Germ cell differentiation of mouse embryonic stem cells can be influenced by the culture medium
Germ cell differentiation of mouse embryonic stem cells can be influenced by the culture medium
azam soleimani,1,*Nehleh Zareifard,2soghra bahmanpour,3
1. Shiraz University of Medical Sciences, Shiraz, Iran 2. Shiraz University of Medical Sciences, Shiraz, Iran 3. Shiraz University of Medical Sciences, Shiraz, Iran
Introduction: Because embryonic stem cells (ES) can develop into germ cells, optimizing culture conditions for differentiation of ES is highly desirable. In practice, ES cell differentiation protocols for generating germ cell-like cells use either traditional media such as Dulbecco’s modified Eagle’s medium (DMEM or basal ES cell media, such as knockout Dulbecco’s modified Eagle’s medium (KODMEM). We have found no standardized culture medium for induction of germ cell differentiation from ES cells despite the fact that the effect of culture medium on development of gonadal germ cells in vitro has been investigated extensively. We compared two commonly used commercial media, high glucose DMEM and KO-DMEM, and assessed the germ cell differentiation from mouse ES cells by evaluating germ cell expression using EB or monolayer culture systems.
Methods: The mouse ES cell line R1(XY) was cultured on mitomycin C treated mouse embryonic fibroblast in KO-DMEM containing 15% ES qualified FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol and 1000 U/ml LIF. Half of the medium was replaced each day and the cells were passaged every second day. 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. Cell phenotypes were checked every day, using an inverted microscope (Olympus). Their growth was evaluated using trypsin/EDTA (0.05%) digestion for about 5 min to form a single cell suspension of the EBs. EB volumes and the dark areas at the center of each EB were estimated by measuring the mean value of the four diameters of each EB using the formula r3 π4/3, where r = radius.
Results: 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.
Conclusion: We found that KO-DMEM is suitable for both monolayer and EB differentiation conditions. KODMEM is the superior medium for male and female germ cell differentiation. We found that the EB protocol, in the presence of KODMEM, is more efficient for promoting the expression of germ cell markers than the monolayer condition in the same medium. we speculate that this biomimicry might be reinforced in KO-DMEM. We also demonstrated that the volume of the dark area of EBs increased and number of viable cells decreased when EBs were cultured in DMEM. Commercial culture media and serum supplements contain different amounts of reactive oxygen species (ROS). Therefore, the lower number of viable cells and larger dark areas seen after using the traditional DMEM protocol could be due to increased ROS production. On the other hand, it has been shown that loss of Oct4 in PGCs was correlated with apoptosis. Our findings show that Oct4 expression by cells differentiated using DMEM was extremely low, which suggests that our results might explain the lower cell viability in traditional media. Unknown components in proprietary media might be responsible for reduction in cell numbers in the outcome of PGC differentiation. We found a higher rate of germ cell differentiation in the ES cells cultured in KO-DMEM compared to those incubated in DMEM both in the presence or absence of BMP4. Our findings are consistent with earlier reports that optimization of culture conditions and appropriate choice of medium are critical for each differentiation protocol.