Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena)
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This study was conducted to identify embryonic stem cell (ESC) activities of a long-term cultured embryonic cell line previously derived from blastula-stage Oryzias dancena embryos. Five sub-cell lines were established from the embryonic cell line via clonal expansion of single cells. ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation capacity, were examined in the five sub-cell lines. We observed both clonogenicity and AP activity in all five sub-cell lines, but the proportion of cells that exhibited both properties was significantly different among them. Even though we detected different formation rates and sizes of embryoid body (EB) among these cells, all lines were stably able to form EBs and further induction for differentiation showed their capability to differentiate into other cell types in a spontaneous manner. From this study, we determined that the embryonic cell lines examined possessed heterogeneous ESC activities and can be utilized as a marine model system for fish ESC-based research.
KeywordsMarine model Oryzias dancena Embryonic stem cells Heterogeneity
Embryonic stem cells (ESCs) are a representative cell type possessing both continuous self-renewal activity and the potential to differentiate and give rise to all three germ layers (Evans and Kaufman 1981). These two properties make ESCs a valuable tool for cell replacement therapy in humans (Doss et al. 2004) and for transgenic animal research (Camper et al. 1995). Thus, many fish biotechnology scientists have attempted to establish ESCs or ESC-like cells from several fish species, such as zebrafish (Danio rerio; Ho et al. 2014), medaka fish (Oryzias latipes; Hong et al. 1996), sea perch (Lateolabrax japonicus; Chen et al. 2007), Asian seabass (Lates calcarifer; Parameswaran et al. 2007), red seabream (Pagrosomus major; Chen et al. 2003), gilt-head seabream (Sparus aurata; Béjar et al. 2002), Indian major carp (Catla catla; Dash et al. 2010), and marine flatfish (Scophthalmus maximus; Holen and Hamre 2003). Specifically, O. latipes haploid ESCs exhibiting both haploidy and pluripotency may allow for direct genetic analysis to evaluate recessive and disease phenotypes (Yi et al. 2009). Nevertheless, a marine model system is lacking for fish ESC-based genetic analysis and biotechnology. Marine medaka (Oryzias dancena) may be a good marine fish model because it lives in brackish water and is able to acclimate to a wide range of salinities (Inoue and Takei 2002, 2003). In addition, this fish species has characteristics similar to those of Japanese medaka (O. latipes), in that they are able to spawn daily, have transparent bodies during fetal development, grow rapidly and thus have a short generation time, and are easy to manage on a laboratory scale (Cho et al. 2011; Lee et al. 2013). However, ESCs derived from marine medaka have not been reported yet. Previously, we established an embryonic cell line derived from O. dancena blastulas and analyzed its basic cellular characteristics, such as growth rate, chromosomal normality, and response to medium components (Lee et al. 2013). In addition, optimal freezing conditions were developed for this cell line (Kim et al. 2014). In this study, considering the importance of a marine model for fish ESC-based research, we attempted to identify the ESC activities of our previously established embryonic cell line. Based on a previous report that provided a standard protocol to obtain fish ESCs (Yi et al. 2010), we first established five sub-cell lines through clonal expansion of embryonic cells cultured long-term, in an effort to identify a cell line possessing ESC activities. The five established sub-cell lines were characterized subsequently, and their in vitro ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation potential, were compared. The chromosomal normality of each sub-cell line was also identified and compared.
Materials and methods
The O. dancena embryonic cell line that we established in a previous report (Lee et al. 2013) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 4.5 g/L d-glucose, 20 mM HEPES, 1 % (v/v) nonessential amino acids (Gibco), 15 % (v/v) fetal bovine serum (FBS; Cellgro, Manassas, VA, USA), 1 % (v/v) fish serum, 50 μg/mL embryo extract, 1 % (v/v) penicillin–streptomycin mixture (Gibco), 10 ng/mL recombinant human basic fibroblast growth factor (bFGF; Gibco), 100 μM β-mercaptoethanol (Gibco), 2 nM sodium selenite (Sigma-Aldrich, St. Louis, MO, USA), and 1 mM sodium pyruvate (Gibco). The fish serum and embryo extracts were prepared as described previously (Lee et al. 2013). The cells were cultured on 0.1 % gelatin-coated (Sigma-Aldrich) tissue culture plates in culture medium in a 28 °C incubator in air. Cells were sub-cultured every 2 or 3 days once the cells reached 80–90 % confluency.
Derivation of sub-cell lines and clonogenicity testing
To derive sub-cell lines, a total of 3600 embryonic cells were seeded initially in a 60-mm tissue culture plate (SPL Life Sciences, Pocheon, Korea) and cultured in medium for 10 days under an air atmosphere at 28 °C. The medium was replaced every 3 days. Ten clonally expanded colonies formed by the end of the culture period were collected separately and amplified until we obtained a sufficient cell population for further analysis. To test the clonogenicity of each sub-cell line, the same protocol was used to derive clonally expanded colonies, and the colony number was counted visually by crystal violet (Sigma-Aldrich) staining at the end of the culture period.
The clonally expanded colonies from each sub-cell line were fixed with 4 % formaldehyde (Sigma-Aldrich) for 10 min, and the fixed colonies were incubated with AP staining solution consisting of 2 mg naphthol AS-MX phosphate (Sigma-Aldrich) dissolved in 200 μl N,N-dimethylformamide (Sigma-Aldrich), 9.8 ml 0.1 M Tris–HCl (Bioneer, Daejeon, Korea) diluted in Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich), and 10 mg Fast Red TR salt (Sigma-Aldrich) for 30 min. After washing three times with DPBS, color formation indicative of AP activity in the cells was identified under an inverted microscope (TS100-F, Nikon, Tokyo, Japan).
In vitro differentiation
To induce embryoid body (EB) formation, 7 × 105 cells were seeded in a 60-mm petri dish (SPL Life Sciences) in culture medium containing a reduced concentration of bFGF (4 ng/mL) and embryo extract (10 μg/mL). After the formation of EBs, images of 20 EBs derived from each sub-cell line were acquired and the sizes were measured using the TSview program (Tucsen Imaging Technology Co., Ltd., Fujian, China). The size was defined as an average of the length, width and height. All EBs formed were allowed to attach to the bottom of a 100-mm culture plate by incubating the plate at 28 °C for 1 day, and the EB number was counted visually after crystal violet staining. To induce spontaneous differentiation, 10 EBs attached to the bottom of a 60-mm culture dish (SPL Life Sciences) were cultured for 2–3 weeks. The medium used was the same as that used for EB formation and was changed every 3 days. Two different sets of experiments, with and without 10 μM retinoic acid (RA; Sigma-Aldrich) treatment, were performed to induce spontaneous differentiation of the sub-cell lines. These experiments were conducted three times each in an independent manner.
Metaphase chromosomes were prepared as described previously with slight modification (Gong et al. 2014). The cells were washed in DPBS and treated with a 0.075-M KCl (Sigma-Aldrich) solution for 10 min at 28 °C. The swollen cells were fixed using a cold fixative solution comprised of methanol (Sigma-Aldrich) and acetic acid (Sigma-Aldrich) at a ratio of 3:1, and the fixative solution was changed three times by centrifugation at 400g for 5 min. Metaphase chromosomes were spread onto ethanol-treated slides and stained with Giemsa stain solution containing 10 % (v/v) KARYOMAX® Giemsa stain (Gibco) in Gurr’s buffer (Gibco). After washing in distilled water, the slides were air-dried, and the number of chromosomes was counted.
Statistical Analysis System (SAS) software was used to analyze the data. When analysis of variance (ANOVA) identified a significant main effect, treatments were analyzed subsequently by the least squares method or Duncan’s method. Significant differences among treatments were defined by a P value <0.05.
Clonogenicity and AP activity of sub-cell lines
EB formation and in vitro differentiation
In this study, we demonstrated the presence of ESC activities in an O. dancena long-term cultured embryonic cell line. Five sub-cell lines were successfully derived from this embryonic cell line and cultured stably. Each line displayed ESC activities, including clonal expansion ability, AP activity, and in vitro differentiation potential. On the other hand, the proportion of cells bearing each ability within a population differed among the sub-cell lines, and different chromosomal normality was also observed.
To identify ESC cell populations, cellular pluripotency must be demonstrated following rigorous testing standards (Yi et al. 2010; Wobus and Boheler 2005; Zhao et al. 2012). In the present study, we established the presence of several characteristics in the embryonic cell line analyzed in this study, including AP activity, EB formation, and in vitro differentiation into specific cell types, all of which are strong markers of pluripotency, confirming that this cell line authentically possessed ESC activities. However, we could not fully test all parameters due to a lack of information and technical limitations of this fish species. For this reason, this cell line can be labeled as ESC-like cells in current status, similarly to many embryo-derived cell cultures in other fish species given the same identification label (Ho et al. 2014; Parameswaran et al. 2007; Béjar et al. 2002; Dash et al. 2010; Holen and Hamre 2003). Efforts should be made to further characterize the ESC properties of this cell line, such as identification of pluripotency genes in O. dancena and chimera formation through inoculation of cells in developing embryos.
As mentioned above, the primary purpose of establishing sub-cell lines was to identify a superior cell population possessing ESC activity derived from the original cell line. Similar clonal expansion and AP staining were identified among the five sub-cell lines, but no correlations with EB formation, EB size, and differentiation capacity were detected. These results suggest that pluripotency markers are not linked directly to a specific sub-cell line, and thus sub-cell line derivation is not sufficient to indicate superiority of a certain sub-cell line.
Conversely, we identified cellular heterogeneity using sub-cell line derivation experiments. Different levels of cellular capabilities among each sub-cell line indicated heterogeneity from the original cell line. Moreover, partial AP activity within a cell line is indicative of de novo heterogeneity generation during sub-cell line derivation, since each sub-cell line was derived from the clonal expansion of a single cell. Similarly, the karyotype results for each sub-cell line, which indicated partial normality in chromosome number despite the single-cell origins, confirm this phenomenon as well. Similar to our results, previous studies in mice and humans reported morphological or phenotypic heterogeneity among ESCs (Hayashi et al. 2008; Hong et al. 2011; Stewart et al. 2006); thus, heterogeneity may be a general phenomenon of ESC cultures regardless of species. Nonetheless, considering the significance of securing a homogeneous cell population for biotechnological application of the cells, determining the precise explanation for the cellular heterogeneity and subsequently establishing optimal conditions to overcome this are priorities.
As an agent used to induce in vitro lineage-specific differentiation of ESCs, RA induces in vitro differentiation of ESCs into a number of cell types, including neural (Kim et al. 2009; Wichterle et al. 2002), mesodermal (Kennedy et al. 2009; Torres et al. 2012), epithelial (Metallo et al. 2008), pancreatic (Shim et al. 2007), and germ cell lineages (Chen et al. 2012). Our results showed that the differentiation patterns of O. dancena ESC-like cells were distinct at the morphological level depending on the presence or absence of RA. Although additional assessments at the gene and protein levels are required, these results suggest that RA acts as a signaling molecule to direct or inhibit differentiation into specific lineages in O. dancena ESC-like cells, in addition to mammals. Moreover, differentiation pattern biased toward non-neuronal lineage cells suggests that specific signaling pathways different from those in mammals may be involved in the differentiation mechanisms of fish ESC-like cells. Additional studies using O. dancena ESC-like cells as a model would provide more information regarding cellular differentiation in fish.
In conclusion, we report that a previously established O. dancena embryonic cell line possesses major ESC activities in a heterogeneous fashion. These O. dancena ESC-like cells are likely a valuable marine model for fish ESC research.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A1011572).
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