Isolation, cultivation, and differentiation of neural stem cells from adult fish brain

In contrast to mammals, teleost fish are distinct in their ability to continuously produce a tremendous number of new neurons in many regions of the adult brain. In the present study, we have isolated intrinsic stem cells from the telencephalon, corpus cerebelli, and valvula cerebelli of the teleost Apteronotus leptorhynchus and examined their properties in vitro. After 3–4 days in culture, neurospheres developed that grew through cell proliferation and reached diameters of up to 140 μm within 3 weeks. An increase in the number of developing neurospheres could be promoted by addition of epidermal growth factor or basic fibroblast growth factor, but no additive effect was observed after combined treatment. The number of neurospheres could furthermore be enhanced by seeding brain cells at densities of approximately 1 × 106. Differentiation conditions were optimal by exposing neurospheres to 10% fetal bovine serum and laminin as coating substrate. Neurosphere cells gave rise to both neurons, immunopositive for Hu-C/D or MAP2 (2a + 2b), and glial cells, immunopositive for glial fibrillary acidic protein or vimentin. Since, in addition to their multipotency, the cells isolated from the adult teleostean brain exhibited the ability for self-renewal, we hypothesize that they are true stem cells.