An efficient method for the culturing and generation of neurons and astrocytes from second trimester human central nervous system tissue

The isolation, culturing and expansion of human neural progenitors cells ha s important potential clinical applications in cellular transplantation str ategies as well as in developmental studies involving the central nervous s ystem (CNS). This study describes an efficient method to culture neurons an d astrocytes as primary cultures, as well as from proliferative progenitor cells derived from second trimester fetal CNS tissue. Second trimester feta l human tissue was mechanically dissociated and subjected to trypsin-dissoc iation and trituration. The resulting suspension was passed over a Percoll density gradient The middle (second) fraction of cells was centrifuged to y ield a homogenous population of cells with 80%-90% viability. These cells w ere either cultured directly on laminin coated dishes with defined medium s upplemented with fetal bovine serum or in defined medium supplemented with growth factors including epidermal growth factor, basic fibroblast growth f actor and leukemia inhibitory factor. The primary cell cultures yielded neu rons and astrocytes after 3-5 days in vitro verified by immunostaining with MAP2ab and GFAP. Cells exposed to growth factor supplemented medium formed free-floating spheres within one week. Upon growth factor removal and plat ing on laminin-coated dishes, brain derived spheres gave rise to neurons, a strocytes and oligodendrocytes; spinal cord derived spheres generated only astrocytes. This protocol describes an efficient method to generate and cul ture neurons and astrocytes from second trimester human CNS tissue that may be useful in transplantation and developmental studies.