Frosty
NAME
Cryopreservation
TYPE
Mr. Frosty
BRAND
C1562-1EA
SKU
https://www.sigmaaldrich.com/US/en/product/sigma/c1562?srsltid=AfmBOop_0lwqk5VONiPEPXRsJIThyq_U1vmTjaIeiS_dhTHbNn_UcFG1
LINK
atTemperature-80 °C   for Duration24:00:00  .

Store at Temperature-150 °C  .

Mouse Embryonic Stem Cells Thawing

Thaw vial in a Temperature37 °C   water bath.

Prepare a tube with Amount9 mL  ES-DMEM + LIF.

Once the cells have thawed, transfer them to the tube and centrifuge for Centrifigation1000 rpm, 00:05:00 .

Aspirate supernatant.

Resuspend the pellet in ES-DMEM + LIF.

Transfer to a gelatinized plate.

Conversion to Naïve Pluripotency (2i + LIF)

1d 0h 10m

Protocol based on the following literature:

CITATION
Nichols J, Jones K, Phillips JM, Newland SA, Roode M, Mansfield W, Smith A, Cooke A (2009). Validated germline-competent embryonic stem cell lines from nonobese diabetic mice..LINK
https://doi.org/10.1038/nm.1996

CITATION
Mulas C, Kalkan T, von Meyenn F, Leitch HG, Nichols J, Smith A (2019). Defined conditions for propagation and manipulation of mouse embryonic stem cells..LINK
https://doi.org/10.1242/dev.173146

Resuspension of 2i kinase inhibitors (to make Concentration10 millimolar (mM)   stocks):
ReagentCHIR99021Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1046  
Molecular weight:  Concentration465.34 g/mol   
Resuspend Amount5 mg    in Amount1074.5 µL   DMSO.

ReagentPD 0325901Merck MilliporeSigma (Sigma-Aldrich)Catalog #PZ0162  
Molecular weight:  Concentration482.19 g/mol   
Resuspend Amount5 mg    in Amount1036.9 µL   DMSO.

For preparation of Amount250 mL   N2B27 Media (Concentration1 % (v/v)   B27 supplement, Concentration0.5 % (v/v)   N2 supplement , Concentration2 millimolar (mM)   glutamine ; Concentration0.1 millimolar (mM)   β-mercaptoethanol;  Concentration100 U/mL    penicillin/streptomycin in 1:1 DMEM/F12:Neurobasal medium)
In a Amount250 mL   Stericup.
Equipment
Millipore® Stericup® Quick Release Vacuum Filtration System
NAME
0.22 um polyethersulfone membrane filtration system
TYPE
Millipore
BRAND
All Photos(3) Key Documents COA/COQ View All Docum
SKU
https://www.sigmaaldrich.com/US/en/product/mm/s2gpu05re
LINK
0.22 um filter
SPECIFICATIONS
add:

ReagentDMEM/F-12, no glutamineThermo FisherCatalog #21331020  : Amount125 mL   .

ReagentNeurobasal™ MediumGibco - Thermo FisherCatalog #21103049  : Amount125 mL   .

ReagentN2 supplement (100x supplement)Gibco, ThermoFisherCatalog #17502048  : Amount1.25 mL   .

ReagentB-27™ Supplement (50X), serum freeGibco - Thermo FisherCatalog #17504044  : Amount2.5 mL  . 

ReagentPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122  : Amount2.5 mL   .

ReagentL-Glutamine (200 mM)Thermo FisherCatalog #25030149  : Amount2.5 mL   .

Reagent2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500  : Amount1.75 µL   .

For preparation of Amount30 mL   2i + LIF Media.  To Amount30 mL   of N2B27 in Amount50 mL   conical tube add :

 Amount9 µL   of Concentration10 millimolar (mM) stock  CHIR99021 (from step 22) toConcentration3 micromolar (µM)   final concentration.

Amount3 µL   of Concentration10 millimolar (mM) stock  PD0325901 (from step 22) to Concentration1 micromolar (µM)   final concentration.

ReagentGST-LIF recombinant proteinMRC-PPU Reagents and ServicesCatalog #DU1715  to Concentration50 ng/mL final  concentration.

Note
From Concentration3.12 mg/mL stock  , dilute 1/10 in ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV  
and add Amount5 µL   of this dilution to the 2i + LIF media mix.

Filter through syringe filter into conical tube. Store atTemperature4 °C  .

2i + LIF conversion (Do in 6-well or equivalent size plate).

In gelatinized plates, seed Amount80000 cells   per cm2 in 6-well plates in ES-DMEM + LIF, and incubate Duration24:00:00   at Temperature37 °C  .

Wash with Amount1 mL   ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV  twice.

Add Amount200 µL   ReagentStemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo FisherCatalog #A1110501  and incubate at Temperature37 °C   for Duration00:05:00  .

Resuspend in Amount400 µL   2i+LIF media.

CentrifugeCentrifigation0.7 x g, Room temperature, 00:05:00 .

Aspirate supernatant and resuspend in Amount500 µL   2i+LIF media.

AddAmount100 µL   of cell suspension (or Amount80000 cells  per cm2) to new gelatinized plate.

Add Amount2 mL   2i+LIF media.

After 3 splits (steps 33.2 - 33.8 three times) cells are now converted to 2i + LIF (naïve pluripotency).

mESCs converted to naïve (2i + LIF) Pluripotency

Mouse Neural Stem Cell Differentiation from mESCs

1w 6d 3h 5m

Protocol based on the following literature:

CITATION
Pollard SM, Benchoua A, Lowell S (2006). Neural stem cells, neurons, and glia..LINK
https://doi.org/

CITATION
Pollard SM (2013). In vitro expansion of fetal neural progenitors as adherent cell lines..LINK
https://doi.org/10.1007/978-1-62703-574-3_2

For preparation of Amount530 mL (approximately)  NSC media (Concentration1 % (v/v)   B27 supplement, Concentration0.5 % (v/v)   N2 supplement , Concentration2.9 g/L   glucose,  Concentration0.1 millimolar (mM)   minimum essential media (MEM) Non-essential amino acids,  Concentration0.012 % w/v   Bovine Albumin Fraction V,  Concentration0.1 millimolar (mM)   β-mercaptoethanol;  Concentration100 U/mL    penicillin/streptomycin in DMEM/F-12, HEPES).
To a Amount500 mL   bottle of ReagentGibco™ DMEM/F-12 HEPESFisher ScientificCatalog #11-330-032  
add:

Amount7.25 mL   ReagentGlucose SolutionThermo FisherCatalog #A2494001 . 

Amount5 mL   ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050  .

Amount800 µL   ReagentGibco™ Bovine Albumin Fraction V (7.5% solution)Thermo Fisher ScientificCatalog #15260037  .

Amount5 mL   ReagentPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122  .

Amount3.5 µL   Reagent2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500  .

Amount2.5 mL   ReagentN2 supplement (100x supplement)Gibco, ThermoFisherCatalog #17502048  .

Amount5 mL   ReagentB-27™ Supplement (50X), serum freeGibco - Thermo FisherCatalog #17504044  .

Filter in Stericup in biosafety cabinet to sterilize.
Equipment
Millipore® Stericup® Quick Release Vacuum Filtration System
NAME
0.22 um polyethersulfone membrane filtration system
TYPE
Millipore
BRAND
All Photos(3) Key Documents COA/COQ View All Docum
SKU
https://www.sigmaaldrich.com/US/en/product/mm/s2gpu05re
LINK
0.22 um filter
SPECIFICATIONS

Supplements:

Epidermal growth factor (EGF) Concentration20 μg/mL stock   ReagentGibco™ Mouse EGF Recombinant Protein, PeproTech®Fisher ScientificCatalog #31509100UG : Resuspend Amount100 µg    in Amount200 µL   sterile ultrapure H2O. Then add Amount4800 µL   of Concentration0.1 % w/v   BSA.

For a final concentration of Concentration10 ng/mL   use  Amount1 µL   of stock per Amount2 mL   media (1:2000).

Fibroblast growth factor 2 (FGF-2/bFGF)   Concentration20 μg/mL stock   ReagentGibco™ Mouse FGF-basic (FGF-2/bFGF) Recombinant Protein, PeproTech®Fisher ScientificCatalog #4503310UG 
Resuspend Amount10 µg    in Amount100 µL   sterile ultrapure H2O. Then add Amount400 µL   of Concentration0.1 % w/v   BSA.

For a final concentration of Concentration10 ng/mL   use  Amount1 µL   of stock per Amount2 mL   media (1:2000).

ReagentMouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020  stock is ~Concentration2 mg/mL  

For a  final concentration of Concentration2 μg/mL   use Amount10 µL   of stock per Amount10 mL   of NSC media.

NSC differentiation.

From 2i+LIF cells.

In gelatinized plates, seed Amount1250 cells   per cm2 in 2i + LIF media, and incubate for Duration24:00:00   at Temperature37 °C   in a 6-well plate.

Wash gently with ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV  twice.

Change media for NSC media (without supplements), and incubate at Temperature37 °C   for Duration24:00:00   .

Change media every Duration24:00:00   for Duration144:00:00 (6 days) .

Replate Amount2000000 cells   in a nongelatinized 10-cm plate (or less in a proportionally-sized plate) in NSC complete media: NSC media + EGF + FGF2 (Amount1 µL   of diluted stock of each per Amount2 mL   media).

Grow neurospheres for Duration24:00:00  -Duration72:00:00   (until large spheres are formed).

Harvest floating aggregates by transferring media with floating cells to a Amount15 mL    conical tube.

Centrifuge Centrifigation2000 rpm, 00:05:00 .

Aspirate media (leave a little media on pellet).

Resuspend pellet in NSC complete media + Laminin.

Plate in 6-well plate coated with Laminin.

Note
Laminin coating (1/200):

Make 1X PBS calcium, magnesium (PBS Ca2+,Mg2+):
Dilute ReagentGibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055   from 10X to 1X. Mix  Amount50 mL   ReagentGibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055  in Amount450 mL   ultrapure H2O. Filter in Stericup in biosafety cabinet to sterilize.
Equipment
Millipore® Stericup® Quick Release Vacuum Filtration System
NAME
0.22 um polyethersulfone membrane filtration system
TYPE
Millipore
BRAND
All Photos(3) Key Documents COA/COQ View All Docum
SKU
https://www.sigmaaldrich.com/US/en/product/mm/s2gpu05re
LINK
0.22 um filter
SPECIFICATIONS
  For coating solution: add Amount5 µL   of ReagentMouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020  to Amount995 µL    1X PBS Ca2+,Mg2+ in TC hood.

Incubate Duration03:00:00   atTemperatureRoom temperature   or Temperature37 °C   before plating cells.

Grow until NSCs have migrated out of neurospheres, replacing media every day.

Split using Accutase to a new Laminin-coated plate/well.

For established NSCs, media will be NSC complete media + Laminin (optional if plate is laminin-coated).

Typical NSCs

Freezing Mouse Neural Stem Cells

Release with ReagentStemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo FisherCatalog #A1110501  as above, but resuspend in freezing medium: Concentration90 % (v/v)   NSC media + Concentration10 % (v/v)  ReagentDMSO ≥ 99.9%VWR International (Avantor)Catalog #MK494802 .

Use Amount500 µL   of suspension per freezing vial ReagentExternally and Internally Threaded Cryogenic Storage VialsThermo FisherCatalog #12567501 .

Place vials in a
Equipment
Freezing container, Nalgene® Mr. Frosty
NAME
Cryopreservation
TYPE
Mr. Frosty
BRAND
C1562-1EA
SKU
https://www.sigmaaldrich.com/US/en/product/sigma/c1562?srsltid=AfmBOop_0lwqk5VONiPEPXRsJIThyq_U1vmTjaIeiS_dhTHbNn_UcFG1
LINK
atTemperature-80 °C   before moving to Temperature-150 °C   long-term.

Thawing Mouse Neural Stem Cells

Thaw vial in a Temperature37 °C   water bath.

Prepare a tube with  Amount5 mL   NSC media.

Once the cells have thawed, transfer them to the tube and centrifuge for Centrifigation1000 rpm, 00:05:00 .

Aspirate supernatant.

Resuspend the pellet in NSC complete media + Laminin.

Transfer to a ReagentMouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020 (LAM1)-coated well.

Protocol references

Pollard SM. In vitro expansion of fetal neural progenitors as adherent cell lines. Methods Mol Biol. 2013;1059:13-24. doi: 10.1007/978-1-62703-574-3_2. PMID: 23934830.

Pollard SM, Benchoua A, Lowell S. Neural stem cells, neurons, and glia. Methods Enzymol. 2006;418:151-69. doi: 10.1016/S0076-6879(06)18010-6. PMID: 17141035.

Mulas C, Kalkan T, von Meyenn F, Leitch HG, Nichols J, Smith A. Defined conditions for propagation and manipulation of mouse embryonic stem cells. Development. 2019 Mar 26;146(6):dev173146. doi: 10.1242/dev.173146. Erratum in: Development. 2019 Apr 11;146(7):dev178970. doi: 10.1242/dev.178970. PMID: 30914406; PMCID: PMC6451320.

Nichols J, Jones K, Phillips JM, Newland SA, Roode M, Mansfield W, Smith A, Cooke A. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nat Med. 2009 Jul;15(7):814-8. doi: 10.1038/nm.1996. Epub 2009 Jun 2. PMID: 19491843.

Villa F, Fujisawa R, Ainsworth J, Nishimura K, Lie-A-Ling M, Lacaud G, Labib KP. CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle. EMBO Rep. 2021 Mar 3;22(3):e52164. doi: 10.15252/embr.202052164. Epub 2021 Feb 15. PMID: 33590678; PMCID: PMC7926238.

Bustos F, Segarra-Fas A, Chaugule VK, Brandenburg L, Branigan E, Toth R, Macartney T, Knebel A, Hay RT, Walden H, Findlay GM. RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation. Cell Rep. 2018 May 8;23(6):1599-1611. doi: 10.1016/j.celrep.2018.04.022. PMID: 29742418; PMCID: PMC5976579.

Citations

Step 1

Bustos F, Segarra-Fas A, Chaugule VK, Brandenburg L, Branigan E, Toth R, Macartney T, Knebel A, Hay RT, Walden H, Findlay GM. RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation.

https://doi.org/10.1016/j.celrep.2018.04.022

Step 1

Villa F, Fujisawa R, Ainsworth J, Nishimura K, Lie-A-Ling M, Lacaud G, Labib KP. CUL2(LRR1) , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.

https://doi.org/10.15252/embr.202052164

Step 29

Nichols J, Jones K, Phillips JM, Newland SA, Roode M, Mansfield W, Smith A, Cooke A. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice.

https://doi.org/10.1038/nm.1996

Step 29

Mulas C, Kalkan T, von Meyenn F, Leitch HG, Nichols J, Smith A. Defined conditions for propagation and manipulation of mouse embryonic stem cells.

https://doi.org/10.1242/dev.173146

Step 35

Pollard SM, Benchoua A, Lowell S. Neural stem cells, neurons, and glia.

https://doi.org/

Step 35

Pollard SM. In vitro expansion of fetal neural progenitors as adherent cell lines.

https://doi.org/10.1007/978-1-62703-574-3_2

Acknowledgements

We thank Charles Williams, PhD (University of Edinburgh, UK) for protocols and Intisar Koch (Sanford Research) for critical reading of the protocol.

Public workspaceMouse Embryonic Stem Cells Culture and Differentiation to Neural Stem Cells

Mouse Embryonic Stem Cells Culture and Differentiation to Neural Stem Cells