Establishment of the Long‐Term In Vitro Culture System for Chicken Primordial Germ Cells

Abstract

The objective of this study was to establish the long-term in vitro culture system for chicken gonadal primordial germ cells (gPGCs). Primitive gonads collected from 5.5-day-old chicken embryos were dissociated and explanted onto plates pre-coated with 0.1% gelatin. Each of the four different conditioned media from proliferating and mitotically inactivated chicken embryonic fibroblast (CEF) cells and murine embryonic fibroblasts (STO cells, CRL-1053, ATCC, USA), respectively, was supplemented with growth factors and used to support the growth of gPGCs. The result showed that all the conditioned media could promote the growth and colony formation of gPGCs in vitro, in particular the medium conditioned by inactivated CEF cells. The gPGC-derived colonies maintained in inactivated CEF cells-conditioned medium up to 281 days were positively stained by periodic acid Schiff reaction and antibodies specific to anti-SSEA-1, SSEA-3, SSEA-4, integrin alpha6 and integrin beta1. Their capacities of migration via vascular system and taking up residence in the primary gonadal ridge were further demonstrated by transferring to the dorsal aorta of stage 17 recipient embryos. These results suggested that our culture system is able to maintain chicken gPGCs for long-term in vitro culture without losing their capacity to express pluripotent markers and to integrate into the gonads.

Spermatogonial Stem Cells

“…PGCs isolated from embryonic blood were transferred to the stage X blastoderm of recipient embryos, and they again entered the bloodstream and colonised the gonads . Gonadal germ cells isolated from 6.5 -20.5 day incubated embryos and newly hatched chicks and adult chickens entered the germline when they were transferred to the bloodstream of recipient embryos (Chang et al, 1997;Tajima et al, 1998;Han et al, 2002;Minematsu et al, 2004;Naito et al, 2007;Shiue et al, 2009). Furthermore, when spermatogonial stem cells or germline stem cells derived from chicken testes were transferred to the stage X blastoderm or bloodstream of recipient embryos, they contributed to the germline and part of them gave rise to functional gametes (Jung et al, 2010).…”

Embryo Manipulation in Chickens

“…The germline transmission rate of cultured PGCs was low, but subsequently the germline transmission rate of the cultured PGCs was greatly improved (Naito et al, 2015). PGCs tend to form colonies and proliferate on chicken feeder cells in culture (Park and Han, 2000;Han et al, 2002;Shiue et al, 2009;Naito et al, 2010Naito et al, , 2012, while PGCs proliferate without forming colonies on mouse or rat feeder cells in culture (van de Lavoir et al, 2006a;Macdonald et al, 2010;. The characteristics of these cultured PGCs might be different between the two kinds of culture systems.…”

“…PGCs isolated from embryonic gonads were cultured on chicken embryonic fibroblast cells or gonadal stroma cells for more than 2 months, and produced germline chimeric chickens after transfer to the recipient embryos (Park and Han, 2000;Han et al, 2002;Park et al, 2003ab;Shiue et al, 2009). Similarly, PGCs isolated from embryonic blood were cultured on chicken feeder cells and successfully produced germline chimaeric chicken after transfer to the recipient embryos .…”

Transfer and Detection of Freshly Isolated or Cultured Chicken (Gallus gallus) and Exotic Species' Embryonic Gonadal Germ Stem Cells in Host Embryos

“…If the germplasm from removed embryos could be cryopreserved or cultured, and in the future reintroduced into the gene pool, the population would recover that genetic diversity. Previous groups have demonstrated that neither cryopreservation (Naito et al, 1994a; Takei et al, 1997; Tajima et al, 1998; Moore et al, 2006) nor in vitro culture (Chang et al, 1995; Chang et al, 1997; Park et al, 2003a; Park et al, 2003b; Ge et al, 2009; Shiue et al, 2009) impacts the ability of transferred chicken embryonic primordial germ stem cells (PGCs) or embryonic gonadal germline stem cells (GGCs) to produce donor-derived offspring. In addition, several studies have demonstrated the ability of one species’ PGCs orGGCs to colonize the gonadal ridge of another species, including chicken ( Gallus gallus ) PGCs to helmeted guinea-fowl ( Numidea meleagris ) hosts (van de Lavoir et al, 2006), common pheasant ( Phasianus colchicus ) GGCs to chicken hosts (Kang et al, 2008), and chicken PGCs to domestic duck ( Anas domesticus ) hosts (Liu et al, 2012).…”