Effects of Feeder Cells on the Primary Culture of Ovarian Cell Populations from Adult Japanese Medaka (Oryzias latipes)

Jun Hyung Ryu,Seung Pyo Gong 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.1

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew 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.

Wip1-expressing Feeder Cells Retain Pluripotency of Co-cultured Mouse Embryonic Stem Cells Under Leukemia Inhibitory Factor- Deprivated Condition

Jin-Ju Kim,이지선,Bo-Hyun Moon,이미옥,Seun-Hyun Song,Henghong Li,Albert J Fornace,차혁진 대한약학회 2010 Archives of Pharmacal Research Vol.33 No.8

The optimization of in vitro culture conditions for embryonic stem cells (ESCs) is a matter of critical importance; a prompt supply of a sufficient population of cells that retain their pluripotency capabilities must be secured in order to make possible future cell therapies. Despite a number of reports asserting that a variety of cytokines, signaling ligands, and small molecules can help in maintaining the pluripotency of ESCs, mammalian feeder cells continue to be broadly accepted as the method of choice for ESC cultures. This appears to be because mammalian feeder cells seem to produce some as-yet-unidentified factor that makes them very effective as feeder cells. In this study, we investigated wild-type p53 inducible phosphatase (Wip1), the knockdown of which increases Wnt inhibitory factor-1 expression, in its feeder functions toward mouse embryonic stem cells, lowering the effect of Wnt, one of key signaling in maintaining stemness of ESCs. For this purpose, Wip1 was stably expressed in mouse embryonic fibroblast cell line (STO) using retro-viral gene delivery system and then the function as a feeder cell was monitored either with or without leukemia inhibitory factor (LIF) in culture medium. We demonstrated that mouse embryonic stem cells grown with Wip1expressing STO showed higher alkaline phosphatase activity and sustained Oct-4 expression level even under LIF deprivation condition compared to both control and Wip1 phosphatase activity dead mutant expressing STO. These results imply that Wip1 phosphatase activity in feeder cells is important to retain pluripotency of mouse embryonic stem cells under LIF deprivation conditions. These results indicate that genetically engineered feeder cells such as Wip1 expressing cell lines, are alternative strategy for the optimization of maintenance and expansion of mouse embryonic stem cells.

배아줄기세포에 의한 영양세포의 지방 분화

이순례 ( Sun Ray Lee ),박현숙 ( Hyun Sook Park ) 한국조직공학·재생의학회 2010 조직공학과 재생의학 Vol.7 No.1

Human embryonic stem(hES) cells have generally been cultured on mouse embryonic fibroblast feeder cells. Paracrine factors from feeder cells have drawn much attention in their relation to the maintenance of selfrenewal and pluripotency of hES cells. There has however been no attention on factors from hES cells and their effects on feeder cells. In the present study, we examined the effect of hES cells on feeder cells, mouse embryonic fibroblasts, human mesenchymal stem cells and human skin fibroblasts, in indirect co-culture system using porous membrane. We observed lipid droplet formation in feeder cells as early as 2 days after co-culture and lipid vacuole matured with time in the influence of hESCs. Only with hES-conditioned medium adipogeneisis of feeder cells occurred but co-culture with hES cells dramatically increased adipogenesis of feeder cells. This implied adipogenesis of feeder cells occurred via paracrine communication between hES cells and feeder cells. Further investigation on how adipogenesis of feeder cells affects the niche of human embryonic stem cells is required.

A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells

최나영,박요셉,유재성,이혜정,Marcos J. Araúzo-Bravo,고기성,한동욱,Hans R. Schöler,고기남 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.6

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells

Choi, Na Young,Park, Yo Seph,Ryu, Jae-Sung,Lee, Hye Jeong,Arauzo-Bravo, Marcos J.,Ko, Kisung,Han, Dong Wook,Scholer, Hans R.,Ko, Kinarm Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.6

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost-effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

Effects of Feeder Cells on the Primary Culture of Ovarian Cell Populations from Adult Japanese Medaka (Oryzias latipes)

Ryu, Jun Hyung,Gong, Seung Pyo The Korean Society of Animal Reproduction and Biot 2020 한국동물생명공학회지 Vol.35 No.1

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew 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.

The Investigation of Cell Culture Conditions to Maintain Chicken Embryonic Stem Cells as Totipotent Cells

Du, Lixin,An, Jing Asian Australasian Association of Animal Productio 2003 Animal Bioscience Vol.16 No.8

The ES cell can provide a useful system for studying differentiation and development in vitro and a powerful tool for producing transgenic animalds. To investigate the culture condition of chicken embryonic stem (CES) cells which can retain their multipotentiality or totipotency, three kinds of feeder layer cells, SNL cells, primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells, were used as the feeder cells in media of DMEM supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and stem cell factor (SCF) for co-culture with blastoderm cells from stage X embryos of chicken. The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The results showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The typical CES cells clone shape revealed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong alkaline phosphatase (AKP) reactive cells were observed in the fourth passage cells. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2% (22/269) survived to hatching, 5 feather chimeras had been produced. This suggests that an effective culture system established in this study can promote the growth of CES cells and maintain them in the state of undifferentiated and development, which lays a solid foundation for the application of CES cells and may provide an alternative tool for genetic modification of chickens.

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

Bae, Duk Seong,Lee, Jae Kwon Korean Society of Hematology; Korean Society of Bl 2014 Blood Research Vol.49 No.3

<P><B>Background</B></P><P>Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, <I>ex vivo</I> NK cell expansion is one of the important steps for developing NK cell therapeutics.</P><P><B>Methods</B></P><P>CD3<SUP>+</SUP> depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines.</P><P><B>Results</B></P><P>We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naïve NK cells.</P><P><B>Conclusion</B></P><P>Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.</P>

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

Duk Seong Bae,이재권 대한혈액학회 2014 Blood Research Vol.49 No.3

Background Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics. Methods CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines. Results We compared feeder activities of three different cells−PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naïve NK cells. Conclusion Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.

Effect of Tissue Specific Feeder Layer as Feeder Cells on the Proliferation of Murine Spermatogonial Stem Cell In Vitro

Myeong-Geun Oh,Yong-Hee Kim,Polash Chandra Karmakar,Sang-Eun Jung,Seok-Man Kim,Ju-Hee Jin,Buom-Yong Ryu 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male. Among the testicular germ cells, SSCs are considered as an important population because of their capability to multiply in numbers which is also known as self-renewal, and to differentiate into spermatocytes by subsequent meiotic processes finalized with the production of spermatozoa. As SSCs are present in a very small proportion among the total testis cells, in vitro proliferation of SSCs in undifferentiated state is very important in the field of experiments related to germ cell culture. The objective of the study was to establish an effective feeder cell for murine germ cell development in vitro. We used three types of feeder cells to grow murine germ cells, such as STO, tissue specific feeder layer, and C166 feeder cells and green fluorescence protein (GFP) rat SSCs were used to grow on them. A continuous culture was performed for each feeder groups, but C166 feeder cells found unable to proliferate germ cells even after several weeks. We observed a comparatively better proliferation rate of rat germ cells grown on tissue specific feeder layer where cultured germ cell colony and cell count were higher than STO feeder. Immunocytochemistry of undifferentiated SSC marker showed significantly higher expression for germ cell cultured on tissue specific feeder layer. Similarly, mRNA level of undifferentiated spermatogonia were found higher in case of tissue specific feeder layer and STO, although the expression level of differentiated spermatogonia was similar. Finally, the functional characteristics of SSCs were studied by cultured germ cell transplantation into recipient mice. We used donor rat germ cells cultured on both feeder groups for 4 months, where SSCs from tissue specific feeder layer showed higher functional and stemness activity. Transplantation of SSCs cultured for 8 months from tissue specific feeder layer group were performed to recipient rat and we could produce GFP offspring from those recipients. Thus the experiment conclude that tissue specific cells could be an effective feeder layer for murine SSCs culture based on their survivability and proliferation along with retaining the perfect functional characteristics and stemness properties.