Investigating The Effect Of Serum Concentration On The Expression Pattern Of Pluripotency And Lineage-Specific Markers In Mouse Embryoid Bodies Using Immunofluorescence

Abstract

Embryonic stem cells (ESCs) have the ability of unlimited self-renewal in vitro and differentiate into cells composed of all three primitive germ layers. The renewable character of ESCs makes it a good source for cell transplantation and tissue engineering applications. ES cells when allowed to differentiate in suspention culture without anti-differentiation factors, 3D multicellular aggregates known as embryoid bodies (EBs) developed, these represent a good model for studying early embryonic development. ES cells differentiation within EBs is affected by many physical and chemical factors, one of these factors is the serum in the maintenance medium, as it contains hormones and growth factors. In this study, the influence of different serum concentrations on EB differentiation and the difference between the mRNA and protein expression of pluripotent and lineage specific markers of day 4 mouse EBs have been investigated by using immunostaining. The difference of expression of these factors between undifferentiated  mouse ESCs and differentiated day 4 mouse EBs has been examined. The findings were the protein expression of Oct4, Nanog, and Bry were downregulated in EBs plated in high serum, while Gata6 expression was more this is consistent with expression level of mRNA and is in agreement with the evidence of down regulation of pluripotent markers in extraembryonic endoderm. In addition the Oct4 was positively expressed in undifferentiated mouse ESCs, which then reduced in differentiated day 4 mouse EBs.