Genetic and morphologic similarities between mouse embryonic stem cell (escs) and primordial germ cell (pgcs) make it difficult to distinguish the two cell types in in vitro differentiation. using the expression of specific markers of germ cells that are not expressed or expressed at low levels in escs, can help recognizing in vitro differentiated cells.
Methods
In the present study, we attempted to differentiate the mouse embryonic stem cells, oct4-gfp into germ cell-like cells (gclcs) spontaneously in two different ways:1- spontaneous differentiation of escs without lif is its feeder cells (mef) for 14 days as a monolayer culture (sp) group. 2- spontaneous differentiation of escs using eb method. after 3 days culture for hanging drop and 4 days in bacterial plate, totally 7 days as (eb7), then single cell ebs culture for 7 more days without lif, totally 14 days, this group was named as eb culture methods (eb +sp).we tried to evaluate and compared the expression level of germ cell specific genes during in vitro culture duration in both groups.
Results
In both groups, mov10l1 was down regulated (p=0.3) and tex13 and riken were up regulated (p=0.3, p=0.04), respectively. fkbp6 and stra8 were decreased in eb+sp and increased in sp group with no significant differences between them (p=0.1, p=0.07). additionally, in sp group, gene expression of mvh and scp3 were up regulated and had significant differences compared with eb+sp group (p=0.00, p= 0.01), respectively. oct4 was down regulated in both groups. flow cytometry analysis showed that the mean fluorescent intensity (mfi) of the cells for mvh showed that more mvh positive cells were observed in the sp group (87.2±2.61) with significant differences compared to with undifferentiated esc (71±3.02), eb+sp (75.74±3.90), (p=0.00, p=0.01 respectively). but increased mfi of the cells for mvh was not significant differences compared with eb7 group (82±2.61), (p=0.3). immonostaning analyziz showed that positive expression of the mvh protein was examined with a florescence microscope with a representative red color. the round cells that defined as gclcs shown by dapi nuclei counter staining. we observed that round shape cells were red, indicating the expression of the vasa protein. this coloration in the cells of sp group was greater than that of cells in eb+sp group.
Conclusion
Oct4 down regulation as pluripotency factor and expression of meiosis markers indicated that escs differentiated successfully in to germ-cell like cells in both groups. evaluation of gene expression patterns in both groups demonstrated that monolayer culture was more efficient to produce germ cell-like cells in compression with eb methods. still further utilization of culture conditions and optimization will be needed for successful and high quality in vitro germ cells differentiation.
Keywords
Embryonic stem cells, differentiation, germ cell-like cell, monolayer, embryoid body
