Effects of Different Feeders on Cultured Chicken Embryonic Stem Cells
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Objective:To research the optimal culture condition of chicken embryonic stem cells,compare the effect of different feeders on cultured chicken embryonic stem cells in vitro.Methods:The second generation chicken embryonic fibroblast(CEF) and duck embryonic fibroblast(DEF) were used to make feeders after treated with mitomycin C.The state of chicken embryonic stem cells were observed and the number of cell clones was counted on the culture system with those two feeder layers or feeder layer free.Results:The results showed that chicken embryonic stem cells growth well both on the CEF and DEF feeder,and the number of cell clones was no distinct difference between CEF and DEF(P0.05).Conclusion:The results indicated that both of CEF and DEF can be used as feeders to culture chicken embryonic stem cells.Keywords:
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Objective: To investigate the isolation, purification and culture methods of chicken embryonic germ cells(EGCs) on feeder layer cells of mice embryonic fibroblasts(MEFs). Methods: Mice embryonic fibroblasts from the mice embryos of 12.5～13.5 days were cultured for over 18 passages(4 months). Numerous primordial germ cells colonies were derived from gonadal ridges of 5.5-day-old chicken embryos. At primary culture, chicken primordial germ cells(PGCs) were co-cultured with the gonadal stromal cells. For subculture the PGCs were plated with mitotically inactive feeder layer cells of mice embryonic fibroblasts. The medium composed of growth factors and differentiation inhibitory factors. Results: The cultures had some characteristics of embryonic germ cells, such as the ability to be continuously passed(passage 15) and the morphology of periodic acid-Shiff reaction(PAS) positive. The cultures would differentiated into several kinds of cells under the condition of devoid of LIF and feeder layer cells, such as fibroblast cells, neurocytes, autorhythmic cells, etc. When cultured in suspention, they successfully formed embryoid bodies. All of which indicate that the cells obtained were EGCs. Conclusion: Continuously proliferating EGCs can be obtained on feeder layer cells of MEFs.
Embryoid body
Subculture (biology)
Germ layer
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Gonadal ridge
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Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast feeders were seen on the mass-like colonies and round or oval images, and more significant growth in the total number of colonies ( p <0.05) and viable cells in the colonies ( p <0.01) than that from goat embryonic fibroblast feeders, and viable cells in the colonies ( p <0.05) than that from porcine embryonic fibroblast feeders. The feeders from bovine embryonic fibroblasts also reduced viable cells in the colonies, but were not significantly different in the total number of colonies and viable cells in the colonies with mouse embryonic fibroblast feeders. mESCs on the different embryonic fibroblast feeders were expressed as stem cell-specific markers Oct 4 and stage-specific embryonic antigen 1 (SSEA 1). Here, our results indicate that the feeders from goat, porcine and bovine embryonic fibroblasts inhibit the proliferation of mESCs. Key words : Domestic animals, feeders, mouse embryonic stem cells (mESCs), growth.
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Objective:To establish feeder layer for mouse embryonic stem cell. Methods:Isolated and culturied mouse embryonic stem cell using 5 different kinds of mouse embryonic fibroblasts as feeder layers,and the effects of different feeder layers on the development of mouse blastocyst,the proliferation of inner cell mass and the culture of mouse embryonic stem cell were observed. Results:The feeder layer of the primary or the thawed primary mouse embryonic fibroblast improved the hatching of blastocyst and the proliferation of inner cell mass into nest like clone. After being disaggregated,ES clones appeared. Moreover it maintained the stem cell like morphology in a short period which was evidently different with other 3 groups. Conclusion:The feeder layer of the primary or the thawed primary mouse embryonic fibroblast is a effective culture system of the isolation and culture of mouse embryonic stem cell.
Inner cell mass
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Embryoid body
clone (Java method)
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Objective: To investigate the culture and biological characteristics of mouse embryonic stem(ES) cell as a foundational work for its application.Methods: Mouse ES cells were observed in two culture systems: 1) on feeder cells of the primary mouse embryo fibroblasts and 2) in leukemia with inhibititory factor but without feeder cells.Results: The primary mouse embryo fibroblast growth trended radially or circinately.The ES cell clones under the two conditions appeared typical characteristics and maintained un-differentiation state.Conclusion: The ES cells can grow well under those conditions.
KOSR
Primary cell
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Objective To establish a stable and effective system of mouse embryonic fibroblasts(MEFs) feeder layer in order to culture mouse embryonic stem cells(ESC)in vitro.Methods Embryonic fibroblast cells for primary culture were derived from mouse embryos(pregnant 13-15 days) through trypsin digestion.Results After being treated with mitomycin C(10μg/mL),MEFs proliferation could be efficiently repressed and be made into the feeder layer for ESC Undifferentiated growth.Conclusion MEFs feeder layer could effectively support mouse embryonic stem cells to grow.
Mitomycin C
Primary culture
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To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.
Homeobox protein NANOG
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Embryoid body
Rex1
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Two methods were used to prepare the mouse embryonic fibroblast(MEF) feeder layers,and the MEF feeder layers were used to isolate and culture the embryonic stem cells.It was carried out observation to the effect of the various methods on the MEF feeder layers,the effects of the various MEF feeder layers on the development of embryos,the growth of the ICM and the isolation of the embryonic stem(ES)cells,and the optimal passage of the MEF feeder layers were filtered out for preparation of the feeder layers in this experiment.It was find in this experiment that the method for preparation of the MEF feeder layers could simplify the preparation for the MEF feeder layers,but there had no impacts on the isolation the ES cells.
Isolation
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This study was conducted to evaluate whether culture of chicken fibroblasts could induce cell aggregation leading to embryonic stem cell (ESC)-like, colony-formation. Gradual increase in expression of leukemia inhibitory factor (LIF) gene were detected as retrieval age of chicken embryonic fibroblasts (CEFs) was increased from 5- to 8-day-old, which accompanied increase in cell proliferation after culture in LIF-free medium. Colony-like cell aggregation was detected after culture of the 8-day-old fibroblasts, while the exposure to 0.5, 1 or 2 mM glutathione did not increase the cell aggregation. The cell aggregates were positive for periodic acid Schiff solution, and the antibodies of anti-stage specific embryonic antigen (SSEA)-1 and anti-SSEA-3. However, they were negative for alkaline phosphatase and anti- SSEA-4 antibody. Expression of chicken Vasa homolog did not detect in the aggregated CEFs. Continuous culture of the aggregates in LIF- and CEF monolayer-free condition resulted the formation of embryoid bodies possessing the cells being positive for three germ layer makers (Nestin, S-100, smooth muscle actin, desmin, alpha-fetoprotein and Troma-1). In conclusion, our results demonstrate cell aggregates having ESC-like activity can be derived from the culture of chicken fibroblasts on CEF monolayer, which strongly supports the possibility on deriving chicken stem cells from non-germline tissue.
Embryoid body
Stem cell marker
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