In Vitro Culture of Chicken Circulating and Gonadal Primordial Germ Cells on a Somatic Feeder Layer of Avian Origin

Abstract

The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo's feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.

Keywords: cPGCs; chicken; embryo; feeder cell layer; gPGCs; primordial germ cells.

Figure 1

Fresh gonadal primordial germ cells (gPGCs) and circulating blood primordial germ cells (cPGCs) observed under an inverted phase-contrast microscope; gPGCs and cPGCs are visualized in darkfield illumination. The PGCs are distinguished as bright, illuminated cells among other cell types (P.434242 patent application number). Magnification with 40× objective (Leica DMI8, Germany).

Figure 2

The gPGCs cultured for 7 and 14 days, observed under an inverted phase-contrast microscope and in darkfield illumination. This technique allows us to clearly distinguish PGCs from the feeder layer and other cells. The PGCs are visible as bright, illuminated cells among the other cell types (P.434242 patent application number). Magnification with 40× objective (Leica DMI8).

Figure 3

The cPGCs cultured for 7 days observed under an inverted phase-contrast microscope and darkfield illumination (P.434242 patent application number). The cells were stained after 7 days of culture using PKH26 fluorescent dye. Magnification of the objective 40× (Leica DMI8).

Figure 4

Left panel: Male gonad observed under the microscope (Leica DMI8) and stained with PKH26. Right panel: Dispersed cells from the recipient gonads and stained with PKH26.

Figure 5

Proliferation curve of gPGCs cultivated on a chicken embryo feeder layer at pH 8.0. The live gPGCs cells were counted directly in the culture by using the darkfield illumination method.

Figure 6

Left panel: The male gonad observed under the microscope (Leica DMI8) and stained with PKH26. Right panel: Dispersed cells from the recipient gonads and stained with PKH26.

Figure 7

Proliferation curve of cPGCs cultivated on a chicken embryo feeder layer at pH 8.0. The live cPGCs were counted directly in the culture by using the darkfield illumination method.