Feb 20, 2025
Mouse Embryonic Stem Cells Culture and Differentiation to Neural Stem Cells
- Abhinav Anand1,2,
- Francisco Bustos1,2,3
- 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
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, mouse embryonic stem cells culture, differentiation to neural stem cell, mouse embryonic stem cell, neural stem cell, developing mouse embryo, stem cell pluripotency, mouse embryo at the blastocyst stage, property of stem cell pluripotency, neural lineage, induction of neural differentiation, neural differentiation, derived mouse nsc, fetal bovine serum, cell, cultured from this process, cultured in gelatinized plate, blastocyst stage, heterogeneous pluripotency state
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
Mouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020
DMEM, high glucose, no glutamineThermo FisherCatalog #11960069
KnockOut™ Serum ReplacementThermo FisherCatalog #10828028
Fetal Bovine Serum, Regular, USDA Safety TestedCorningCatalog #35-010-CV
MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050
L-Glutamine (200 mM)Thermo FisherCatalog #25030149
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500
ESGRO® Recombinant Mouse LIF ProteinMerck MilliporeSigma (Sigma-Aldrich)Catalog #ESG1107
Human LIF Recombinant Protein, PeproTech®Thermo Fisher ScientificCatalog #300-05-25UG
GST-LIF recombinant proteinMRC-PPU Reagents and ServicesCatalog #DU1715
Gelatin from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890
PBS (10X), pH 7.4Thermo FisherCatalog #70011069
0.05% Trypsin-EDTA, phenol redInvitrogen - Thermo FisherCatalog #25300054
DMSO ≥ 99.9%VWR International (Avantor)Catalog #MK494802
Externally and Internally Threaded Cryogenic Storage VialsThermo FisherCatalog #12567501
DMEM/F-12, no glutamineThermo FisherCatalog #21331020
Neurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049
N2 supplement (100x supplement)Gibco - Thermo Fisher ScientificCatalog #17502048
B-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #17504044
Glucose SolutionThermo FisherCatalog #A2494001
Gibco™ Bovine Albumin Fraction V (7.5% solution)Thermo Fisher ScientificCatalog #15260037
StemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo Fisher ScientificCatalog #A1110501
Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV
CHIR99021Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1046
PD 0325901Merck MilliporeSigma (Sigma-Aldrich)Catalog #PZ0162
Gibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055
Gibco™ Mouse FGF-basic (FGF-2/bFGF) Recombinant Protein, PeproTech®Fisher ScientificCatalog #4503310UG
Gibco™ Mouse EGF Recombinant Protein, PeproTech®Fisher ScientificCatalog #31509100UG
Gibco™ DMEM/F-12 HEPESFisher ScientificCatalog #11-330-032
Troubleshooting
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
LINK
Citation
LINK
5d
ESCs must be passed every 48:00:00 and media changed every 24:00:00 . However, occasionally cells can be grown for 48:00:00 without a media change if the plate is in low confluency.
5d
ESCs can be grown in 10 mL of ES-DMEM + LIF in a 10 cm plate.
1000000 cells typically give a confluent plate after 48:00:00 , after which a 1/10 or 1/20 splitting is normally sufficient for keeping stocks.
2d
Preparation of ES-DMEM + LIF
For preparation of 600 mL ES-DMEM +LIF: DMEM containing 10 % (v/v) fetal bovine serum (FBS), 5 % (v/v) Knock-Out serum replacement, 50 ng/mL GST-tagged leukemia inhibitory factor (LIF), 100 U/mL penicillin/streptomycin, 2 millimolar (mM) glutamine, 0.1 millimolar (mM) minimum essential media (MEM) Non-essential amino acids, 1 millimolar (mM) sodium pyruvate and 0.1 millimolar (mM) β-mercaptoethanol (BME).
To a 500 mL DMEM, high glucose, no glutamineThermo FisherCatalog #11960069 bottle add:
Fetal Bovine Serum, Regular, USDA Safety TestedCorningCatalog #35-010-CV : 60 mL .
KnockOut™ Serum ReplacementThermo FisherCatalog #10828028 : 30 mL .
MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050 : 6 mL .
L-Glutamine (200 mM)Thermo FisherCatalog #25030149 : 6 mL .
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122 :6 mL .
2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500 : 4 µL .
GST-LIF recombinant proteinMRC-PPU Reagents and ServicesCatalog #DU1715 to 50 ng/mL final concentration : 10 µL of 3.12 mg/mL stock.
Note
Alternative LIF sources:
- ESGRO® Recombinant Mouse LIF ProteinMerck MilliporeSigma (Sigma-Aldrich)Catalog #ESG1107 use 500 U/mL final .
- Human LIF Recombinant Protein, PeproTech®Thermo Fisher ScientificCatalog #300-05-25UG use 50 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
LINK
0.22 um filter
SPECIFICATIONS
Preparation of 0.1% Gelatin in PBS
Prepare 1% Gelatin.
Dissolve 10 g ofGelatin from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890 powder in 1000 mL ultrapure water.
Autoclave.
Prepare 0.1% Gelatin in PBS.
To a 500 mL bottle add:
45 mL PBS (10X), pH 7.4Thermo FisherCatalog #70011069 .
425 mL ultrapure water.
50 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
LINK
0.22 um filter
SPECIFICATIONS
Mouse Embryonic Stem Cells Passaging (10cm plate)
15m
Aspirate the media.
Wash cells in 5 mL Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV
twice.
Aspirate and add 2 mL 0.05% Trypsin-EDTA, phenol redInvitrogen - Thermo FisherCatalog #25300054 .
Incubate 00:05:00 at 37 °C .
5m
Add 4 mL of ES-DMEM +LIF to the trypsinized cells and gently pipette 10 times to break up any remaining cell clumps.
Transfer to a 15 mL conical tube.
Centrifuge 1000 rpm, 25°C, 00:05:00 , aspirate, and resuspend in 1 mL of media.
5m
Gelatinize plates: add 5 mL per 10 cm plate of 0.1 % (v/v) gelatin in PBS, spread and incubate for 00:05:00 , and aspirate.
5m
Count cells and plate desired seeding density in 7 mL ES-DMEM +LIF per 10 cm plate.
mESCs in ES-DMEM + LIF Media
Mouse Embryonic Stem Cells Freezing
1d
Prepare Freezing media (80% ES-DMEM, 10% DMSO, 10% FBS).
For 10 mL of freezing media add:
8 mL ES-DMEM + LIF.
1 mL DMSO ≥ 99.9%VWR International (Avantor)Catalog #MK494802 .
1 mL Fetal Bovine Serum, Regular, USDA Safety TestedCorningCatalog #35-010-CV .
Split cells normally (steps 7-12), centrifuge 1000 rpm, 00:05:00 , and gently resuspend in freezing media.
Transfer to Externally 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
LINK
at-80 °C for 24:00:00 .
1d
Store at -150 °C .
Mouse Embryonic Stem Cells Thawing
Thaw vial in a 37 °C water bath.
Prepare a tube with 9 mL ES-DMEM + LIF.
Once the cells have thawed, transfer them to the tube and centrifuge for 1000 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
LINK
Citation
LINK
Resuspension of 2i kinase inhibitors (to make 10 millimolar (mM) stocks):
CHIR99021Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1046
Molecular weight: 465.34 g/mol
Resuspend 5 mg in 1074.5 µL DMSO.
PD 0325901Merck MilliporeSigma (Sigma-Aldrich)Catalog #PZ0162
Molecular weight: 482.19 g/mol
Resuspend 5 mg in 1036.9 µL DMSO.
For preparation of 250 mL N2B27 Media (1 % (v/v) B27 supplement, 0.5 % (v/v) N2 supplement , 2 millimolar (mM) glutamine ; 0.1 millimolar (mM) β-mercaptoethanol; 100 U/mL penicillin/streptomycin in 1:1 DMEM/F12:Neurobasal medium)
In a 250 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
LINK
0.22 um filter
SPECIFICATIONS
add:
DMEM/F-12, no glutamineThermo FisherCatalog #21331020 : 125 mL .
Neurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049 : 125 mL .
N2 supplement (100x supplement)Gibco - Thermo Fisher ScientificCatalog #17502048 : 1.25 mL .
B-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #17504044 : 2.5 mL .
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122 : 2.5 mL .
L-Glutamine (200 mM)Thermo FisherCatalog #25030149 : 2.5 mL .
2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500 : 1.75 µL .
For preparation of 30 mL 2i + LIF Media. To 30 mL of N2B27 in 50 mL conical tube add :
9 µL of 10 millimolar (mM) stock CHIR99021 (from step 22) to3 micromolar (µM) final concentration.
3 µL of 10 millimolar (mM) stock PD0325901 (from step 22) to 1 micromolar (µM) final concentration.
GST-LIF recombinant proteinMRC-PPU Reagents and ServicesCatalog #DU1715 to 50 ng/mL final concentration.
Note
From 3.12 mg/mL stock , dilute 1/10 in Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV
and add 5 µL of this dilution to the 2i + LIF media mix.
Filter through syringe filter into conical tube. Store at4 °C .
2i + LIF conversion (Do in 6-well or equivalent size plate).
In gelatinized plates, seed 80000 cells per cm2 in 6-well plates in ES-DMEM + LIF, and incubate 24:00:00 at 37 °C .
1d
Wash with 1 mL Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV twice.
Add 200 µL StemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo Fisher ScientificCatalog #A1110501 and incubate at 37 °C for 00:05:00 .
5m
Resuspend in 400 µL 2i+LIF media.
Centrifuge0.7 x g, Room temperature, 00:05:00 .
5m
Aspirate supernatant and resuspend in 500 µL 2i+LIF media.
Add100 µL of cell suspension (or 80000 cells per cm2) to new gelatinized plate.
Add 2 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
LINK
Citation
LINK
For preparation of 530 mL (approximately) NSC media (1 % (v/v) B27 supplement, 0.5 % (v/v) N2 supplement , 2.9 g/L glucose, 0.1 millimolar (mM) minimum essential media (MEM) Non-essential amino acids, 0.012 % w/v Bovine Albumin Fraction V, 0.1 millimolar (mM) β-mercaptoethanol; 100 U/mL penicillin/streptomycin in DMEM/F-12, HEPES).
To a 500 mL bottle of Gibco™ DMEM/F-12 HEPESFisher ScientificCatalog #11-330-032
add:
7.25 mL Glucose SolutionThermo FisherCatalog #A2494001 .
5 mL MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050 .
800 µL Gibco™ Bovine Albumin Fraction V (7.5% solution)Thermo Fisher ScientificCatalog #15260037 .
5 mL Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122 .
3.5 µL 2-Mercaptoethanol, 99%, pureThermo Fisher ScientificCatalog #AC125472500 .
2.5 mL N2 supplement (100x supplement)Gibco - Thermo Fisher ScientificCatalog #17502048 .
5 mL B-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #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
LINK
0.22 um filter
SPECIFICATIONS
Supplements:
Epidermal growth factor (EGF) 20 μg/mL stock Gibco™ Mouse EGF Recombinant Protein, PeproTech®Fisher ScientificCatalog #31509100UG : Resuspend 100 µg in 200 µL sterile ultrapure H2O. Then add 4800 µL of 0.1 % w/v BSA.
For a final concentration of 10 ng/mL use 1 µL of stock per 2 mL media (1:2000).
Fibroblast growth factor 2 (FGF-2/bFGF) 20 μg/mL stock Gibco™ Mouse FGF-basic (FGF-2/bFGF) Recombinant Protein, PeproTech®Fisher ScientificCatalog #4503310UG
Resuspend 10 µg in 100 µL sterile ultrapure H2O. Then add 400 µL of 0.1 % w/v BSA.
For a final concentration of 10 ng/mL use 1 µL of stock per 2 mL media (1:2000).
Mouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020 stock is ~2 mg/mL
For a final concentration of 2 μg/mL use 10 µL of stock per 10 mL of NSC media.
NSC differentiation.
From 2i+LIF cells.
In gelatinized plates, seed 1250 cells per cm2 in 2i + LIF media, and incubate for 24:00:00 at 37 °C in a 6-well plate.
1d
Wash gently with Phosphate-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 37 °C for 24:00:00 .
1d
Change media every 24:00:00 for 144:00:00 (6 days) .
1w
Replate 2000000 cells in a nongelatinized 10-cm plate (or less in a proportionally-sized plate) in NSC complete media: NSC media + EGF + FGF2 (1 µL of diluted stock of each per 2 mL media).
Grow neurospheres for 24:00:00 -72:00:00 (until large spheres are formed).
4d
Harvest floating aggregates by transferring media with floating cells to a 15 mL conical tube.
Centrifuge 2000 rpm, 00:05:00 .
5m
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 Gibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055 from 10X to 1X. Mix 50 mL Gibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055 in 450 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
LINK
0.22 um filter
SPECIFICATIONS
- For coating solution: add 5 µL of Mouse LamininMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2020 to 995 µL 1X PBS Ca2+,Mg2+ in TC hood.
- Incubate 03:00:00 atRoom temperature or 37 °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 StemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo Fisher ScientificCatalog #A1110501 as above, but resuspend in freezing medium: 90 % (v/v) NSC media + 10 % (v/v) DMSO ≥ 99.9%VWR International (Avantor)Catalog #MK494802 .
Use 500 µL of suspension per freezing vial Externally 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
LINK
at-80 °C before moving to -150 °C long-term.
Thawing Mouse Neural Stem Cells
5m
Thaw vial in a 37 °C water bath.
Prepare a tube with 5 mL NSC media.
Once the cells have thawed, transfer them to the tube and centrifuge for 1000 rpm, 00:05:00 .
5m
Aspirate supernatant.
Resuspend the pellet in NSC complete media + Laminin.
Transfer to a Mouse 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.2018.04.022Step 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.202052164Step 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.1996Step 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.173146Step 35
Pollard SM. In vitro expansion of fetal neural progenitors as adherent cell lines.
https://doi.org/10.1007/978-1-62703-574-3_2Acknowledgements
We thank Charles Williams, PhD (University of Edinburgh, UK) for protocols and Intisar Koch (Sanford Research) for critical reading of the protocol.