Ascorbate effect on serum free cultured hESC

ABSTRACT: Ascorbate activates CD30 expression and causes widespread specific demethylation of the epigenome of serum free cultured hESC. The genetic and epigenetic integrity of human Embryonic Stem cells (hESCs) is critical to their future applications in research and medicine. hESC cultured in serum free media can accumulate point mutations, aneuploidy and progressive epigenetic changes over prolonged culture in vitro. We have identified ascorbate as the only molecule in the very widely used Knock-out serum replacement medium that is sufficient to induce expression of CD30, a biomarker for aneuploidy in hESCs. In fact, we show that hESC cultured in the presence of ascorbate for 20 passages not only display demethylation of the CD30 locus, but exhibit widespread and remarkably specific and consistent demethylation of 1,847 genes in both HES2 and HES3 cells. The specific ascorbate induced demethylation changes in the hESC epigenome, of which 86% are shared between the two lines, identify a subset of genes in hESC, including CD30, that are sensitive to serum free culture medium induced epigenetic changes. Experiment. HES2 cells were maintained on 20% Fetal calf serum on mouse embryonic fibroblast feeder layers with mechanical dissection. Using HES2 after 99 mechanical passages (P99), hESC were grown for 17-20 (+17 - +20) passages in the presence of 20% Knock-Out Serum Replacement (Invitrogen) either with (+ASC) or without (-ASC) Ascorbate with enzymatic culturing. RNA (FACs sorted for the presence of the pluripotent stem marker TG30) from three passages (17, 19 and 20) (replicates) for both +ASC and –ASC samples were arrayed.

ORGANISM(S): Homo sapiens

SUBMITTER: Gabriel Kolle 

PROVIDER: E-GEOD-16590 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress