Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons

Abstract

NTera 2/cl.D1 (NT2) cells, a human teratocarcinoma cell line, were manipulated following retinoic acid treatment to yield greater than 95% pure cultures of neuronal cells (NT2-N cells). The commitment of NT2-N cells to a stable neuronal phenotype is irreversible as judged by the lack of mitotic activity or phenotypic reversion over a period of 2 months in culture. Furthermore, NT2-N cells express a variety of neuronal markers including many neuronal cytoskeletal proteins, secretory markers, and surface markers. NT2-N cells resemble primary neuronal cultures from rodents morphologically and in density of process outgrowth and, like primary neurons, go on to elaborate processes that differentiate into axons and dendrites. This culture method yields sufficient highly differentiated postmitotic NT2-N cells for both biochemical and molecular biological studies. Indeed, when undifferentiated NT2 cells were stably transfected with a beta-galactosidase (beta-gal) expression plasmid, beta-gal expression was shown to be present in both undifferentiated NT2 and postmitotic NT2-N cells. Thus, the ability to transfect expression plasmids into undifferentiated NT2 cells will allow the introduction of normal and mutant gene products into cells that can then be induced to become stable, postmitotic human neurons. We conclude that NT2 cells and NT2-N cells represent a unique model system for studies of human neurons, and a novel vehicle for the expression of diverse gene products in terminally differentiated polarized neurons.

Differential Cellular Expression of Neurotrophins in Cortical Tubers of the Tuberous Sclerosis Complex

“…NTera 2 human teratocarcinoma cells were differentiated into postmitotic neurons (NT2N) as previously described. 23 Differentiation was induced by culturing cells (3.2 ϫ 10 3 cells/cm 2 ) in the presence of 10 mol/L all-trans retinoic acid for 5 weeks followed by replating at 1:6 density without retinoic acid in the presence of mitotic inhibitors (10 mol/L 5-fluoro-2Ј-deoxyuridine, 10 mol/L uridine, 1 mol/L cytosine-␤-D-arabinose furanoside). One week later, a final replating was done using culture vessels coated with Matrigel (Collaborative Biomedical Products, Bedford, MA), and cells were maintained in the presence of mitotic inhibitors for an additional 2 to 3 weeks.…”

“…Although the phenotypic distinctions between these cells remain a source of debate, we attempted to devise a strategy to classify these cells based on size and morphology. Neurotrophin mRNA expression was also evaluated in human NTera2 neurons (NT2N), 23 a human neuronal cell line that expresses p75NGFR, trkA, trkB, and trkC mRNAs. 24 These neurons were examined in their native state and after stable transfection with a tuberin antisense construct that reduced tuberin expression.…”

ZPK inhibits PKA induced transcriptional activation by CREB and blocks retinoic acid induced neuronal differentiation

“…The growth and di erentiation of NT2 cells was carried out as described previously (Pleasure et al, 1992).…”

“…NTera2/cl.D1(NT2), a human teratocarcinoma cell line, when properly manipulated provides an excellent model system to study retinoic acid (RA) induced di erentiation of neuronal cells (NT2-N) (Pleasure et al, 1992). Evidence suggests that neurotrophins such as brain derived nerve growth factor (BDNF) and trk receptors are upregulated during retinoic acid induced di erentiation of NT-2 cells (Cheung et al, 1996).…”

Hypoxia-inducible transgene expression in differentiated human NT2N neurons – a cell culture model for gene therapy of postischemic neuronal loss

“…The combination of five HRE with a cytomegalovirus (CMV) minimal promoter exhibited excellent hypoxia responsiveness with over 500-fold induction in some cell types. 10 Ntera2c1.D2 (NT2), a human embryonic carcinoma cell line, terminally differentiates into postmitotic neurons (NT2N) by treatment with retinoic acid, [11][12][13] and may be used as a cell culture model for human neurons. Nondifferentiated NT2 cells ( Figure 1A) are rapidly dividing and may be conveniently expanded before treatment with retinoic acid, which then induces typical morphological changes with differentiation to postmitotic neurons (NT2N) ( Figure 1B).…”