TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing
Figure 4
Methylcellulose differentiation of stably transfected ES cells in the presence of hematopoietic cytokines. E14 ES cells were stably transfected as described and grown to confluency. Triplicate 10 cm gelatin coated tissue culture plates containing 4000 cells each were plated of both the TET2-pCDNA3 (TET2 stable transfect) and pCDNA3 control (EV stable transfect). Cells were differentiated using MethoCult GF H4034 (Stem Cell Technologies), a combination of methylcellulose and granulocyte-colony stimulating factor (G-CSF) in the media and colonies analysed according to the manufacturer’s instructions at day 22 after plating for differentiation. A total of 229 colonies were classified in the TET2 stable transfect condition and 173 colonies for the EV stable transfect condition. (a) Relative differentiation levels of TET2 stable transfects and EV stable transfects at 22 days under methylcellulose condition. TET2 stable transfects were on average 8% less differentiated at the time of analysis. (b) Photomicrographs of embryoid bodies derived from stably transfected ES cells.
