Enhanced cultivation of chicken primordial germ cells

Abstract

The cultivation and expansion of chicken primordial germ cells (cPGCs) are of critical importance for both biotechnological applications and the management of poultry genetic biodiversity. The feeder-free culture system has become the most popular approach for the cultivation and expansion of cPGCs. However, despite some success in the cultivation of cPGCs, the reproducibility of culture conditions across different laboratories remains a challenge. This study aimed to compare two defined and enriched media for the growth of cPGCs originating from the Hubbard JA57 broiler. To this end, cPGCs were isolated from the embryonic blood of Hamburger-Hamilton (HH) stages 14-16 and cultured at various time points. The Growth properties and characteristics of these cells were evaluated in two different culture conditions (the defined or enriched medium) and their migratory properties were assessed after genetic engineering and injection into the vasculature of 2.5-day-old chicken embryos. The main finding of this study was that the use of an enriched medium (the defined medium with Knock-Out Serum Replacement; KOSR) resulted in improved growth properties of cPGCs originating from the Hubbard JA57 broiler compared to a defined medium. The ability to cultivate and expand cPGCs is crucial for the generation of both genetically engineered birds and breeds of interest from local or commercial origins. Therefore, these results highlight the importance of choosing an appropriate culture medium for cPGCs growth and expansion.

Figure 1

Real and schematic depiction of cPGCs, growth factors involved in cPGCs survival and proliferation in-vitro, and stem-cell/germ-cell specific markers in cPGCs. (A) 120-day-old cPGCs culture with lots of doublet cells and prominent eccentric refractive granules (left) and several important growth factors involved in the survival and proliferation of cPGCs are illustrated (right). (B-a) Immunocytochemistry was performed using primary antibodies against DDX4 and DAZL proteins as well as EMA1 and SSEA1 cell surface markers. Conjugated secondary antibodies were used to label the primary antibodies. (B-b) Flow cytometry was performed using primary EMA1 antibody which was labeled using conjugated secondary antibody and conjugated primary antibody was used for detecting SSEA1 cell surface marker. (IgG: Immunoglobulin G; IgM: Immunoglobulin M; FITC: Fluorescein isothiocyanate; AF647: Alexa Fluor 647).

Figure 2

Enriched medium improves derivation, expansion, long-term culture, and proliferation rates of cPGCs. (A) Components of the cPGC basic medium were supplemented with several types of growth factors to make an enriched medium (specifically containing lipid-rich albumin provided by KOSR) or defined medium (without KOSR). (B) The rate of derived (a), expanded (b), long-term cultured (c), 70-day-old (d), and 120-day-old (e) cPGCs were compared in enriched and defined media. The number of derived (f) and expanded (g) cPGCs in the enriched medium were compared with those in the defined medium. (C) The specific features of cPGCs cultured in enriched and defined media were compared. The oval dash lines indicate the doublet form of cPGCs (a, b, c). In the insets, the eccentric refractive granules (white arrows) and dead cells (red arrows) are depicted (E: enriched, D: defined, M: male, F: female, 14,15,16: HH stages). (D) Comparison of the number of dead (red arrows) and clumpy (yellow arrows) cells in different cell lines of cPGCs cultured in enriched and defined media. *: p < 0.05, **: p < 0.01 are statistically significant.

Figure 3

Enriched medium enhances the expression of stem-cell and germ-cell specific markers and transcription factors in cPGCs. DAZL-positive (A-a, B-a) and DDX4-positive (A-b, B-b) cPGCs, as well as unstained cPGCs (Ac, Bc; Negative control: it was only stained using secondary antibody) imaged by confocal microscopy are shown. The promoter assay shows the expression of tdTomato controlled by DAZL (A-d, B-d; top) and DDX4 (A-d, B-d; bottom) promoters in cPGCs. Panels A-e and B-e show the expression analysis of EMA1 and SSEA1 in cPGCs cultured in enriched and defined media by flow cytometry at day 70. Panels A-f and B-f show the expression analysis of EMA1 and SSEA1 in cPGCs cultured in enriched and defined media by flow cytometry at day 120. The cell membrane localization of EMA1 and SSEA1 on cPGCs are shown in panels A-g, A-h, B-g, and B-h. Also, no green fluorescence signals were observed in the negative groups (A-i and B-i). A DAPI staining was performed for each group (A-j, B-j). Panel C shows the comparison of the expression level of cNANOG and cOCT4 transcripts between 70- and 120-day-old cPGCs cultured in defined (C-a, C-c) and enriched (C-b, C-d) media. Panel D shows the comparison of the expression level of cNANOG and cOCT4 transcripts between cPGCs cultured in the defined and enriched media at day 70 (D-a, D-c) and 120 (D-b, D-d).

Figure 4

cPGCs expanded in the enriched medium have the competence of homing and colonization in the embryonic gonads. (A) schematic illustration showing co-electroporation of transposon/transposase vectors into the cPGC, injection of the td-Tomato-expressing cPGCs into the chicken embryo (HH stage 13–15), and localization of these cPGCs in the chicken gonads (HH stage 26–28). (B–D) The td-Tomato-expressing cPGCs that were electroporated in 650v, 850v, and 1050v, respectively. (E–G) Analysis of the electroporation efficiency by flow cytometry. H) Negative control (cells that were not electroporated). (I,J) Localization of td-Tomato-expressing cPGCs in the 6-day-old embryo gonads (HH stage 26–28). (I-a, J-a) Bight field images of dissected chicken embryo gonads (HH stage 26–28). (I-b, J-b) The td-Tomato-expressing cPGCs localized in the chicken embryo gonads (HH stage 26–28) imaged by red fluorescence. (I-c, J-c) Merged images of chicken embryo gonads at HH stage 26–28. v: volt, ms: millisecond, p: pulse.