Development. 2019 Mar 15; 146(6): dev173146.

Published online 2019 Mar 26. doi: 10.1242/dev.173146

PMCID: PMC6451320

Defined conditions for propagation and manipulation of mouse embryonic stem cells

Carla Mulas,^1,* Tüzer Kalkan,¹ Ferdinand von Meyenn,² Harry G. Leitch,^3,4 Jennifer Nichols,^1,5 and Austin Smith^1,6,*

ABSTRACT

The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.