Germ cell differentiation of mouse embryonic stem cells can be influenced by the culture medium

Abstract

ABSTRACTAlthough embryonic stem (ES) cells can differentiate into germ cells, little is known about the influence of culture media on this process. We investigated the effect of two culture media on the capacity of ES cells to differentiate into germ cells using embryoid body (EB) and monolayer culture protocols. Germ cell differentiation was induced in mouse ES cells under four experimental conditions: EB/Dulbecco’s modified Eagle’s medium (EB/DMEM), EB/knockout Dulbecco’s modified Eagle’s medium (EB/KO-DMEM), monolayer/Dulbecco’s modified Eagle’s medium (monolayer/DMEM), and monolayer/knockout Dulbecco’s modified Eagle’s medium (monolayer/KO-DMEM). After incubation for 6 days, quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess expression of the germ cell markers, Mvh, Oct4, Rec8, Scp1, Scp3 and Stra8. Also, Oct4 and Mvh expressions at the protein level were assessed using immunocytochemistry; we evaluated alkaline phosphatase activity in addition to cell number and viability. Germ cell-specific marker expression was increased significantly in cells differentiated in KO-DMEM for both EB and monolayer protocols; the highest level was in cultures using the EB protocol. The highest cell proliferation rate was observed using the monolayer/KO-DMEM protocol and the lowest using the EB/DMEM protocol. Generally, KO-DMEM exhibited the greatest impact on germ cell differentiation and cell proliferation. Optimization of germ cell differentiation of ES cells requires careful selection of culture medium.