Establishment of long term cultures of neural stem cells from adult sea bass, Dicentrarchus labrax

Abstract

Long term cell cultures could be obtained from brains of adult sea bass (Dicentrarchus labrax) up to 5 days post mortem. On three different occasions, sea bass brain tissues were dissected, dispersed and cultured in Leibovitz's L-15 media supplemented with 10% fetal bovine serum. The resulting cellular preparations could be passaged within 2 or 3 weeks of growth. The neural cells derived from the first trial (SBB-W1) have now been passaged over 24 times within two years. These cells have been cryopreserved and thawed successfully. SBB-W1 cells are slow growing with doubling times requiring at least 7 days at 22 °C. These long term cell cultures could be grown in suspension as neurospheres that were immunopositive for nestin, a marker for neural stem cells, or grown as adherent monolayers displaying both glial and neural morphologies. Immunostaining with anti-glial fibrillary acidic protein (a glial marker) and anti-neurofilament (a neuronal marker), yielded positive staining in most cells, suggesting their possible identity as neural stem cells. Furthermore, Sox 2, a marker for neural stem cells, could be detected from these cell extracts as well as proliferating cell nuclear antigen, a marker for proliferating cells. SBB-W1 could be transfected using pEGFP-N1 indicating their viability and suitability as convenient models for neurophysiological or neurotoxicological studies.

Introduction

The capacity for self-renewal and the ability to reconstitute a tissue are the classical definitions of a stem cell, and it appears that stem cells are present in most fish tissues, including the brain (Hinsch and Zupanc, 2006, Chapouton et al., 2007, Kaslin et al., 2008). This may explain why fish show “indeterminate growth”, or the ability to grow unrestricted given optimal conditions as opposed to the restrictive growth shown by mammals. As fish grow, new cells are generated and this is paralleled throughout all organs including neural tissues. In contrast to mammals, adult fish have the capacity to generate significant amounts of nervous tissue following injury (Nona, 1995, Caminos et al., 1999, Zupanc, 2006, Chapouton et al., 2007, Udvadia, 2008). This replacement is associated with a high degree of functional recovery (Larner et al., 1995), which is attributed to several factors in fish, including the persistence of proliferative zones containing neural stem cells (Zupanc and Clint, 2003, Grandel et al., 2006, Hinsch and Zupanc, 2006). Recently, using a combination of bromodeoxyuridine (BrdU) treatment and immunohistochemical techniques, Pellegrini et al. (2007) demonstrated that radial glial cells have mitotic activity and represent progenitors or precursor cells in adult zebrafish brain. These radial glial cells can divide to generate newborn cells in many brain regions and such newborn cells can further divide, migrate and differentiate into neurons as shown by BrdU, proliferating cell nuclear antigen, Hu, and acetylated tubulin immunostaining (Pellegrini et al., 2007). This is in contrast to that observed in lampreys and amphibians, in which most of the new neurons that are added to the central nervous system appear to be derived from late differentiation of immature neurons (Meeker and Farel, 1997, Farel, 2001, Vidal Pizarro et al., 2004). The persistence of proliferating cells in the central nervous system of fishes may account for the ready establishment of brain derived cell lines from fish including TB2, a neural progenitor cell line from tilapia brain (Wen et al., 2008a), GB cell line, derived from grouper brain (Lai et al., 2003), BB cell line, established from the brain tissue of barramundi (Chi et al., 2005), as well as many short and long term neural cell cultures (De Boni et al., 1976, Anderson and Waxman, 1985, Anderson, 1993, Hinsch and Zupanc, 2006). It is surprising, however, that so few adult fish brain cell lines exist, as neurogenesis continues at a high rate throughout the adult life in many different areas of the fish CNS (Hinsch and Zupanc, 2006).

Understanding of physiological processes in fish species, especially for those with increasing commercial value in aquaculture such as the sea bass (D. labrax) has prompted both applied and basic studies involving fish neural tissues (Cerdá-Reverter et al., 2000, González-Martínez et al., 2002, González-Martínez et al., 2004, Bayarri et al., 2004, Moles et al., 2007), and the need for available cultured neural cells of fish origin. The emergence of neural diseases caused by viruses within the intensive aquaculture practices (Chi et al., 2005, Parameswaran et al., 2006b, Cutrín et al., 2007) also highlights the need for cell cultures from brain tissues of fish as hosts for tissue specific viruses. In this study, we report on the in vitro culture of sea bass brain cells derived from various brain regions including telencephalic, diencephalic, mesencephalic, metencephalic and myelencephalic areas as well as from the pituitary and pineal glands. Proliferative cells were obtained from each zone but most consistently from the mesencephalon and metencephalon, and a cell line, SBB-W1, was developed from adult sea bass cerebellar–tegmental areas. These cells were large and irregular in shape with highly euchromatic nuclei and numerous cytoplasmic processes. Immunocytochemical characterization points to a population of neural stem cell progenitors. The cells' transfection capability demonstrate potential for their use to investigate differential responses to toxicants, nutrients and growth factors, and could serve as models for neural regeneration studies as well as for elucidating mechanisms of neurotoxicity, neurophysiology and neuroendocrinology.