Effects of Extracellular Signaling on the Endogenous Expression of Self-Renewal-Stimulating Factor Genes in Mouse Embryonic Stem Cells

공승표,이승태 사단법인 한국동물생명공학회 2012 Reproductive & developmental biology Vol.36 No.1

In order to provide the basis for developing practical mouse embryonic stem cells (mESCs) culture method, how the endogenous level of self-renewal-stimulating factor genes was altered in the mESCs by different extracellular signaling was investigated in this study. For different extracellular signaling, mESCs were cultured in 2 dimension (D),3D and integrin-stimulating 3D culture system in the presence or absence of leukemia inhibitory factor (LIF) and transcriptional level of Lif, Bmp4 and Wnt3a was evaluated in the mESCs cultured in each system. The expression of three genes was significantly increased in 3D system relative to 2D system under LIF-containing condition, while only Wnt3a expression was increased by 3D culture under LIF-free condition. Stimulation of integrin signaling in mESCs within 3D system with exogenous LIF significantly up-regulated transcriptional level of Bmp4, but did not induce transcriptional regulation of Lif and Wnt3a. In the absence of LIF inside 3D system, the expression of Lif and Bmp4was significantly increased by integrin signaling, while it significantly decreased Wnt3a expression. Finally, the signal from exogenous LIF significantly caused increased expression of Lif in 2D system, decreased expression of Bmp4 in both 2D and 3D system, and decreased expression of Wnt3a in integrin-stimulating 3D system. From these results,we identified that endogenous expression level of self-renewal-stimulating factor genes in mESCs could be effectively regulated through artificial and proper manipulation of extracellular signaling. Moreover, synthetic 3D niche stimulating endogenous secretion of self-renewal-stimulating factors will be able to help develop growth factor-free maintenance system of mESCs

Establishment and Characterization of Permanent Cell Lines from Oryzias dancena Embryos

이동욱,공승표,김민성,남윤권,김동수 한국수산과학회 2013 Fisheries and Aquatic Sciences Vol.16 No.3

The development of species-specific fish cell lines has become a valuable tool for biological research. In recent years, marine medaka Oryzias dancena has been recognized as a good experimental model fish but there are no reports of establishment of cell lines from this fish. In this study, two cell lines from O. dancena blastula embryos were established from 41 total trials (4.9%). The two cell lines displayed typical in vitro morphology and have been cultured for >121 passages, which corresponds to 293 days. The doubling times of the cell lines were 29.84 and 28.59 h, respectively, and both possessed the potential to expand in a clonal manner, albeit with significant differences between the two cell lines. The absence of any of the four main medium supplements; i.e., fish serum, fetal bovine serum, basic fibroblast growth factor, and medaka embryo extract, significantly inhibited growth. The proportion of cells possessing normal chromosome number was 45% and 46.7% of the cell lines, respectively. Taken together, two cell lines that proliferate continuously were established from marine medaka and these cell lines may provide a basic tool for characterizing the unique features of this fish species.

Ovarian cell aggregate culture in teleost, marine medaka (Oryzias dancena): basic culture conditions and characterization

최재훈,공승표 사단법인 한국동물생명공학회 2024 Journal of Animal Reproduction and Biotechnology Vol.39 No.1

Background: Although an understanding of the proliferation and differentiation of fish female germline stem cells (GSCs) is very important, an appropriate threedimensional (3D) research model to study them is not well established. As a part of the development of stable 3D culture system for fish female GSCs, we conducted this study to establish a 3D aggregate culture system of ovarian cells in marine medaka, Oryzias dancena. Methods: Ovarian cells were separated by Percoll density gradient centrifugation and two different cell populations were cultured in suspension to form ovarian cell aggregates to find suitable cell populations for its formation. Ovarian cell aggregates formed from different cell populations were evaluated by histology and gene expression analyses. To evaluate the media supplements, ovarian cell aggregate culture was performed under different media conditions, and the morphology, viability, size, gene expression, histology, and E2 secretion of ovarian cell aggregates were analyzed. Results: Ovarian cell aggregates were able to be formed well under specific culture conditions that used ultra-low attachment 96 well plate, complete mESM2, and the cell populations from top to 50% layers after separation of ovarian cells. Moreover, they were able to maintain minimal ovarian function such as germ cell maintenance and E2 synthesis for a short period. Conclusions: We established basic conditions for the culture of O. dancena ovarian cell aggregates. Additional efforts will be required to further optimize the culture conditions so that the ovarian cell aggregates can retain the improved ovarian functions for a longer period of time.

Suspension Culture-Mediated Tetraploid Formation in Mouse Embryonic Stem Cells

이재희,공승표,임정묵,이승태 사단법인 한국동물생명공학회 2012 Reproductive & developmental biology Vol.36 No.1

Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained in vitro for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.

Effects of Temperatures and Basal Media on Primary Culture of the Blastomeres Derived from the Embryos at Blastula Stage in Marine Medaka Oryzias Dancena

최재훈,공승표 사단법인 한국동물생명공학회 2018 한국동물생명공학회지 Vol.33 No.4

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including 28°C, 31°C, 34°C, and 37°C and 2 basal media including Dulbecco’s modified eagle’s medium (DMEM) and Leibovitz’s L-15 medium (L15). With the exception of a case cultured in L15 at 31°C, the rate of primary cell adherence reached 100% when the blastomeres were cultured over 31°C. The period for primary adherence was significantly shorter in the groups cultured in 34°C and 37°C than in the ones in 28°C and 31°C. The proportion of subculture was significantly high in the group cultured in DMEM at 31°C compared to the other groups. Collectively, we demonstrated that the culture in DMEM at 31°C was effective to primary culture of the blastomeres derived from blastula embryos.

Cloning and characterization of a cDNA encoding a paired box protein, PAX7, from black sea bream, Acanthopagrus schlegelii

최재훈,한단희,공승표 사단법인 한국동물생명공학회 2021 한국동물생명공학회지 Vol.36 No.4

Paired box protein, PAX7, is a key molecule for the specification, maintenance and skeletal muscle regeneration of muscle satellite cells. In this study, we identified and characterized the cDNA and amino acid sequences of PAX7 from black sea bream (Acanthopagrus schlegelii ) via molecular cloning and sequence analysis. A. schlegelii PAX7 cDNA was comprised of 1,524 bp encoding 507 amino acids and multiple sequence alignment analysis of the translated amino acids showed that it contained three domains including paired DNA-binding domain, homeobox domain and OAR domain which were well conserved across various animal species investigated. Pairwise Sequence Alignment indicated that A. schlegelii PAX7 had the same amino acid sequences with that of yellowfin seabream (A. latus ) and 99.8% identity and similarity with that of gilt-head bream (Sparus aurata ). Molecular phylogenetic analysis confirmed that A. schlegelii PAX7 formed a monophyletic group with those of teleost and most closely related with those of the fish that belong to Sparidae family including A. latus and S. aurata . In the investigation of its tissue specific mRNA expression, the expression was specifically identified in skeletal muscle tissue and a weak expression was also shown in gonad tissue. The cultured cells derived from skeletal muscle tissues expressed PAX7 mRNA at early passage but the expression was not observed after several times of subculture.

Establishment and long-term culture of the cell lines derived from gonad tissues of Siberian sturgeon (Acipenser baerii)

류준형,남윤권,공승표 한국수산과학회 2016 Fisheries and Aquatic Sciences Vol.19 No.2

To culture germline stem cells in vitro, establishment of the cell lines that can be used as the feeder cells is a prerequisite. In this study, we tried to establish gonad-derived cell lines in Siberian sturgeon (Acipenser baerii). Five 1-year-old A. baerii were used as a donor of gonad tissues, and gonad-dissociated cells were cultured in vitro. Subsequently, determination of growth conditions, long-term culture, characterization, and cryopreservation of the cell lines were also conducted. Five gonad-derived cell lines were stably established and cultured continuously over at least the 73th passage and 402 culture days under the media containing 20 % fetal bovine serum at 28 °C. All cell lines consisted of two main cell types based on morphology even if the ratio of the two cell types was different depending on cell lines. Despite long-term culture, all cell lines maintained diploid DNA contents and expression of several genes that are known to express in the A. baerii gonad. After freezing and thawing of the cell lines, post-thaw cell viabilities between 57.6 and 92.9 % depending on cell lines were indentified, suggesting that stable cryopreservation is possible. The results and the cell lines established in this study will contribute to the development of an in vitro system for A. baerii germline stem cell culture.

Factors Affecting Primary Cultures of Haliotis discus hannai Ovary-dissociated Cells and General Culture Aspects

류준형,남윤권,공승표 한국수산과학회 2015 Fisheries and Aquatic Sciences Vol.18 No.1

We investigated factors affecting primary cultures of Pacific abalone Haliotis discus hannai ovary-dissociated cells to identify general aspects of their early-phase culture. Ninety-seven cell populations derived from 30 individuals were cultured in different media with varying compositions of medium supplements, and initial attachment, subculture, and survival for ≥10 weeks were assessed according to medium composition and individual. We also examined the time required for subculture and the rate of cell death according to both culturing period and passage number within 10 weeks. A lack of fetal bovine serum (FBS) and hemolymph significantly inhibited the growth of cultured cells, while we detected no significant effect of medium composition on initial cell attachment. Through data reallocation, with the omission of data from cell populations cultured in FBS-free and hemolymph-free media, we showed that growth inhibition was also affected by individual differences among the abalones used. During the culture, we observed four different types of cell morphology. Moreover, considerable time was required for subculture—18.4 and 19.5 days for first and second subcultures, respectively—and cell death did not occur within 30 days or for passage 0. Our results will provide valuable information for developing universal cell culturing guidelines in abalone species and suggest the feasibility of culturing abalone ovary-dissociated cells.

In Vitro Culture of Primary Testicular Stromal Cells derived from Mouse with Different Genetic Background : Optimization of Culture Temperature

박혜진,윤정임,최정훈,이은송,공승표,이승태 사단법인 한국동물생명공학회 2013 한국동물생명공학회지 Vol.28 No.4

Spermatogonial stem cells (SSCs) developed into sperms through spermatogenesis have been utilized as a usefultool in the field of regenerative medicine and infertility. However, a small number of highly qualified SSCs are residedin the seminiferous tubule of testis, resulted in developing effective in-vitro culture system of SSCs for solving simultaneouslyquantitative and qualitative problems. Presently, SSCs can be enriched on testicular stromal cells (TSCs),but there are no systematic researches about TSC culture. Therefore, we tried to optimize culture condition of TSCsderived from mouse with different strains. For these, proliferation and viability were measured and compared byculturing ICR outbred or DBA/2 inbred mouse-derived TSCs at 35 or 37℃. In case of ICR strain, primary TSCscultured at 37℃ showed significantly higher proliferation and viability than those at 35℃ and significant increase ofproliferation and viability in sub-passaged TSCs was detected in the 35℃ culture condition. Moreover, sub-passageof primary TSCs at 35℃ induced no significant effects on proliferation and viability. In contrast, in case of DBA/2strain, significantly improved proliferation were detected in the primary TSCs cultured at 35℃, which showed nosignificant difference in the viability, compared to those at 37℃. Furthermore, sub-passaged TSCs cultured at 37℃showed no significant differences in proliferation and viability, compared to those at 35℃. However, with significantdecrease of proliferation induced by sub-passage of primary TSCs at 35℃, no significant effects on proliferation andviability were resulted from sub-passage of primary TSCs at 37℃. From these results, culture temperature of primaryTSCs derived from outbred and inbred strain of mouse could be separately optimized in primary culture and subculture

Initial Culture Conditions for Primary Cell Populations Derived from Radula Tissue in Abalone Haliotis discus hannai

김민성,남윤권,김동수,공승표 한국수산과학회 2014 Fisheries and Aquatic Sciences Vol.17 No.3

Abalone immortal cell lines can be used to study the physiological properties and disease mechanisms of abalone at the cellular and molecular level. As a first step for the final goal to establish abalone immortal cell lines, we examined various initial culture conditions for primary cell populations derived from Haliotis discus hannai radula tissue. The survival rate after cell isolation procedures using the enzymatic method was as low as 9.95 ± 2.37%. Based on three different experimental conditions for H. discus hannai radula-derived cell culture, we found that the salinity of the media and the presence of growth-promoting factors were important to support radula-derived primary cell populations during the initial culture. The growth factor-containing media adjusted to 35 psu salinity could induce 100% (8 out of 8 trials) initial cell attachment, and the rate of cell attachment reached 50–70%. The data obtained from this study will provide useful information for developing immortal cell lines from abalone species.