Possibility to Establish Chicken Stem Cell from Non-germline Tissue; Detection of Colony-forming Cells after Chicken Fibroblast Culture and Subsequent Stem Cell Characterization

Abstract

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.