The Isolation And Characterization Of Porcine Oogonial Stem Cells From Ovaries

Here we present the derivation of the OSC lines from Inner Mongolian white gilt ovaries and the characterization of the OSC lines.Ovarian tissues collected from a local abattoir were digested with collagenase to single cells.The methods including the pre-plating and antibody coated magnetic beads were used to enrich OSCs.Initially,the single cells were cultured overnight to remove the cells attached to the culture dishes.Then the suspension cells were incubated with the anti-Fragilis antibody coated magnetic beads,and the cells were collected in the magnetic standard.After washing three times with medium,the cell pellets were resuspended in medium and cultured in the medium with serum,EGF,bFGF and LIF at 38.5?,5%CO2.And the biological characteristics of the obtained oogonial stem cells were verified.The colonies were appeared after culture 5-7 days,then the cells were passaged every 2 or 3 day.Our result showed that porcine OSCs could be maintained in our culture system for more than 56 passages without losing the capability of self-renewing and proliferating.The appearance of the colonies of porcine OSCs is similar to the mouse OSCs with a clearly cell boundary.Like other stem cells,the ratio of nuclear and cytoplasm is very high.However,after 6 passages the oocyte-like cells were appeared with a zona pellucida like structure around the cells which specifically binded to anti-ZP3 antibodies.Those oocyte-like cells can grow to 60?m in diameter.The immunofluorescence chemistry assay showed that those cells were expressed Scp3 genes,which was indicated those oocyte-like cells in meiophase.The immunofluorescence chemistry assay and RT-PCR analysis showed that the porcine OSCs were expressed Oct4,DDX4,C-kit,Nanog,Ifitm3 and Blimpl genes.Further it was demonstrated that the porcine OSCs possessed 38 autochromosmes and 2 X chromosmes.These results showed that the cell line had all characteristics of the oogonial stem cells,demonstrated that the porcine oogonial stem cell line was successfully constructed in vitro.