Description: Haploid embryonic mouse stem cell line established from inner cell mass derived from activated unfertilized oocytes from superovulated 129/Sv female mice

Also Known As:

Species: Mouse

Tissue: Embryo

Growth Properties: Adherent. Use gelatin (porcine)-coated tissue culture plastic. Feeder layers recommended for optimal growth (see H129-1 – Instructions for Culturing in the Product Documents section below)

Morphology: Small cells growing in large dense round colonies

Growth Medium: For 100ml of (2i) growth medium use:
100 ml NDiff 227 (Takara Clontech, catalogue number Y40002);
30 μl CHIR99021 (10 mM stock, Reagents Direct catalogue number 27-H76, 2mg);
10 μl PD0325901 (10 mM stock, store at -20°C; Reagents Direct catalogue number 39-C68, 2mg);
100,000 units LIF (=1 µg; eBioscience catalogue number 14-8521-80, mouse recombinant LIF, 25 µg);
5 ml 7.5% BSA (Life Technologies catalogue number 15260-037)

Subculturing Procedure: At sub-confluence incubate using Accutase solution for 5 minutes at 37°C. Add 10 ml DMEM/F12 with 3 ml 7.5% BSA per 500 ml and pipette up and down 5 times to make a single cell suspension. Spin at 300 x g for 5 minutes and re-suspend in fresh 2i medium and plate into new tissue culture flask (at a ratio of 1:5 to 1:10). Establish new cultures on mitotically-arrested mouse embryonic fibroblasts monolayers or gelatin coated cell culture plastic as above or at 4-5 x 10e4 cells/cm2. A complete media change must be performed at least every other day and cells subcultured every 2-3 days. Cultures must be kept at a low density with small colony sizes. Growth to high density or large colony size or prolonged subcultures will result in loss of haploid phenotype and increase in number of diploid cells as well as differentiation. Cultures must be incubated in a humidified 5% CO2/95% air incubator at 37°C. Vials contain > 10e6 cells. Contents can be thawed counted and placed in a 25cm2 flask. The haploid mouse stem cells upon resuscitation will have a viability of ~80% and a count of ~2 x 10e6 cells per ampoule. Upon cultivation with recommended culture conditions in a 25cm2 flask H129-1 cells will attain a viability of ~95% and and a cell yield of approximately 1 x 10e6 cells.

Release Conditions: Yes

Depositor: Dr Martin Leeb, Centre for Stem Cell Research, Wellcome Trust and Medical Research Council Stem, Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

Originator: Yes

References: Leeb, M and Wutz, A (2011). Derivation of haploid ebryonic stem cells from mouse embryos. Nature 479, 131-134 PMID: 21900896

Hyperlink to ECACC Cell Line Data Sheet: 14040203