Establishment Of Embryonic Germ Cells Derived From Primordial Germ Cells In Rabbit And Cattle

The paper reported here the isolation of rabbit pluripotent ES-like cells from PGCs of 15-28dpc (day post coidum) ftuses of NZW, SBR and DEB rabbits. Animals were slaughtered from day 15 to day 28 of gestation, and fetuses were dissected from the uteri. Isolated tissues (genital ridges and surrounding tissues) contained PGCs were washed several time with PBS (-) and incubated in 0.04% EDTA + 0.25% trypsin for 20 mm at room temperature. Then collected PGCs with the average diameter of rabbit PGC at 10.5 ii in, that of the cow PGC at 18 i were cultured on niitotically inactivated mouse, rabbit and cattle fibroblasts in the presence of 2Ong/mL LW 65ng/mL SCF and 2Ong/niL Forskolun. After 24hrs culture, the proliferation of PGCs was observed from l5dpc-24dpc rabbit fetuses. Rabbit PGCs seldom showed the typical vesicle and pseudopodial fonnation. In addition, its negative AKP-stauning makes some trouble for the primary identification. However, at 48 hour of primary culture, PGCs in division and its grape-mass-shaped clumps were observed. PGCs proliferated rapidly and formed island-like or nest-like colonies. Their growth mode is same to the ES-like cells derived from rabbit blastocysts (Graves, 1993; Nieinan, 1994). In NZW rabbits, ES-like colonies were observed from l5dpc fetuses to 24dpc fetuses and were subloned up to S passages consisting of 28 second passage, 3 third passage and 2 fourth passage and 2 fifth passage. Under the same culture condition, nest-like colonies were observed only from l9dpc and 2odpc fetuses in SBR and DEB rabbits when rabbit PGCs were seeded onto homologous feeder layers. Compared with this, no ES-like cells were obtained when PGCs were cultured onto bEF feeder layers. Furthermore, these nest-shaped or island-shaped ES-like colonies differentiated into other cell type rapidly. In cattles, PGCs were obtained from two lO6dpc (including a male and a female) and one 74dpc female fetuses treated with the same method. After 72 hours culture, ES-like colonies were observed. The subclone was unsuccessful. Under certain culture conditions, rabbit ES-like cells can be induced to aggregate, a process that results in embryonic body (EB) formation. After long-time culture, these EBs begin to form terminally differentiated cell types, including epithlial-lilce cells and fibroblast-like cells, autorhytbmic cells. In conclusion, this is the first report, as far as we know, on the isolation and establishment of EG cells derived from rabbit PGCs. Both subclone culture and in vitro difference evidence demonstrate that the isolated EG cells were pluripotent. These cells are potentially useful for further study in this field.