NTera 2 Cells: A human cell line which displays characteristics expected of a human committed neuronal progenitor cell

Abstract

We have identified a human cell line with a phenotype resembling committed CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expressed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule-associated protein (MAP) expressed in human neuroepithelium. NT2/D1 cells also expressed the cell adhesion molecules NCAM and N-cadherin which are thought to be important in cell-cell interactions within the neuroepithelium. 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 treatment, a proportion of NT2/D1 cells terminally differentiate into neurons and that this occurs via an asymmetric stem cell mode of differentiation. In light of the identification of the neuroepithelial phenotype of NT2/D1 cells we decided to examine more closely the relationship of in vitro neurogenesis in NT2/D1 cells, during RA treatment to that of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and increased phosphorylation of NF-M. By 10-14 days, these cells began to resemble neurons morphologically, i.e., with rounded cell bodies and processes. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons began to express MAP2b and tau and extinguished their expression of nestin. Recently, we developed a method for obtaining pure cultures of neurons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cells. Given the data obtained in this study and the characterization of the neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons.

Antitumor Activity of a Kinesin Inhibitor

“…Tissues were obtained from the Cooperative Human Tissue Network (Philadelphia, PA) and National Disease Research Interchange (Philadelphia, PA). NT-2 cells and differentiated NT2-N neurons were obtained from Layton BioScience (Atherton, CA) and prepared as described by Pleasure and Lee (18). Total RNA was extracted with a ToTALLY RNA kit (Ambion; Austin, TX) according to the manufacturer's protocol.…”

“…KSP mRNA levels were expressed relative to endogenous levels of ␤-glucuronidase mRNA and normalized to the KSP:␤-glucuronidase expression level in proliferating HeLa cells (HeLa ϭ 1000). Extracts of NT2 and postmitotic NT2-N cells (18) were immunoblotted using antibodies directed against KSP/Eg5 (19), against ␣-tubulin (DM1-␣), and against proliferating cell nuclear antigen (PC10; Sigma-Aldrich, Inc.). SE were calculated for each group of samples measured.…”

Production of Human CNS Neurons from Embryonal Carcinoma Cells Using a Cell Aggregation Method

“…Neuronal differentiation of NT2 cells maintained as monolayer cultures were previously demonstrated to be influenced by the types of substrates (16). We have compared the effects of various extracellular matrix on the differentiation of NT2 aggregates.…”

“…Because undifferentiated NT2 cells resemble neuroepithelial cells and retain properties of stem cells (16), it is possible that cell/cell interaction can be enhanced by forming cell aggregates as in the P19 cells (11). In the present study, we have examined whether cell aggregation can promote the RA-induced neuronal differentiation of NT2 cells.…”

“…Among the human cell types known to respond to RA, the human embryonal carcinoma (EC) cell line, NTera2 cl.D/1 (NT2), is one of the most extensively characterized (2,16). Following treatment with RA for four weeks, NT2 cells, as monolayer cultures, undergo terminal differentiation and become postmitotic central nervous system (CNS) neurons (1).…”