NTERA-2 CELLS - A HUMAN CELL-LINE WHICH DISPLAYS CHARACTERISTICS EXPECTED OF A HUMAN COMMITTED NEURONAL PROGENITOR-CELL

We have identified a human cell line with a phenotype resembling commi tted CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expres sed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule-a ssociated protein (MAP) expressed in human neuroepithelium. NT2/D1 cel ls also expressed the cell adhesion molecules NCAM and N-cadherin whic h are thought to be important in cell-cell interactions within the neu roepithelium. These NT2/D1 cells also expressed small amounts of NF-L, alpha-internexin, NF-M, and MAP2c, indicating that they are committed to a neuronal fate. Previous studies have shown that, following RA tr eatment, a proportion of NT2/D1 cells terminally differentiate into ne urons and that this occurs via an asymmetric stem cell mode of differe ntiation. In light of the identification of the neuroepithelial phenot ype of NT2/D1 cells we decided to examine more closely the relationshi p of in vitro neurogenesis in NT2/D1 cells, during RA treatment to tha t of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and incre ased phosphorylation of NF-M. By 10-14 days, these cells began to rese mble neurons morphologically, i.e., with rounded cell bodies and proce sses. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons b egan to express MAP2b and tau and extinguished their expression of nes tin. Recently, we developed a method for obtaining pure cultures of ne urons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cell s. Given the data obtained in this study and the characterization of t he neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some ste m cell characteristics and is capable only of terminal differentiation into neurons. (C) 1993 Wiley-Liss, Inc.