Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Abstract

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGCLCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.

Poultry genetic resource conservation using primordial germ cells

“…Chicken PGCs isolated from embryonic blood can be expanded in a complex medium containing chicken serum, fetal bovine serum (FBS), fibroblast growth factor 2 (FGF2), and buffalo rat liver (BRL) cell-conditioned medium on a feeder of either BRL cells or Sandoz inbred mouse-derived thioguanine-resistant and ouabain-resistant (STO) fibroblasts [9]. Several research groups revealed that FGF signaling is required for chicken PGC proliferation in vitro [18, 48, 49]. A recent study has suggested that the membrane-bound form of chicken stem cell factor 2 (SCF2), but not the secreted form of chicken SCF1, enhanced the propagation of chicken PGCs in cooperation with FGF2 [50].…”

“…3. Outline of a poultry PGC-bank program., the procedures of ex situ conservation of poultry genetic resources consist of five steps; 1) collection of embryonic tissues containing PGCs from target lines or breeds, 2) purification or culture of PGCs, 3) PGC storage in liquid nitrogen, 4) PGC transplantation to sterilized recipient embryos, and 5) recovery of populations by mating of male and female recipients. To date, successful long-term culture of PGCs from various chicken breeds including indigenous breeds has been reported [9, 18, 22, 49]. Therefore, at least in the chicken, the major advantage of PGCs for use as a source of gene banking is that PGCs obtained from a single donor embryo would be amplified significantly in vitro prior to cryopreservation.…”

Perspectives on avian stem cells for poultry breeding

“…Poultry regeneration using embryonic and somatic stem cells (Usui et al 2009), poultry cloning and generation of transgenic poultry (Leighton et al 2008;van de Lavoir et al 2006;Zhu et al 2005) should be one of the most important subjects (Park et al 2014). Improvement of the cell culture should also be critical (Miyahara et al 2014). Scientific knowledge (Nakamura et al 2007) and technological refinement (Barrangou et al 2015;Yamamoto et al 2007a) should open up new frontiers for poultry breeding.…”

Embryo Manipulation in Chickens

“…They cultured PGCs isolated from embryonic blood on feeder cells derived from Sandoz inbred mouse-derived thioguanine-resistant and ouabain-resistant (STO) cells or Buffalo Rat Liver (BRL) cells in a culture medium containing basic fibroblast growth factor (bFGF) and stem cell factor (SCF). The usefulness of this culture method was confirmed by Macdonald et al (2010) and Miyahara et al (2014). Choi et al (2010) devised a feeder-free culture method for PGCs and found that bFGF is one of the key factors for the maintenance and proliferation of PGCs.…”