바다송사리(Oryzias dancena) 단일 포배 유래 배아세포주 확립과 특성분석

Abstract

Embryonic stem cells (ESCs) are derived from early developing embryos containing pluripotent cells such as the inner cell mass of mammalian blastocysts or blastomeres from fish blastula embryos. They have the ability to differentiate into almost all adult cell types and self-renew. Accordingly many studies have been conducted on the in vitro culture and application of ESCs in various animals, including fish. Fish ESCs are ideal experimental tools for the generation of desired animal models not only for many research areas such as basic studies in molecular, cellular, and developmental biology but also for commercial interest. But, most of fish ESCs developed so far are ESC-like cell lines and there is a lack of definite ESCs. In current, the culture conditions used universally for fish ESCs derivation were largely derived from the study of ESCs derivation from Japanese medaka (Oryzias latipes). However, it is difficult to apply those conditions to all fish species and thus more suitable culture conditions customized to each fish species need to be developed. So far, the blastomeres for primary culture of fish ESCs derivation have been derived from about 10 blastula stage embryos in most cases. This culture method gives intercellular genetic heterogeneity to the cultured cells until clone cell lines will be established, which can be a major obstacle for developing stable ESCs culture conditions. Therefore, in order to establish ESC lines that have genetic homogeneity in the early stage of culture, I evaluated the feasibility of ESCs derivation from a single blastula-derived blastomeres of marine medaka (Oryzias dancena) by using feeder cells. Then, the established cell lines were characterized in terms of morphology, growth rate, alkaline phosphatase activity, differentiation potential and pluripotency gene expression. As a result, I established 22 embryonic cell lines using single blastula of marine medaka confirming that use of basic fibroblast growth factor-expressing feeder cells during primary culture significantly enhance the efficiency of cell line derivation. Through the characterization assay of the established cell lines, it was confirmed that they were ESC-like cells. The results from this study will provide basic data for the study of fish ESCs.