In vitro culture and characterization of duck primordial germ cells

Abstract

This study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

Keywords: Muscovy duck (Cairina moschata); Pekin duck (Anas platyrhynchos); mule duck (C. moschata × A. platyrhynchos); primordial germ cell (PGC).

Figure 1.

Generation of Muscovy duck PGCs. (A) Blood was collected from the dorsal aorta of E5 Muscovy duck embryos at stage HH 16. (B) MDcPGCs were obtained after 35 d of culture in FAcs medium. Scale bar: 100 μm. (C) MDcPGCs formed clusters and were highly confluent after 35 d of culture. Scale bar: 50 μm. (D) An E9 Muscovy duck embryo (stage HH 28). (E) Embryonic gonads, indicated by dotted lines, were collected and dispersed to obtain PGCs. Scale bar: 0.5 mm. (F) MDgPGCs were cultured from dispersed gonads and easily isolated from adherent stromal cells after 1 d of culture. Scale bar: 50 μm. (G and H) MDgPGCs remained proliferative in FAcs medium after 5 d of culture. Scale bars: 100 and 50 μm, respectively.

Figure 2.

Growth assay of CcPGCs and MDcPGCs. (A) The total number of CcPGCs and MDcPGCs after 8 d of culture in FAcs medium. (B) Doubling time of CcPGCs and MDcPGCs. A total of 1 × 10⁴ cells were seeded, and the total cell number was counted after 8 d of culture. The doubling time was calculated (Roth V. 2006 Doubling Time Computing, available from http://www.doubling-time.com/compute.php). Data are expressed as mean ± SEM from at least 3 independent experiments. ****P < 0.0001.

Figure 3.

Comparison of MDgPGCs cultured in FAcs and FAot media. (A) Proliferation of MDgPGCs in FAcs and FAot media over 1 wk. Scale bar: 100 μm. (B) Proliferation efficiency of MDgPGCs in FAcs and FAot media over 1 wk. The relative cell number was determined by comparing absorbance of the samples in the CCK-8 assay with a standard curve generated using known numbers of chicken PGCs. Data are presented as mean ± SEM. ****P < 0.0001. (C) Fold change in the relative total cell number compared with the relative number of MDgPGCs seeded. Data are presented as mean ± SEM. ****P < 0.0001.

Figure 4.

Culture of chicken and duck gPGCs in FAot medium. (A) Image of CgPGCs cultured for 105 d (35 passages). Scale bar: 50 μm. (B) Images of MDgPGCs, (C) PDgPGCs, and (D) MUDgPGCs cultured for 1 wk. Scale bar: 50 μm.

Figure 5.

Characterization of chicken and duck gPGCs. (A) Staining of CgPGCs, MDgPGCs, PDgPGCs, and MUDgPGCs with PAS. Scale bar: 50 μm. (B) RT-PCR analysis of CgPGCs, MDgPGCs, PDgPGCs, and MUDgPGCs. Expression of germline-specific markers (DDX4 and DAZL), pluripotency markers (OCT4, SOX2, NANOG, and SALL4), and other PGC-specific markers (PIWIL1, PIWIL2, CXCR4, PRDM14, and PRDM1) was evaluated. The housekeeping gene GAPDH and RPS17 was used as the internal control. EFs from each breed were used as somatic cells for comparison. Water was used as a negative control in the reverse transcription reaction. (C) Immunofluorescence staining of the pluripotency marker SSEA-1 in CgPGCs (scale bar: 10 μm), MDgPGCs (scale bar: 10 μm), PDgPGCs (scale bar: 7.5 μm), and MUDgPGCs (scale bar: 10 μm). SSEA-1-positive cells: green; DAPI nuclear staining: blue. (D) Immunofluorescence staining of the germ cell marker DDX4 in CgPGCs (scale bar: 7.5 μm), MDgPGCs (scale bar: 5 μm), PDgPGCs (scale bar: 7.5 μm), and MUDgPGCs (scale bar: 5 μm). DDX4-positive cells: red; DAPI nuclear staining: blue. (E) Flow cytometric analysis of immunocytochemical staining of SSEA-1 and DDX4 in CgPGCs, MDgPGCs, PDgPGCs, and MUDgPGCs. The percentage of SSEA-1 or DDX4-positive cells is shown in each panel. gPGCs were stained with mouse IgM isotype antibodies or rabbit IgG secondary antibodies as a control. Ten thousand cells were assessed per sample.

Figure 6.

Expression of PGC markers in MDgPGCs during culture in vitro. mRNA expression of germline-specific markers (DDX4 and DAZL), pluripotency genes (OCT4, SOX2, NANOG, and SALL4), and other PGC markers (PIWIL1, PIWIL2, CXCR4, PRDM14, and PRDM1) was examined by RT-PCR. GAPDH and RPS17, both ubiquitously expressed genes, were used as the internal control. All germline-specific markers were expressed in CgPGCs cultured for 30, 125, and 250 d. Expression was analyzed in MDgPGCs cultured for 3, 7, and 14 d. EFs from each breed served as a somatic cell control. Water was used as a negative control in the reverse transcription reaction.

Figure 7.

Homing of transplanted gPGCs to the gonads of recipients. (A) Graphical summary of the procedures used to functionally characterize the germ cell properties of PGCs expanded in vitro. The ability of transplanted chicken and duck PGCs to home to the gonads of recipients was assessed. (B) Structure of pAS7w.EGFP.puro, which had a total length of 3,501 bp. Features were visualized using SnapGene Viewer 4.0 (GSL Biotech LLC, USA). (C) Transplantation of cultured chicken EGF::PGCs into chicken embryos. Chicken male EGF::PGCs (upper panel) had a round shape when grown in suspension and ubiquitously exhibited green fluorescence, demonstrating expression of the transgene. Scale bar: 100 μm. After transplantation of EGF::PGCs into the blood circulation of an E3 chicken embryo and incubation for 7 d, donor cells were detected in the gonadal region (dotted lines). Scale bar: 500 μm. (D) Allogeneic transplantation of virally transduced MDgPGCs into E5 Muscovy duck embryos. Some MDgPGCs were EGFP-positive at 2 d after viral transduction (upper panel). Scale bar: 100 μm. EGFP-expressing MDgPGCs were detected in the gonads of E11 duck embryos (lower panel, dotted lines). Scale bar: 500 μm. (E) The presence of duck genetic material in the gonads of recipient chickens after transplantation of duck gPGCs. (a) Species-specific primer sets were used to amplify a chicken-specific PCR product (221 bp, upper panel) and duck-specific amplicons (189 bp; lower panel) from the 3 duck breeds. (b) Confirmation of the presence of duck gPGCs in the gonads of recipient chicken embryos at 7 d after transplantation. Duck-specific amplicons were observed in the gonads of chicken embryos transplanted with MDgPGCs, PDgPGCs, and MUDgPGCs (lower panel). Chicken-specific amplicons were observed in all samples as a positive control (upper panel).

Figure 8.

Graphical summary of the isolation, in vitro expansion, genetic modification, and functional characterization of duck cPGCs and gPGCs.