Derivation of Neuronal Cells from Fetal Normal Human Astrocytes (NHA)

Abstract

Pathological changes in most neurological diseases are marked by cell loss. To understand the mechanisms of neurogenesis and brain repair at a cellular level, observation on less complex systems provide valuable knowledge which offer the basis for therapeutic interventions. This has been the impetus for neural cell culture studies and the development of in vitro models. Here, we provide protocols for differentiation into neuronal lineage of commercially available normal human astrocytes (NHA) that are isolated from normal fetal human brain (Lonza, Inc.). It is known that some of GFAP positive astrocytic cells have stem/progenitor cell characteristics; however, understanding of the human GFAP positive cells with these characteristics remains limited. The genesis of neuronal lineage cells from the NHA occurs in adherent culture conditions by removal of serum and exposure to bFGF. When transferred to serum-free medium supplemented with bFGF, NHA cells generate neuronal precursors that express doublecortin, nestin and are negative for GFAP. After withdrawal of bFGF they mature into neurons. The average time required for generation of neuronal cells using this protocol is about 3 weeks. Our model of neurogenesis captures a contained in vitro system consisting of both neurons and glia. This “human brain in a dish” model can be used to assay the effects of interventions on developing human neurons at a cellular and molecular level and is also suitable for modeling of various aspects of human diseases and testing of novel therapeutic strategies.