Long term culture and characterization of chicken primordial germ cells

Avian primordial germ cells (PGCs) can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation.
Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. Moreover, we recently isolate and cultivate a new PGC line from turkey. All PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS. RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a progressive drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. At day 6, colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. In order to evaluate the germinal differentiation of cultured PGCs during the gonadal development as well as the germline transmission rate, we established a stably expressing GFP line that was successfully injected in emrbyos. Results are in progress.