Study On Embryonic Stem Cell From Cultured Primordial Germ Cells Of Mongolian Sheep

The primitive germ cell is a kind of new cell resources used as isolation and coloning embryonic stem (ES)cell .In this study,genital ridge and the surrouding tissue from 30-45 day old fetuses of Mongolia sheep were minced with the machine and disaggregated by trypsin/EDTA solution,and then ,PGCS and it's fetal fibroblast were co-cultured at 38 ℃,5%CO2 concentration and saturated humidity in a CO2 incubator. Dublecco's Modified Eagle's Medium(DMEM)with 10% fetal bovine serm (FBS) was used as culture medium without any cell growth factors.The results indicated that at the sixth day of primary culture,we observed nest-like colonies of ES-like cells of sheep divided and proliferated from PGCS.The sixth passage ES-like cells were stained with alkaline phosphatase(APK),and all these ES-like colonies were APK-positive in deep brown .Karyotypic analyses indicated apparently normal sheep chromatosome. These show cultured cells are indeed ES-like cells. Stained with H-E,colonies of ES-like cells are the horse's hoofs form ,and cellscollected and formed several growth division center.ES-like cell have a large nucleus with a little cytoplasm, and the boundary between cells are unclear.Observed with transmission electronic microscope,ES-like cell have obvious nucleolus and a little heterochromatin,and many ribosomes in the cytoplasm with much endoplasmic reticulum and mitochondria. A part of cells appeared alveoli on the nuclear envelope,and chromatin was pycnosis and the . apoptosis body was formed .Through refrigeration and deposit for a month at liquid nitrogen , the ES-like cell was thawed and cultured, survived and developed into colonies,and more than 60 %cells of second passage appared apopsis observed with electron microscope.