Long-term in vitro culture and preliminary establishment of chicken primordial germ cell lines.

Linglin Kong,Guobin Chang,Xin Zhang,Bowen Chen,Lingling Qiu,Yang Zhang,Ying Chen,Qixin Guo,Wei Shen

doi:10.1371/journal.pone.0196459

Abstract

Primordial germ cells (PGCs) are precursors of functional gametes and can be used as efficient transgenic tools and carriers in bioreactors. Few methods for long-term culture of PGCs are available. In this study, we tested various culture conditions for PGCs, and used the optimum culture system to culture chicken gonad PGCs for about three hundred days. Long-term-cultured PGCs were detected and characterized by karyotype analysis, immunocytochemical staining of SSEA-1, c-kit, Sox2, cDAZL, and quantitative RT-PCR for specific genes like Tert, DAZL, POUV, and NANOG. Cultured PGCs labeled with PKH26 were reinjected into Stage X recipient embryos and into the dorsal aorta of Stage 14–17 embryos to assay their ability of migration into the germinal crescent and gonads, respectively. In conclusion, the most suitable culture system for PGCs is as follows: feeder layer cells treated with 20 μg/mL mitomycin C for 2 hours, and with 50% conditioned medium added to the factor culture medium. PGCs cultured in this system retain their pluripotency and the unique ability of migration without transformation, indicating the successful preliminary establishment of chicken primordial germ cell lines and these PGCs can be considered for use as carriers in transgenic bioreactors.

Highlights

Due to the particularity of embryonic development, it is highly difficult to obtain gametes and single cell fertilized eggs from poultry
Feeder layer cells could support Primordial germ cells (PGCs) adherence and proliferation, and morphological differentiation of PGCs with or without feeder cells was observable after culturing for 5 days (Fig 2A and 2B)
The inhibition of mitomycin-treated STO cells was moderate at 20 μg/mL for 2 h, which was the treatment condition used for the feeder layers in the PGC subculture and follow-on experiments

Summary

Introduction

Due to the particularity of embryonic development, it is highly difficult to obtain gametes and single cell fertilized eggs from poultry. Primordial germ cells (PGCs), as precursors of functional gametes, demonstrate unique pluripotency and differentiation potential, as well as migration patterns during development, which are extremely different from those in mammals [1]. PGCs circulate temporarily in the bloodstream at the Hamburger and Hamilton (H&H) Stages 13–15, migrate to the germinal ridges at Stage 27, and differentiate into oocytes and spermatocytes [2,3]. The unique migration pattern of chicken PGCs facilitates their isolation, which is ideal for the establishment of embryonic stem cell lines. PGCs with multi-potential properties can be widely used in many fields such as germ cell and developmental biology, transgenesis and genome editing, conservation of avian genetic.

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Conclusion