Long-Term Proliferation of Chicken Primordial Germ Cells Cultured In Vitro.

Abstract

Abstract.To promote the utilization of chicken primordial germ cells (PGCs) in transgenic manipulation, the in vitro culture of these cells was conducted. The PGCs were isolated from the blood vessels of chicken embryos, labeled with PKH26 red fluorescent cell linker, refined by picking up with micromanipulator attached with a fine glass pipette and cultured on feeder cells derived from chicken germinal ridges in medium DMEM plus F10 supplemented with 10% fetal calf serum (FCS) in 96 well plate at 39 ˚C, 5% CO2 in air. The result of cell counting showed that in this condition chicken PGC number increased at least 29 times when the cells were cultured for up to 17 days and the proliferation of chicken PGCs was directly observed through cell tracing experiment, suggesting that feeder cells from germinal ridges might provide some kinds of essential factor(s) for PGC division.

Population of Circulating Primordial Germ Cells in Early Japanese Quail Embryos

“…PGCs circulate temporarily in the developing bloodstream in avian embryos, and this unique migratory pathway makes PGC collection easy. Basic characteristics of quail PGCs were similar to those of chick embryos which were described previously (Mayer, 1964;Yang and Fujihara, 1999…”

“…During gastrulation, the PGCs migrate anteriorly via the hypoblast and reside in the extraembryonic germinal crescent (Eyal-Giladi et al, 1981;Urven et al, 1988;Han et al, 1994). As soon as the blood vessels form at Hamburger and Hamilton (1951) stage 10, PGCs from the germinal crescent start to circulate temporarily through the bloodstream, and by stages 20-24 these PGCs have migrated into the gonadal anlage where they rapidly proliferate and differentiate into either spermatogonia in the testis or oogonia in the ovary (Mayer, 1964;Fujimoto et al, 1976;Nakamura et al, 1988;Kuwana, 1993;Fujihara, 1999). Migration features of PGCs facilitate their isolation and transfer in early developing avian embryos (Fujimoto et al, 1976;Chang et al, 1992;Yasuda et al, 1992;Ono et al, 1999).…”

Purification of avian circulating primordial germ cells by Nycodenz density gradient centrifugation

“…Of course, there is also a requirement for PGCs to be harvested efficiently from the eggs by a simple method. Previous methods involved difficult and time-consuming work such as aspirating PGCs by a fine glass micropipette to assure high purity (Yang and Fujihara, 1999). In addition, more embryos were required to collect enough blood to yield sufficient PGCs because of the low recovery rate.…”

Embryonic Fibroblast-Conditioned Medium Enhances Viability and Proliferation of Chick Circulating Primordial Germ Cells (cPGCs) in Suspension Culture.

“…Avian cPGCs have hitherto been treated as anchorage-dependent cells, as have mouse PGCs, and have been studied in co-cultures with feeder cells. For example, chicken cPGCs were cultured with stroma cells from the genital ridge as feeder cells, and were shown to proliferate [21,22]. The suspension culture method we employed included no feeder cells, and thus provides a direct way to identify the factors affecting the survival and proliferation of cPGCs.…”