Development of cell culture systems for use in fish embryonic stem cell technology

Advances in embryonic stem (ES) cell research over the past twenty years have raised great interest and hope in this technology as a means for deciphering the human genome and offering cures for human diseases. While the vast majority of research has been done with murine ES cells, efforts to develop similar in vitro cultures in other animals have only yielded limited results. Thus far, the creation of specific genetic mutations through cell-mediated gene-targeting is a powerful method still applicable only to mice. Zebrafish, a small aquarium species, offers several advantages over murine models, including low economic cost, simplicity in maintenance, and most importantly, a period of early development that is much easier to monitor. ES cell technology and the ability to generate targeted knockouts would be an ideal complement to the current research toolset for zebrafish. Zebrafish ES (ZES) cells have been derived and successfully cultivated in vitro using a complex array of supplements as well as a rainbow trout spleen feeder layer. To aid in the development of a method to generate knockout zebrafish, two new zebrafish cell lines have been derived and characterized from the embryo, ZEB2J, and from the spleen, ZSSJ.; ZEB2J was initiated from blastula stage embryos of zebrafish expressing green fluorescent protein, maintained for several passages on monolayers of the rainbow trout spleen stromal cell line, RT34st, and then grown continuously at a high cell density in the absence of feeders in a simple medium, Leibovitz's L-15 supplemented with fetal bovine serum (FBS). During the switch to feeder-free conditions and subsequent subcultivations, the cells retained an epithelial-like shape. Morphological changes in a minority of the cells that would indicate spontaneous differentiation were not observed. ZEB2J grew on conventional tissue culture plastic to form monolayers in which small aggregates of cells would appear and increase in size with time. These aggregates became less obvious after approximately 50 subcultivations. The best growth of ZEB2J cultures occurred with 15 or 20% FBS, and little or no growth occurred in L-15 alone. ZEB2J grew well at 22 to 32°C. Cell number increased slowly in cultures at 18°C and remained unchanged in cultures at 4°C. At 26°C and in 15% FBS, ZEB2J has undergone more than 120 population doublings. Also, no cells stained for beta-galactosidase, which can be a marker of senescent cells, suggesting that ZEB2J is immortal. However, most ZEB2J cells were aneuploid in karyotype. Despite this, ZEB2J expressed several stem cell markers. Most cells stained for alkaline phosphatase (AP) activity, some expressed SSEA-1, and Pou-2 mRNA was detected in cultures by RT-PCR. Both ZEB2J and RTS34st formed spheroids in culture vessels coated with the phospholipid polymer, 2-methacryloxyloxyethyl phosphorylchloline (MPC). ZEB2J and RTS34st also formed mixed or heterotypic spheroids, which could be identified as aggregates of fluorescent and non-fluorescent cells. Alone in conventional tissue culture vessels, ZEB2J grew poorly. When in coculture together with several cell lines, including RTS34st, ZEB2J formed many colonies that were fluorescent and easily scored. Thus, one of many possible uses for ZEB2J is to compare the effectiveness of different cell lines as feeders for ZES cell cultures.; ZSSJ was sought as a potentially superior feeder cell line to RTS34st and was initiated from zebrafish spleens by explant outgrowth. The cells were predominantly epithelial-like and grew best at 26 to 28°C in Leibovitz's L-15 with 15°/o FBS. ZSSJ has undergone around 20 population doublings to date and few ZSSJ stained for senescence-associated beta-galactosidase activity, which suggests that the cell line is immortal. Most cells in ZSSJ cultures stain well for AP activity, as did some RTS34st. As feeder cells for ES cell cultures are often growth-arrested, the dose of gamma-irradiation necessary...