Variability in gene markers expression levels during spontaneous murine embryoid body differentiation

Abstract

Embryonic stem cells (ESC) aggregates in suspension (named embryoid bodies, EB) can further attached to tissue culture plates to allow the emergence of differentiated specific cells. During the differentiation process, Oct4 and Nanog gene expression decreases along with increase of developmental gene markers. Toxicans to embryo development are usually screened in vitro by comparing their effects on expression levels of developmental gene markers during EB differentiation. We test the effect of the attachment of EB to microplates on gene expression differentiation markers to analyze embriotoxicants. Embryoid bodies were formed by the conic method and further transferred to 24 -well tissue culture microplates and incubated with 5-fluorouracil (5-FU). The expression profile of early differentiation gene markers along with gene markers of ESC undifferentiation were analyzed by RT-PCR. Using microscopic evaluation, three distinct morphologies of the attached cells from untreated EBs were observed. Each one showed a distinct pattern of differentiation gene markers and distinct Oct4/Nanog ratios. On the other hand, morphologies of 5-FU treated EBs were similar and it seems to be necrotic. However the profile of the differentiation gene markers from 5-FU treated EBs was similar to untreated EBs but Oct4/Nanog ratio was quite different. These results suggest that embryoid body differentiation after attachment to tissue culture plates can not be easily controlled (even the size before attachment was equal), leading to a wide range of variability in differentiation gene markers levels. So, comparisons between control and toxicans-treated cultures are not adequate. The gene expression Oct4/Nanog ratio is probably an alternative way to detect embryotoxicity.