Mangrove-Crab Blood Cells Proliferate In Vitro and Display Neuronal Proteins Following Pituitary-Extract Stimulus

In this study we propose a cell-culture protocol to better comprehend the involvement of immune/blood cells (hemocytes) in the adult neurogenesis of crustaceans. We examined whether the hemocytes of the crab Ucides cordatus may develop into neural cells in response to an in vitro stimulus. The experiments involved two steps. First, we selected an appropriate substrate for use in the culture medium. Hemocytes proliferated and differentiated most on poly-d-lysine. Second, we added pituitary extract to the poly-d-lysine-coated culture in order to determine the cell types into which hemocytes differentiated. This supplement was chosen due to its mitogenic and cell-differentiation properties. Using cell type-specific antibodies (anti-GFAP, anti-vimentin, anti-beta III Tubulin, and anti-NeuN), we were able to identify putative neural progenitors. This showed that upon stimulation, hemocytes have mitotic activity and can display neural precursor proteins. In contrast to the protocols commonly used for vertebrate cell cultures, the protocol used here proved, for the first time, to be capable of stimulating crustacean blood cells to grow. This method also offers a basis for cultivating crustacean blood cells for various purposes, such as the study of adult neurogenesis.