Functional astrocyte–neuron lactate shuttle in a human stem cell-derived neuronal network

The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte–neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture.     

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 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 cellcell interactions within the neuroepithelium. These NT2/D1 cells also expressed small amounts of NF-L, α-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 mestin. 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. © 1993 Wiley-Liss, Inc.     

Characterization of the NT2‐derived neuronal and astrocytic cell lines as alternative in vitro models for primary human neurons and astrocytes

Primary human fetal neurons and astrocytes (HFNs and HFAs, respectively) provide relevant cell types with which to study in vitro the mechanisms involved in various human neurological diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. However, the limited availability of human fetal cells poses a significant problem for the study of these diseases when a human cell model system is required. Thus, generating a readily available alternative cell source with the essential features of human neurons and astrocytes is necessary. The human teratoma‐derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal and glial cells can be generated. Nevertheless, a direct comparison of NT2 neurons and primary HFNs in terms of their morphology physiological and chemical properties is still missing. This study directly compares NT2‐derived neurons and primary HFNs using immunocytochemistry, confocal calcium imaging, high‐performance liquid chromatography, and high‐content analysis techniques. We investigated the morphological similarities and differences, levels of relevant amino acids, and internal calcium fluctuations in response to certain neurotransmitters/stimuli. We also compared NT2‐derived astrocytes and HFAs. In most of the parameters tested, both neuronal and astrocytic cell types exhibited similarities to primary human fetal neurons and astrocytes. NT2‐derived neurons and astrocytes are reliable in vitro tools and a renewable cell source that can serve as a valid alternative to HFNs/HFAs for mechanistic studies of neurological diseases. © 2014 Wiley Periodicals, Inc.     

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.     

Gap junction-mediated bidirectional signaling between human fetal hippocampal neurons and astrocytes

Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy.     

Evaluation of the Importance of Astrocytes When Screening for Acute Toxicity in Neuronal Cell Systems

Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC₅₀ values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.     

Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells

Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, −32, and −43. Connexin26 and −32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and −32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells. J. Neurosci. Res. 49:19–31, 1997. © 1997 Wiley-Liss Inc.     

Neutrophil-specific chemokines are produced by astrocytic cells but not by neuronal cells

BACKGROUND: Neutrophils have a central role in the inflammatory conditions of the central nervous system (CNS). ELR chemokines direct neutrophil migration, but the source of chemokines in the CNS is unclear. We quantified chemokine production using cell-line models of astrocytic and neuronal cells, specifically NT2.N cells, a human line with characteristics of immature neurons, and NT2.A cells, a line with characteristics of astrocytes. OBJECTIVE: In NT2.N and NT2.A cells, and their parent cell line NT2, we sought to: (1) quantify ELR chemokines, (2) determine receptor (CXCR-1 and CXCR-2) expression, and (3) measure the function of the chemokines generated from these cells. DESIGN/METHODS: NT2 cells were differentiated into NT2.N cells and NT2.A cells with all trans retinoic acid and mitosis inhibitors. Chemokine concentrations in culture supernatants were determined by ELISA. Immunofluorescence was used to detect CXCR-1 and CXCR-2. RT-PCR was used to determine chemokine and chemokine receptor mRNA. Chemotaxis assays were used to assess function. RESULTS: ELR chemokines were not detected in supernatants of NT2 or NT2.N cells, although mRNA for GRO-gamma/CXCL3 was found in both. In contrast, in NT2.A cells, mRNA and protein were present for GCP-2/CXCL6, GRO-alpha/CXCL1, GRO-gamma/CXCL3, and IL-8/CXCL8. CXCR-1 and CXCR-2 were expressed on NT2, NT2.N, and NT2.A cells detected by immunofluorescent staining and RT-PCR. Supernatants of NT2.A cells resulted in neutrophil chemotactic function of 30.5 +/- 3.9%, greater than NT2 cells (12.3 +/- 1.6%, mean +/- SEM, P < 0.01). CONCLUSIONS: We speculate that astrocytes are a source of ELR chemokines in the human CNS and that neurons and astrocytes can respond to those chemokines.     

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

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.     

Expression and regulation of antiviral protein APOBEC3G in human neuronal cells

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) has recently been identified as a potent antiviral protein. Here, we examined the expression and regulation of APOBEC3G in human brain tissues and the cells of central nervous system (CNS). Similar to the immune cells, human brain tissue and the CNS cells expressed APOBEC3G at both mRNA and protein levels. The expression of APOBEC3G could be up-regulated in human neuronal cells (NT2-N) and astrocytes (U87-MG) by interferons (IFN-α, β and γ), interleukin-1 (IL-1), and tumor necrosis factor. Other cytokines (IL-4, IL-6 and transforming growth factor beta1) and CC-chemokines (CCL3, 4 and 5), however, had little impact on the expression of APOBEC3G. In addition, pseudotyped HIV-1 infection and cytokine/chemokine-enriched supernatants from lipopolysaccharide-stimulated macrophage cultures induced APOBEC3G expression in NT2-N cells. APOBEC3G expressed in the neuronal cells and astrocytes was biologically functional, as the suppression of APOBEC3G expression by the specific siRNA led to increase of pseudotyped HIV-1 replication in these cells. These findings provide direct and compelling evidence that there is intracellular expression and regulation of functional APOBEC3G in the neuronal cells, which may be one of innate defense mechanisms involved in the neuronal protection in the CNS.     

Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytes

The formation of functional synapses is a late milestone of neuronal differentiation. The establishment of functional synapses can be used to assess neuronal characteristics of different cell lines. In the present study, we examined the in vitro conditions that influence the ability of human neurons derived from the NT2 cell line (NT2N neurons) to establish synapses. The morphologic, immunologic, and electrophysiologic characteristics of these synapses was examined. In the absence of astrocytes, NT2N neurons rarely formed synapses and their action potentials were weak and uncommon. In contrast, when plated on primary astrocytes, NT2N neurons were able to form both glutamatergic excitatory (71%) and GABAergic inhibitory (29%) functional synapses whose properties (kinetics, ion selectivity, pharmacology, and ultrastructure) were similar to those of synapses of neurons in primary cultures. In addition, coculture of NT2N neurons with astrocytes modified the morphology of the neurons and extended their in vitro viability to more than 1 year. Because astrocyte-conditioned medium did not produce these effects, we infer that direct contact between NT2N neurons and astrocytes is required. These results suggest that NT2N neurons are similar to primary neurons in their synaptogenesis and their requirement for glial support for optimal survival and maturation. This system provides a model for further investigations into the neurobiology of synapses formed by human neurons.     

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury,lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.     

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.     

Bcl-2 expression regulates cell sensitivity to S100beta-mediated apoptosis.

The S100beta protein is overexpressed in the brain of patients with Alzheimer's disease and Down's syndrome and is able to induce apoptosis in neurons at high concentrations. The intracellular events that regulate the apoptotic effect are largely unknown. This study investigates the roles of the bcl-2 proto-oncogene, one of the best-defined apoptotic genes, on cell death induced by S100beta. Human neuronal precursor NT2/D1 cells showed a high degree of cell death by apoptosis after exposure to 2 microM S100beta in serum-free medium. Death was preceded by a down-regulation of the Bcl-2 protein. Gene transfer with a full-length bcl-2 cDNA under the control of a constitutive promoter in NT2 cells elevated Bcl-2 protein levels and repressed S100beta-mediated cell death. When exposed to retinoic acid, the NT2/D1 cells differentiated into a neuronal phenotype. The differentiated cells up-regulated their levels of Bcl-2 and became resistant to S100beta-induced cell death. Downregulation of Bcl-2 by an antisense oligonucleotide in the differentiated cells, however, increased their susceptibility to S100beta-related cytotoxicity. Therefore, apoptosis induced through S100beta signaling is subject to regulation by Bcl-2. A combined alteration such as up-regulation of S100beta together with down-regulation of Bcl-2 may be important in the pathogenesis of Alzheimer's disease and Down's syndrome.     

Retinoic acid induces cholinergic differentiation of NTera 2 human embryonal carcinoma cells

Retinoic acid (RA), a natural metabolite of vitamin A, influences the survival and neurotransmitter phenotype of several classes of vertebrate neurons during development. We now report that RA induces a subpopulation of NTera 2/clone D1 (NT2) human embryonal carcinoma cells to differentiate into postmitotic cells with cholinergic properties (NT2-N cells). After growth for 6 days in the presence of RA (10 μM) low levels of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) were detected in NT2 cell cultures. ChAT activity in the NT2 cell cultures continued to increase for at least an addition 22 days to a final activity of 50 pmol ACh synthesized/min/mg protein. Immunohistochemical staining of RA-treated cultures demonstrated that only those cells with a neuronal morphology (NT2-N cells) expressed the human ChAT protein. Since such cells comprised a small proportion (∼20%) of the population, the ChAT activity per neuronal cell was estimated to approach 250–300 pmol ACh/min/mg protein. Cultures composed of >95% NT2-N cells had significantly lower ChAT specific activities and this could be increased by either ciliary neurotrophic factor or leukemia inhibitory factor, but not by nerve growth factor. We conclude that NT2 cells provide a system in which to study the molecular events that underlie neurotransmitter choice during the differentiation of human cholinergic neurons.     

Telomerase and neuronal marker status of differentiated NT2 and SK-N-SH human neuronal cells and primary human neurons

Upon treatment with retinoic acid, NTera-2 (NT2) human teratocarcinoma and SK-N-SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT-2 cells is partially dependent on the time of differentiation (24-55 days). SK-N-SH cells differentiate into a mixed population of neuronal and epithelium-like cells. Here we report modified protocols that increase the number of differentiated NT-2 and SK-N-SH cells and that establish an enriched neuronal SK-N-SH-derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton-associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down-regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK-N-SH cells may represent an excellent source for studies investigating the role of telomerase or other survival-promoting activities in protecting human neuronal cells from cell death-mediating stresses associated with neurodegenerative diseases.