Fish Physiology and Biochemistry

, Volume 41, Issue 6, pp 1569–1576 | Cite as

Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena)

  • Dongwook Lee
  • Jun Hyung Ryu
  • Seung Tae Lee
  • Yoon Kwon Nam
  • Dong Soo Kim
  • Seung Pyo Gong


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.


Marine 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

Cell culture

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.

AP activity

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.

Karyotype analysis

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

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

Five sub-cell lines were derived successfully from 10 clonally expanded colonies that were collected and amplified separately and then cultured for at least 288 days. Conversely, the other five colonies failed to grow past five sub-culturings. As shown in Fig. 1, all five sub-cell lines had similar cell morphologies and possessed both the ability to grow in a clonal manner and AP activity. However, the rate of colony formation and the number of AP-positive colonies were significantly different among the five sub-cell lines (P < 0.0001, Fig. 1c). The numbers of colonies that formed from each of the five sub-cell lines derived from the 3600 cells initially seeded were 668 (19 ± 1.3 %), 554 (15 ± 2.7 %), 363 (10 ± 0.6 %), 87 (2 ± 0.4 %), and 907 (25 ± 1.9 %), respectively, of which 222 (33 ± 0.3 %), 117 (21 ± 3.8 %), 73 (20 ± 4.3 %), 10 (11 ± 2.8 %), and 343 (38 ± 2.7 %) colonies, respectively, were positive for AP staining.
Fig. 1

Images of cells, colonies, and alkaline phosphatase (AP)-positive colonies. Five sub-cell lines were derived from an Oryzias dancena embryonic cell line. Following low-density cell culture, all five sub-cell lines were able to form colonies (a) exhibiting AP activity (b). Non-stained colony was used as a negative control. c Different colony formation and AP activity capabilities were detected among the five sub-cell lines. Data are presented as mean ± standard deviation (SD) of percentage values from three independent experiments. Scale bar 50 μm for cell morphology and 200 μm for the others

EB formation and in vitro differentiation

To identify in vitro developmental potential, EB formation in the five sub-cell lines was induced by suspension culture in culture media containing reduced concentrations of bFGF and embryo extract. EBs began to appear on culture day 3 and grew continuously thereafter. On culture day 5, EBs exhibited a typical morphology, consisting of a spherical structure composed of tightly packed cells with a clear outline (Fig. 2a). As shown in Fig. 2b, different EB formation rates were identified among the sub-cell lines (P < 0.0001). Sub-cell line 5 formed the highest number of EBs (13.6 ± 1.6 × 103 EBs), while sub-cell line 3 displayed significantly less EB formation (1.6 ± 0.08 × 103 EBs). The other sub-cell lines formed EBs at similar levels (5.6 ± 0.4–6.6 ± 0.1 × 103 EBs, P > 0.05). The average sizes of the EBs that formed on culture day 5 from each sub-cell line ranged from 48.3 ± 1.7 to 68.7 ± 1.9 μm and were significantly different among the sub-cell lines (Fig. 2c, P < 0.0001). When the EBs derived from each sub-cell line were further induced to differentiate spontaneously after attaching to a culture plate for 2–3 weeks, all EBs spread out with cell growth and differentiation regardless of the sub-cell line (Fig. 3a); furthermore, differentiated cells that were clearly distinguishable from the undifferentiated ones were identified along the outer periphery of growing EBs (Fig. 3b, c). They comprised various cell types that were fully differentiated morphologically: neuronal lineage cells that differentiated into neuron-like cells with dendritic structures (Fig. 3d, e) or star-shaped cells resembling oligodendrocytes (Fig. 3f), unidentified migrating and flattened cells (Fig. 3g, h, respectively), fibroblast-like cells (Fig. 3i), and multinucleated cells (Fig. 3j). In addition, differentiation patterns were distinct depending on the presence or absence of RA in the media. In the absence of RA, EBs tended to differentiate into neuronal lineage cells (Fig. 3b, d–f), but this phenomenon was blocked in the presence of RA, which induced the differentiation of EBs into other lineages (Fig. 3c, h–j).
Fig. 2

Embryoid body (EB) formation. To induce EB formation, cells were cultured in suspension in culture medium containing a reduced concentration of basic fibroblast growth factor (bFGF) and embryo extract. a EBs observed on day 5 of culture. Scale bar 100 μm for a1 and 50 μm for a2. The number (b) and size (c) of EBs formed from each sub-cell line were measured, and mean ± SD values from three independent experiments were compared among the five sub-cell lines. Significant differences in both number and size were detected among the five sub-cell lines

Fig. 3

Spontaneous EB differentiation in vitro. EBs further differentiated spontaneously after attaching to a culture plate (a), and differentiated cells were identified along the outer periphery of growing EBs (b, c). Neuronal lineage cells [e.g., neuron-like cells (d, e) and star-shaped cells (f)], unidentified migrating (g) and flattened cells (h), fibroblast-like cells (i), and multinucleated cells (j) were identified after 2–3 weeks of culture. In c, hj, retinoic acids were also added to the cultures during the induction of differentiation. Arrow heads in d indicate dendritic structures projecting from the cells. Representative images of sub-cell line 1 are depicted in this Figure. Nu nucleus, scale bar 200 μm for a, 100 μm for b, and 50 μm for cj

Chromosomal normality

To evaluate the chromosomal normality of the five sub-cell lines, metaphase chromosomes were prepared and counted in each cell line. As a result, we identified cell populations bearing a normal number of 48 chromosomes in all five sub-cell lines (Fig. 4a). The proportions of cells displaying normal chromosome numbers were 32.5, 20.0, 22.5, 25.0, and 35.0 % in the five sub-cell lines, respectively (Fig. 4b).
Fig. 4

Karyotype analysis. Metaphase chromosomes from each cell line were prepared on glass slides, and the chromosome number was counted after Giemsa staining. a Image of metaphase chromosomes with a normal number (n = 48). Representative images from sub-cell line 1 are shown. Scale bar 5 μm. b Chromosome normality of the five sub-cell lines. Forty metaphase chromosome samples from each sub-cell line were selected randomly, and the chromosome number was counted. The number of samples bearing a normal chromosome number was converted into a percentage


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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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Dongwook Lee
    • 1
  • Jun Hyung Ryu
    • 1
  • Seung Tae Lee
    • 2
  • Yoon Kwon Nam
    • 1
    • 3
  • Dong Soo Kim
    • 1
    • 3
  • Seung Pyo Gong
    • 1
    • 3
  1. 1.Department of Fisheries BiologyPukyong National UniversityBusanKorea
  2. 2.Department of Animal Life ScienceKangwon National UniversityChuncheonKorea
  3. 3.Department of Marine Biomaterials and AquaculturePukyong National UniversityBusanKorea

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