The use of the animals described in the sections below has been approved by the Ethics Committee on Animal and Human Research of the Universitat Autònoma de Barcelona and by the Departament d'Agricultura, Ramaderia, Pesca i Alimentació of the Generalitat de Catalunya (protocol #8741).
1. Feeder Cell Inactivation and Storage
NOTE: Human foreskin fibroblasts (HFF-1) were previously frozen in 1 mL of freezing solution consisting of 90% Fetal Bovine Serum (FBS) and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until use.
2. Feeder Cell Culture
3. Embryo Collection and Culture
4. Stem Cell Derivation
5. Stem Cell Culture and Maintenance
Following this protocol, over 95% of outgrowth formation should be achieved after the first week of culture (Figure 1A). Establishment of mESC lines after six passages is variable among experimental replicates, with an average success of 60-80%. The addition of 2i does not significantly improve the results when working with permissive mouse strains, such as those with a 129S2 background, but it is necessary when working with non-permissive strains.
Mouse ESC colonies should present a regular morphology with a flat shape and defined edges when cultured without treatment (Figure 1B-C) or cultured with 2i (Figure 1D). Colonies presenting peripheral differentiation signs should be discarded (Figure 1E-F). If the derivation efficiencies obtained are lower than the expected values, control the growth of mESC colonies and subculture them more often to avoid overgrowth, as this can cause cell death and would promote differentiation (Figure 1G-H).
Figure 1: Representative images of mESC colonies in culture. (A) Outgrowth derived from a blastocyst after the first week of culture (B, C) Several mESC colonies at passage 10 presenting defined edges and a flat shape. (D) Colonies from mESC lines treated with 2i. (E, F) Examples of semi-differentiated mESC colonies presenting peripheral differentiation signs (highlighted with dashed ellipses). (G, H) Examples of overgrown mESC colonies presenting a bad morphology and differentiation signs. Scale bars = 100 µm. Please click here to view a larger version of this figure.
To verify the stemness of the putative mESC lines obtained after six weeks of culture, the presence of pluripotency and differentiation markers should be assessed. The mESC lines should be positive for pluripotency markers such as Oct4 and Sox2 (Figure 2 A-B). Moreover, after induction of spontaneous differentiation by culturing the cells for 10 days without feeder cells in DMEM supplemented with 10% FBS, mESC lines are expected to be positive for the ectoderm marker β-tubulin class III (Tuj1) (Figure 2C), the mesoderm marker α-Smooth Muscle Actin (αSMA) (Figure 2D) and the endoderm marker Alpha-Fetoprotein (AFP) (Figure 2E). Only the lines positive for the five markers assessed should be considered true mESC lines and thus used for the calculation of the derivation efficiency. Usually, this corresponds to virtually all the mESC lines generated using the present protocol.
Figure 2: Representative immunofluorescence against pluripotency and differentiation markers. mESC colonies expressing (A) Oct4 (green) (B) and Sox2 (red). Differentiated mESC colonies expressing (C) Tuj1 (green), (D) αSMA (green) and (E) AFP (green). In all images the nuclear material is counterstained with Hoeschst (blue). Scale bars = 100 µm. Please click here to view a larger version of this figure.
1 mL sryringe | BD | 309628 | |
2-β-mercaptoethanol | Life Technologies | 31350-010 | |
4-well plate | Thermo Scientific Nunc | 176740 | |
Acidic Tyrode's solution | Home-made | See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4ºC. Alternatively, for long-term storage keep it at -20ºC. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M. | |
BSA | Sigma | A3311 | |
Chicken anti-Mouse IgG, Alexa Fluor 488 | Molecular Probes – Invitrogen | A-21200 | 1:500 dilution. Secondary antibody for Oct4, Tuj1, αSMA and AFP mouse antibodies. |
CHIR990212 | Axon Medchem | 1386 | Reconstitute with 2.15 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20ºC. Avoid freeze and thaw cycles. |
CryoTube | Thermo Scientific Nunc | 3754518 | |
DMSO | Sigma | 41640 | |
DMEM | BioWest | L0107 | |
FBS | BioWest | S1810 | Inactivate it prior to use by heating for 30 mins at 56ºC. Aliquot and store at -20ºC |
Flushing needle | BD | 304000 | Cut the end of the needle and ground to a blunt tip on an abrasive stone |
Gelatin from porcine skin | Fluka | 48724 | Dilute at 0,2% in destilled water and autoclave it. Each dilution can be used for a month. |
Goat anti-Rabbit IgG, Alexa Fluor 594 | Molecular Probes – Invitrogen | A-11037 | 1:500 dilution. Secondary antibody for Sox2 rabbit antibody. |
HBSS | BioWest | L0611 | |
Hepes-buffered CZB | Home-made | See composition in Chatot et al, 1989 | |
Human chorionic gonadotropin | Divasa-Farmavic | Veterin-Corion 3000 UI | Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20ºC for up to two months. Once thawed do not freeze again. |
Human foreskin fibroblasts | ATCC | ATCCSCRC-1041 | For long term storage it is recommended to store them in liquid nitrogen. |
KnockOut Serum Replacement | Life Technologies | 10828-028 | Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20ºC (check the expiration date). Avoid freeze and thaw cycles. |
KSOMaag Evolve | Zenith Biotech | ZEKS-050 | Supplement with 4 mg/ml BSA. Equilibrate at 37ºC and 5% CO2 a day prior to use. |
Leukemia Inhibitory Factor | Merk Millipore | ESG1106 | |
Mice | Charles River/Harlan | It is recommended to use mice from a permissive strain in terms of mESC derivation (such as 129S2 or C57BL). However, inbreed strains are less efficient producing embryos. Therefore, it is recomended to use a hybrid strain, such as 129S2 x C57BL or B6CBAF1 to take advantage of the hybrid vigor. | |
Mineral oil | Sigma | M8410 | Embryo tested. Photosensitive. |
Mitomycin C | Serva | 2980501 | Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4ºC protected from light. Attention, it is harmful and suspected of causing cancer. |
Mouse anti-tubulin β III (Tuj1) | Biolegend | MMS-435P | 1:500 dilution |
Mouse monoclonal anti-αSMA | Sigma | A5228 | 1:200 dilution |
Mouse monoclonal anti-AFP | R&D Systems | MAB1368 | 1:50 dilution |
Mouse monoclonal anti Oct3/4 | Santa cruz | Sc-5279 | 1:50 dilution |
Non-Essential Amino Acids | Life Technologies | 11140-035 | |
PD0325901 | Axon Medchem | 1408 | Reconstitute with 2.08 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20ºC. Avoid freeze and thaw cycles. |
Petri dish (35mm) | Thermo Scientific Nunc | 153066 | |
Petri dish (60mm) | Thermo Scientific Nunc | 150288 | |
Pregnant mare's serum gonadotropin | Foligon | Foligon-1000 UI | Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20ºC for up to two months. Once thawed do not freeze again. |
Rabbit policlonal anti-Sox2 | Merck Millipore | AB5603 | 1:200 dilution |
T75 falcon | Thermo Scientific | 130190 | |
Trypsin-EDTA 10x | BioWest | X0930 | Dilute 1:10 in HBSS to obtain a 1x solution |
Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.
Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.
Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.