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.     

Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos

Hepatocyte growth factor (HGF), originally cloned as a hepatocyte mitogen, has recently been reported to exhibit neurotrophic activity in addition to being expressed in different parts of the nervous system. At present, the effects of HGF on neural stem cells (NSCs) are not known. In this study, we first report the promoting effect of HGF on the proliferation of neurospheres and neuronal differentiation of NSCs. Medium containing only HGF was capable of inducing neurosphere formation. Addition of HGF to medium containing fibroblast growth factor 2 or epidermal growth factor increased both the size and number of newly formed neurospheres. More neurons were also obtained when HGF was added in differentiation medium. In contrast, neurosphere numbers were reduced after repeated subculture by mechanical dissociation, suggesting that HGF-formed neurospheres comprised predominantly progenitor cells committed to neuronal or glial lines. Together, these results suggest that HGF promotes proliferation of neurospheres and neuronal differentiation of NSCs derived from mouse embyos.     

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.     

Human NT2 neurons express a large variety of neurotransmission phenotypes in vitro

The NT2 cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristic of a committed neuronal precursor at an early stage of development. NT2 cells can be induced by retinoic acid to differentiate in vitro into postmitotic central nervous system (CNS) neurons (NT2-N cells). The commitment of NT2-N cells to a stable neuronal phenotype is irreversible. Because it may be possible to transplant these human neurons to compensate for neuronal loss after traumatic injuries or neurodegenerative diseases of the CNS, knowledge of their phenotype is essential. This study aimed to characterize in detail the neurotransmission phenotype of NT2-N cells by using immunocytochemical methods. Single peroxidase immunostaining demonstrated that NT2-N cells expressed the gamma-aminobutyric acidergic (GABAergic), catecholaminergic, and cholinergic phenotypes to a large extent and expressed the serotonergic phenotype to a minor extent. NT2-N cells also expressed different neuropeptides, such as neuropeptide Y, oxytocin, vasopressin, calcitonin gene-related peptide, and Met- and Leu-enkephalin. Double fluorescence immunostaining further indicated that a large number of NT2-N cells could express GABA and another neurotransmitter or neuropeptide at the same time. Finally, electron microscopy demonstrated that these NT2 neurons elaborate classical synaptic contacts. The multipotentiality of these neurons, combined with their apparent functionality, suggests that they may represent useful material for a variety of therapeutic approaches aimed at replacing dead neurons after neurodegenerative diseases or lesions of the CNS.     

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.     

Functional properties of neurons derived from fetal mouse neurospheres are compatible with those of neuronal precursors in vivo

Neural stem cells can be propagated in culture as neurospheres, yielding neurons and glial cells upon differentiation. Although the neurosphere model is widely used, the functional properties of the neurosphere-derived neurons have been only partially characterized, and it is unclear whether repeated passaging alters their functional properties. In this study, we analyzed voltage- and transmitter-gated responses in neuron-like cells obtained by differentiating fetal mouse neurospheres at increasing passages in culture. We report that neurons fire overshooting action potentials in response to depolarizing currents up to passage 10 but loose this capability at later passages, as the density of voltage-gated Na(+) and K(+) currents decreases. In contrast, the immunoreactivity for the neuronal marker beta-tubulin remains unaltered up to passage 21, indicating that this marker is not representative of cell function. In almost all neurons, gamma-aminobutyric acid (GABA) evoked bicuculline-sensitive whole-cell currents, resulting from the activation of GABA(A) receptors, which appeared to be excitatory, insofar as the reversal potential of GABA-gated current was about -50 mV. Much smaller currents were elicited by the glutamatergic agonist AMPA, and only occasional responses to glycine were detected. In these functional aspects, neurosphere-derived neurons are similar to immature neurons differentiating in vivo. Therefore, at least for a limited number of passages in vitro, neurospheres provide an adequate model of in vivo neurogenesis.     

Retinoic acid induction of ES-cell-derived neurons: the radial glia connection

Neuronal induction by retinoic acid (RA) is commonly used in embryonic stem (ES) cell differentiation. Two recent papers show that this paradigm induces a population of neurogenic precursors with properties of radial glia. Upon differentiation, RA-treated cells give rise to a defined and developmentally restricted neuronal lineage. This role of RA in cell fate specification provides new perspectives for studying the radial glia-neuron transition and for generating homogenous populations of neurons from ES cells.     

Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers

Neural stem cells (NSCs) with self-renewal and multilineage differentiation properties can potentially repair degenerating or damaged neural tissue. Here, we have enriched NSCs from neurospheres, which make up a heterogeneous population, by fluorescence-activated cell sorting (FACS) with antibodies against syndecan-1, Notch-1, and integrin-beta1, which were chosen as candidates for hematopoietic cell-or somatic stem cell-markers. Antigen-positive cells readily initiated neurosphere formation, but cells lacking these markers did so less readily. Doubly positive cells expressing both syndecan-1 and Notch-1 underwent neurosphere formation more efficiently than did singly positive cells. The progeny of sorted cells could differentiate into neurons and glial cells both in vitro and in vivo. These antibodies were also useful for isolating cells from the murine embryonic day 14.5 brain that efficiently formed neurospheres. In contrast, there was no distinct difference in neurosphere formation efficiency between Hoechst 33342-stained side population cells and main population cells, although the former are known to have a stem cell phenotype in various tissues. These results indicate the usefulness of syndecan-1, Notch-1, and integrin-beta1 as NSC markers.     

Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia

The study tested the hypothesis that transplantation of embryonic stem (ES) cells into rat cortex after a severe focal ischemia would promote structural repair and functional recovery. Overexpression of the human anti-apoptotic gene bcl-2 in ES cells was tested for increasing survival and differentiation of transplanted cells and promoting functional benefits. Mouse ES cells, pretreated with retinoic acid to induce differentiation down neural lineages, were transplanted into the post-infarct brain cavity of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCA). Over 1-8 weeks after transplantation, the lesion cavity filled with ES cell-derived cells that expressed markers for neurons, astrocytes, oligodendrocytes, and endothelial cells. ES cell-derived neurons exhibited dendrite outgrowth and formed a neuropil. ES cell-transplanted animals exhibited enhanced functional recovery on neurological and behavioral tests, compared to control animals injected with adult mouse cortical cells or vehicle. Furthermore, transplantation with ES cells overexpressing Bcl-2 further increased the survival of transplanted ES cells, neuronal differentiation, and functional outcome. This study supports that ES cell transplantation and gene modification may have values for enhancing recovery after stroke.     

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.     

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.     

新生小鼠端脑组织神经干细胞诱导分化为胆碱能神经元的研究

目的探讨新生小鼠端脑组织神经干细胞是否能够分化成胆碱能神经元。方法取新生小鼠端脑组织．用无血清方法分离培养神经干细胞；用克隆培养的方法检验培养细胞的干细胞特性；用免疫荧光细胞化学的方法检测神经干细胞标志巢蛋白（nestin）及干细胞诱导分化后神经元标志微管相关蛋白2（MAP2）、星形胶质细胞标志胶质纤维酸性蛋白（GFAP）、胆碱能标志胆碱乙酰转移酶（CHAT）；比较不同的诱导分化条件（5％胎牛血清、5％胎牛血清＋碱性成纤维细胞生长因子）对胆碱能神经元分化的影响。结果从新生小鼠端脑组织分离培养出具有自我更新、扩增能力的神经球；各培养基中神经球均为nestin阳性。诱导分化后均能够产生MAP2阳性神经元、GFAP阳性星形胶质细胞以及ChAT阳性的胆碱能神经元。分化培养中加入碱性成纤维细胞生长因子能够提高胆碱能神经元分化的比例。结论新生小鼠端脑组织神经干细胞能够分化成胆碱能神经元。     

Dynamic behavior of cells within neurospheres in expanding populations of neural precursors

Large-scale expansion of neural stem and progenitor cells will be essential for clinically treating the large number of patients suffering from neurodegenerative disorders such as Parkinson's disease. Other applications of neural stem cell technology include further research in areas such as neural development or drug testing. Neural stem cells can be grown in vitro as tissue aggregates known as neurospheres, and in the current study, experiments were performed to determine the spatial arrangement and behavior of the cells within the neurosphere structure. A protocol utilizing sulfonated lipophilic fluorescent dyes was developed to effectively label populations of neural stem and progenitor cells without compromising cell density during culture. Cells retained the labels for at least 7 days. Using the labeling protocol, we discovered that the cells within the neurospheres were mobile and, moreover, the cells on the periphery of the neurospheres could migrate into the center of the neurospheres. Most important, the mixing time of two merging neurospheres was observed to be the same order of magnitude as the neural stem cell doubling time (approximately 20 h). This study is the first to show that the neurosphere system is dynamic, and these results will serve as a stepping stone to more in-depth studies of the neurosphere microenvironment.     

Interplay of leukemia inhibitory factor and retinoic acid on neural differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells have great potential for cell replacement in neurodegenerative disorders. Implantation of these cells into the brain, however, requires their prior differentiation. We examined the interplay between leukemia inhibitory factor (LIF) and retinoic acid (RA) on neural differentiation of mouse ES (mES) cells. Mouse embryonic stem cells were allowed to form cell aggregates, the so-called embryoid bodies (EBs), in the absence or presence of LIF. In the absence of LIF, mES cells downregulated the expression of the undifferentiated mES cell marker Oct-3/4, and increased mRNA levels of two neural precursor markers, Sox-1 and Nestin, as well as the neuronal marker beta-tubulin III. This neuronal differentiation was enhanced by treating EBs with RA. Moreover, RA irreversibly increased the number of postmitotic neurons in culture, as shown by the reduction of proliferating mES cells and the increase in beta-tubulin III-positive cells 6 days after RA removal, which in turn affected mES cell viability. The addition of LIF during EBs formation, however, blocked completely this neuronal differentiation. Our findings also showed that pre-differentiation of mES cells in vitro avoided the teratocarcinoma formation observed when proliferating mES cells were grafted into the brain. In addition, mES cells pre-differentiated with RA in culture showed a reduction in proliferation and the presence of neural phenotypes after grafting. In conclusion, the present results indicate that RA enhances neuronal differentiation of mES cells in the absence of LIF, although it compromises cell viability and transplantation.     

Staurosporine is a potent activator of neuronal,glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells

Staurosporine (STS), a broad spectrum protein kinase inhibitor, was previously shown to induce neurite outgrowth in several neuroblastoma cell lines. However, data on the neurotrophic potential of this alkaloid in embryonic stem cell systems were not available. Therefore, three mouse ES cell lines, IB10, RW4, and Bruce 4, were induced to enter neurogenesis in culture at low concentrations of STS. These cells differentiated into epidermal growth factor-responsive neural precursor cells, formed neurospheres, and further developed to neurons and astrocytes. The clonally derived neurospheres consisted of multipotent cells which exhibited some of the classical characteristics of early CNS stem cells and could be propagated in vitro. STS was antagonistic in several ways to retinoic acid (RA), a vitamin A metabolite, which promotes neuritogenesis. Results from RT-PCR experiments and inhibition studies with RA provided evidence that staurosporine exerted its neurotrophic effects through the induction of very late levels of the nerve growth factor and protein kinase C neurogenesis pathways.     

Peripheral glial cell differentiation from neurospheres derived from adipose mesenchymal stem cells

Mesenchymal stem cells derived from bone marrow and adipose tissue are being considered for use in neural repair because they can differentiate after appropriate induction in culture into neurons and glia. The question we asked was if neurospheres could be harvested from adipose-derived stem cells and if they then could differentiate in culture to peripheral glial-like cells. Here, we demonstrate that adipose-derived mesenchymal stem cells can form nestin-positive non-adherent neurosphere cellular aggregates when cultured with basic fibroblast growth factor and epidermal growth factor. Dissociation of these neurospheres and removal of mitogens results in expression of the characteristic Schwann cell markers S100 and p75 nerve growth factor receptor and GFAP. The simultaneous expression of these glia markers are characteristic features of Schwann cells and olfactory ensheathing cells which have unique properties regarding remyelination and enhancement of axonal regeneration. When co-cultured with dorsal root ganglion neurons, the peripheral glial-like cells derived from adipose mesenchymal stem cells aligned with neuritis and stimulated neuritic outgrowth. These results indicate that neurospheres can be generated from adipose-derived mesenchymal stem cells, and upon mitogen withdrawal can differentiate into peripheral glial cells with neurotrophic effects.