Journal of Animal Reproduction and Biotechnology 2020; 35(1): 65-72
Published online March 31, 2020
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Jun Hyung Ryu1,† and Seung Pyo Gong1,2,*
1Department of Fisheries Biology, Pukyong National University, Busan 48513, Korea
2Department of Marine-Biomaterials and Aquaculture, Pukyong National University, Busan 48513, Korea
Correspondence to: Seung Pyo Gong
†Current address: Institute of Marine and Environmental Technology, University of Maryland Baltimore County, Baltimore, MD, USA
Fish ovarian germline stem cells (OGSCs) that have the abilities to selfrenew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.
Keywords: feeder cells, in vitro culture, Japanese medaka, ovarian germline stem cells
Ovarian germline stem cells (OGSCs) differentiate into oocytes before birth and thus, their existence in the postnatal ovaries is controversial in most mammalian species (Telfer et al., 2005). In contrast, the existence of OGSCs in adult fish ovaries has been demonstrated (Nakamura et al., 2010) and the production of offspring through transplantation of adult female-derived OGSCs has been achieved in zebrafish (Wong et al., 2011; Wong et al., 2013) and trout (Yoshizaki et al., 2010; Lee et al., 2016). Thus, fish OGSCs can be a useful resource for the study of developmental biology, genetics and biotechnology like primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). However, their utilization in the fields has been restricted due to a lack of knowledge regarding their in vitro culture implying that the effort should be made to establish a stable fish OGSC culture system.
Previously, Wong et al. demonstrated that rainbow trout splenic feeder cells expressing zebrafish leukemia inhibitory factor (LIF) and zebrafish ovarian somatic feeder cells expressing zebrafish fibroblast growth factor 2 (FGF2), glial cell derived neurotrophic factor (GDNF), gonadal soma derived factor (GSDF) or LIF could not only maintain germline competency of zebrafish OGSCs but also support their growth
For this reason, this study was performed to investigate the effects of feeder cells on the primary culture of
Adult Japanese medaka (
In order to establish feeder cell lines for
An established embryonic cell line was transfected with 1 μg FGF2 expression vectors using 1 μL lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and the transfected cells were selected by treating 600 μg/mL hygromycin (Invitrogen) for 4 weeks. The colonies formed were harvested using cloning cylinders (Corning Life Sciences, Corning, NY, USA) and cultured individually. After establishment of stable cell lines, FGF2 mRNA expression was confirmed by RT-PCR analysis. To detect the protein expression, a cell line, of which FGF2 mRNA expression was confirmed, was used. Cells harvested from a 100 mm culture dish (SPL Life Sciences) were washed twice with DPBS. Then cell lysates were obtained by a sonication in 1% (w/v) triton X-100 (Sigma-Aldrich) in DPBS and protein concentration of the cell lysates was measured using Pierce BCA protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. The denatured protein was loaded on 15% acrylamide SDS gels, electrophoresed for 1.5 h at 100 V and transferred to PVDF transfer membranes (Bio-Rad, Hercules, CA, USA) for 1 h at 100 V. After blocking with 5% (w/v) skim milk (Sigma-Aldrich) in PBS with 0.1% (v/v) Tween 20 (Sigma-Aldrich) (PBST) for 1 h at room temperature, the membranes were incubated with monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich) at a 1:1,000 dilution for 15 h at 4°C. After washing the membranes three times with PBST for 3 min, HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo Scientific) was applied with 1:20,000 dilution for 1 h at room temperature. The membranes were washed three times with PBST for 3 min and signals of the proteins were visualized using enhanced chemiluminescence solution (Bio-Rad) and X-ray films (Kodak, Tokyo, Japan) by exposure for 3 min.
To obtain ovarian cell populations, 10 ovaries were subjected to mechanical and enzymatic dissociation as mentioned in the part of ovarian cell isolation. Then, to enrich germ cell populations by removing red blood cells and anchorage-dependent cells, the retrieved cells were washed twice with DPBS containing 1% (v/v) P/S and loaded onto the top of layered Percoll (Sigma-Aldrich) solution consisting of 20, 25, 30, 35, 40, 50 and 60% in a 15 mL conical tube (Falcon). After centrifugation at 800×g for 30 min, density fractions of 20-40% were harvested. After being washed twice with DPBS, the cells were resuspended in L15 supplemented with 10% (v/v) FBS and 1% (v/v) P/S. Then, 2-5 × 106 cells were seeded on a 35 mm petri dish (SPL Life Sciences) coated with 0.1% (w/v) gelatin and incubated for 15 h at 28°C. The cells that were floating or loosely bound were harvested and used for further experiments. These cells were designated as “enriched ovarian cell population” after this.
To investigate the effect of feeder cells on the growth of ovarian cells, 1.5 × 104 live cells from the enriched ovarian cell populations were seeded on feeder cells or feeder-free condition. For the culture on feeder cells, 5 × 105 feeder cells were seeded on a well of 24-well plates (SPL Life Sciences) and mitotically inactivated by treating 10 μg/mL mitomycin-C (Sigma-Aldrich) for 3 h before use. Prior to initiation of culture, all culture plates with or without feeder cells were incubated with ovarian germline stem cell medium (OGSM) for 24 h at 28°C in an air atmosphere. Enriched ovarian cell populations were labeled with 6 μM PKH26 (Sigma-Aldrich) for 3 min and cultured in OGSM consisting of L15 supplemented with 25 mM HEPES (Sigma-Aldrich), 6 mg/mL D-(+)-glucose (Sigma-Aldrich), 1% (v/v) glutamax (Gibco), 1% (v/v) non-essential amino acids (Gibco), 0.5% (w/v) BSA (Sigma-Aldrich), 5% (v/v) FBS, 0.25% (v/v) trout serum (Caisson Laboratories, Smithfield, UT, USA), 1 μg/mL medaka embryo extract, 1% (v/v) P/S, 25 μg/mL bovine insulin (Sigma-Aldrich), 100 μg/mL recombinant human apo-transferrin (Sigma-Aldrich), 10 ng/mL recombinant human basic fibroblast growth factor (bFgf; Gibco), 10 ng/mL recombinant human glial cell-derived neurotrophic factor (Peprotech, Rocky Hill, NJ, USA), 5% (v/v) Knockout serum replacement (Gibco), 50 μM ascorbic acid (Sigma-Aldrich), 50 μM β-mercaptoethanol (Gibco), and 2 nM sodium selenite (Sigma-Aldrich) at 28°C in an air atmosphere. Medaka embryo extract was extracted as described previously (Choi and Gong, 2018). Half of the culture medium was replaced with fresh one every three days. For measurement of the cell number after culture for 10 days, the cells were harvested by treatment with 0.05% trypsin-EDTA (Gibco) and the number of fluorescent cells was counted using a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) under a TS-100F microscope equipped with a fluorescent unit (Nikon, Tokyo, Japan).
The statistical analyses were performed using SPSS version 18 (IBM-SPSS, Chicago, IL, USA). The data were analyzed by One way-ANOVA or t-test followed by Duncan’s method. Significant differences among groups were determined when
To find optimal tissue source to be able to supply feeder cell lines, culture of the cells from three tissues including embryonic tissue, fin, and ovary was performed. All cell populations regardless of tissue sources showed primary adherence to substrates and of those, more than 80% were subcultured at least once (Table 2). However, cell death was observed after the removal of fin fragments and after 2 weeks of culture in fin- and ovary-derived cells, respectively. The cell populations derived from fins and ovaries could not reach to passage 5 in culture and were just maintained for 18.2 ± 6.0 and 20.8 ± 13.4 days, respectively. On the contrary, 18 (66.7%) out of 27 cell populations from embryonic tissues was cultured to more than passage 5 and 9 (33.3%) out of 27 was cultured to more than passage 20. Twenty two (81.5%) out of 27 cell populations was maintained in culture for more than 50 days. These indicated that the embryonic tissues were an optimal tissue source to derive the cell lines that can be used as feeder cells among three tissues tested. Three embryonic cell lines (named as EC1, EC2, and EC3) were used as feeder cells in this study. These ECs contained two cell types that were morphologically different; fibroblast-like cells and epithelial-like cells. In EC1 and EC3, fibroblast-like cells were predominant whereas epithelial-like cells were predominant in EC2 (Fig. 1A). These three ECs did not express germline stem cell-specific Nanos2 and germ cell-specific Vasa genes indicating that germline cells were not included in these ECs (Fig. 1B).
To examine the effects of feeder cells on the culture of ovarian cells, enriched ovarian cell populations that were labeled with fluorescent PKH26 were cultured on feeder cells derived from each of three ECs (EC1, EC2, and EC3) and the change of cell number was investigated after culture for 10 days. The enriched ovarian cell populations cultured on feeder cells formed cell aggregates unlike those cultured under feeder-free condition and those cultured on EC1 and EC3 showed a significant increase in cell number after culture for 10 days compared to those cultured on feeder-free condition (
The FGF2 expression vector was transfected into EC3 and stable cell line expressing FGF2 was subsequently established by antibiotic selection. RT-PCR analysis confirmed that the established cell line expressed the mRNA of foreign FGF2 whereas it did not express endogenous FGF2 (Fig. 3A). Moreover, the expression of FLAG-tagged FGF2 (18.6 kDa) was detected in the stable cell line by western blot analysis (Fig. 3B). To test the effects of FGF2-expressing feeder cell line (named as EC3F) on ovarian cell culture, enriched ovarian cell populations were cultured on EC3 or EC3F feeder cells. Furthermore, the effects of recombinant human bFGF as a medium supplement were also tested by removing it from OGSM. As the results, the cultured ovarian cells formed cell aggregates during in vitro culture regardless of treatment groups (Fig. 4A). After culture for 10 days, the number of cells increased from 1.08 to 1.37-fold relative to the initial cell number seeded according to the experimental groups (Fig. 4B). No significant difference was detected among experimental groups indicating that both human recombinant bFGF and medaka FGF2 produced by EC3F were not effective to induce ovarian cell proliferation.
To develop a suitable culture condition for a specific cell type, several points including physical aspects, nutrients, signaling molecules such as hormones and cytokines, substrate condition and culture method should be considered (Freshney, 2010). Feeder cells have been often utilized to develop or improve the culture condition for certain cells, as they can supply several soluble signaling molecules and extracellular matrices to target cells during in vitro culture (Hongisto et al., 2012; Villa-Diaz et al., 2013). In mammals, the cells derived from embryos such as mouse embryonic fibroblasts have been frequently used for the culture of GSCs (Kanatsu-Shinohara et al., 2003; Kubota et al., 2004). In the present study, embryonic cell lines were established and two of them significantly promoted the growth of enriched ovarian cell populations when used as feeder cells. This would have been due to the extracellular matrices and cytokines secreted from the feeder cells. For establishment of embryonic cell lines, the embryos at stages 32 to 36 were used and they are known to express various kinds of hormones and growth factors such as GSDF and insulin (Assouline et al., 2002; Shibata et al., 2010). Based on this, the specific molecules that positively influenced to ovarian cells in culture needs to be found for the optimization of OGSC culture system.
To achieve more active proliferation of ovarian cells, a feeder cell line artificially-expressing FGF2 was developed. Because of the amino acid dissimilarities between mammals and fish, mammalian growth factors supplemented in culture media may have low or no activity on fish GSCs (Kawasaki et al., 2012). Thus, testing fish growth factors instead of mammalian ones is a major step for the establishment of a more suitable culture condition for fish OGSCs. FGF2, a member of the FGF family, is an important factor for the self-renewal of mammalian SSCs (Ishii et al., 2012; Takashima et al., 2015). The protein identity of FGF2 between human and medaka is 73% and it was reported that recombinant human FGF2 supports SSCs from fish including medaka, trout and zebrafish at 1-100 ng/mL concentrations (Hong et al., 2004; Shikina et al., 2008; Kawasaki et al., 2012). In addition, it was reported that zebrafish FGF2 supports the colony formation and proliferation of zebrafish SSCs and OGSCs during
In the present study, the effects of feeder cells on the culture of medaka ovarian cell populations were examined. The feeder cells derived from medaka embryonic tissues were able to support ovarian cell proliferation during short culture period. The results from this study will provide fundamental information for development of the optimal conditions for OGSC culture and will contribute to the utilization of OGSCs in various areas.
This work was supported by a Research Grant of Pukyong National University (2019).
No potential conflict of interest relevant to this article was reported.
JH Ryu performed the experiments and wrote the paper; SP Gong conceived and designed the experiments and wrote the paper.
JH Ryu, Ph.D., Researcher,
SP Gong, Professor,