Glial-restricted precursors are derived from multipotent neuroepithelial stem cells

Neuroepithelial cells in the developing ventricular zone differentiate into neurons, astrocytes, and oligodendrocytes. It is not known, however, whether this differentiation occurs in a single step or is a pathway utilizing intermediate more restricted precursor cells. To characterize the generation of glial cells from multipotent stem cells we have cultured neuroepithelial (NEP) cells from E10.5 rat embryos. Cultured NEP cells do not express any glial differentiation markers when grown on fibronectin/laminin under nondifferentiation conditions. NEP cells, however, differentiate into A2B5 immunoreactive cells which can subsequently give rise to oligodendrocytes and astrocytes. Clonal analysis of NEP cells demonstrates that the A2B5 immunoreactive cells arise in clones that contain neurons and astrocytes, indicating that A2B5(+) cells arise from multipotent NEP precursor cells. A2B5(+) cells, maintained as undifferentiated cells over multiple passages, can subsequently give rise to both oligodendrocytes and astrocytes. A2B5(+) cells, however, do not generate neurons. Thus A2B5(+) cells represent a restricted progenitor cell population that differentiates from a multipotent NEP cell. Based on our results we propose that differentiation of the multipotential NEP cells to terminally differentiated glial cells occurs via intermediate restricted precursors.     

Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into gamma-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.     

Retinoic acid treated P19 embryonal carcinoma cells differentiate into oligodendrocytes capable of myelination

Retinoic acid treatment of P19 embryonal carcinoma cells induces their differentiation into cultures containing neurons and astrocytes. We present two lines of experimentation indicating that oligodendrocytes also develop from retinoic acid-treated P19 cells. We isolated an immortal cell line from retinoic acid-treated P19 cell cultures whose proliferation is dependent upon epidermal growth factor. Upon removal of the growth factor these cells differentiate into both astrocytes and oligodendrocytes as determined by immunostaining with antibodies to the astrocyte marker glial fibrillar acidic protein and the oligodendrocyte markers, myelin associated glycoprotein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase. This cell line appears to be a bi-potential glial precursor. We also found that oligodendrocytes developed directly from P19 cells when retinoic acid-treated cells were transplanted into the brains of neonatal rat pups. Cells that developed into oligodendrocytes migrated into fiber bundles up to several millimeters from the site of the graft. These P19-derived oligodendrocytes appeared to myelinate axons from host neurons. Thus, retinoic acid-treated P19 cells differentiate into neurons, astrocytes and oligodendrocytes, the three cell types that normally develop from embryonic neuroectoderm, indicating that these cell cultures differentiate in a fashion closely resembling that of embryonic neuroectoderm.     

Cytokines affecting survival and differentiation of an astrocyte progenitor cell line

The effects of various cytokines on survival and differentiation of an astrocyte progenitor cell line (AP-16) were examined. Epidermal growth factor (EGF) deprivation caused death of AP-16 cells by apoptosis. Transforming growth factor-alpha (TGF-alpha) and basic fibroblast growth factor (bFGF) prevented the apoptosis occurring in the absence of EGF. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) induced glial fibrillary acidic protein (GFAP) and decreased A2B5 antigen in AP-16 cells, indicating that these cytokines induced AP-16 cells to differentiate into astrocytes.     

Expression of two neuronal markers, growth-associated protein 43 and neuron-specific enolase, in rat glial cells

Recent studies have revealed that proteins such as growth-associated protein 43 (GAP-43) and neuron-specific enolase (NSE), believed for many years to be expressed exclusively in neurons, are also present in glial cells under some circumstances. Here we present an overview of these observations. GAP-43 is expressed both in vitro and in vivo transiently in immature rat oligodendroglial cells of the central nervous system, in Schwann cell precursors, and in non-myelin-forming Schwann cells of the peripheral nervous system. GAP-43 mRNA is also present in oligodendroglial cells and Schwann cells, indicating that GAP-43 is synthesized in these cells. GAP-43 is also expressed in type 2 astrocytes (stellate-shaped astrocytes) and in some reactive astrocytes but not in type 1 astrocytes (flat protoplasmic astrocytes). These results suggest that GAP-43 plays a more general role in neural plasticity during development of the central and peripheral nervous systems. NSE enzymatic activity and protein and mRNA have been detected in rat cultured oligodendrocytes at levels comparable to those of cultured neurons. NSE expression increases during the differentiation of oligodendrocyte precursors into oligodendrocytes. In vivo, NSE protein is expressed in differentiating oligodendrocytes and is repressed in fully mature adult cells. The upregulation of NSE in differentiating oligodendrocytes coincides with the formation of large amounts of membrane structures and of protoplasmic processes. Similarly, NSE becomes detectable in glial neoplasms and reactive glial cells at the time when these cells undergo morphological changes. The expression of the glycolytic isozyme NSE in these cells, which do not normally contain it, could reflect a response to higher energy demands. This expression may also be related to the neurotrophic and neuroprotective properties demonstrated for this enolase isoform. NSE activity and protein and mRNA have also been found in cultured rat type 1-like astrocytes but at much lower levels than in neurons and oligodendrocytes. Thus GAP-43 and NSE should be used with caution as neuron-specific markers in studies of normal and pathological neural development.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.     

In vitro and in vivo analysis of a rat bipotential O-2A progenitor cell line containing the temperature-sensitive mutant gene of the SV40 large T antigen

Primary cultures from neonatal optic nerve contain pluripotential O-2A progenitor cells that are capable of differentiating into oligodendrocytes, type-2 astrocytes or adult O-2A progenitors (O-2Aadult). Since primary optic nerve cultures contain a mixture of glial cell types of which only a small number are O-2A progenitors, experiments on cell lineage and differentiation carried out using these cultures are both intrinsically limited and difficult to interpret. Ideally, cells from a clonal cell population would provide the optimal starting material for biological studies. In this paper we describe the creation of an O-2A progenitor cell line using a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen gene. This cell line has provided sufficient numbers of cells to allow analysis of their in vitro properties and their behaviour following transplantation into an in vivo environment. At the non-permissive temperature (39 degrees C), these cells differentiate into oligodendrocytes and type-2 astrocytes in a similar fashion to O-2A progenitor cells from primary cultures (O-2Aprim). When grown in media containing platelet-derived growth factor and basic fibroblast growth factor, the cell numbers can be expanded in culture without differentiating, consistent with the behaviour of O-2Aprim progenitor cells. By exploiting this property, it has been possible to culture large numbers of O-2A progenitors for in vivo analysis. In this study we have shown that transplantation of this O-2A cell line into glia-free areas in adult rat spinal cord results in differentiation of a proportion of cells into oligodendrocytes which are capable of myelinating axons. Furthermore, differentiation of O-2A cells into astrocytes was also observed, indicating that the bipotentiality of these cells in vitro can also be demonstrated in vivo.     

The adult rat hippocampus contains primordial neural stem cells

Adult-derived hippocampal progenitors generate neurons, astrocytes, and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal, it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking, we have followed individual cells through the process of proliferative expansion, commitment, and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid, elevated cyclic AMP, or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain.     

Central nervous system stem cells in the embryo and adult

The central nervous system is generated from neural stem cells during embryonic development. These cells are multipotent and generate neurons, astrocytes and oligodendrocytes. The last few years it has been found that there are populations of stem cells also in the adult mammalian brain and spinal cord. In this paper, we review the recent development in the field of embryonic and adult neural stem cells.     

FGF and EGF are mitogens for immortalized neural progenitors

Individual neural progenitors, derived from the external germinal layer of neonatal murine cerebellum, were previously immortalized by the retrovirus-mediated transduction of avian myc (v-myc). C17-2 is one of those clonal multipotent progenitor cell lines (Snyder et al., 1992, Cell 68: 33-51; Ryder et al., 1990, J. Neurobiol. 21:356-375). When transplanted into newborn mouse cerebellum (CB), the cells participate in normal CB development; they engraft in a cytoarchitecturally appropriate, nontumorigenic manner and differentiate into multiple CB cell types (neuronal and glial) similar to endogenous progenitors (Snyder et al., 1992, as above). They also appear to engraft and participate in the development of multiple other structures along the neural axis and at multiple other stages (Snyder et al., 1993, Soc. Neurosci. Abstr. 19). Thus conclusions regarding these immortalized progenitors may be applicable to endogenous neural progenitors in vivo. To help identify and analyze factors that promote differentiation of endogenous progenitors, we first investigated the ability to maintain C17-2 cells in a defined, serum-free medium (N2). The cells survive in vitro in N2 but undergo mitosis at a very low rate. Addition of epidermal growth factor (EGF), however, either from mouse submaxillary gland or the human recombinant protein, appreciably stimulates thymidine incorporation and cell division approximately threefold. Basic fibroblast growth factor (bFGF) is an even more potent mitogen, promoting thymidine incorporation, cell division, and a net increase in cell number equal to that in serum. Both EGF and bFGF are active at very low nanomolar concentrations, suggesting that they interact with their respective receptors rather than a homologous receptor system. The findings demonstrate that C17-2 cells can be maintained and propagated in a fully defined medium, providing the basis for analysis of other growth and differentiation factors. That EGF and particularly bFGF are mitogenic for these cells is in accord with recent observations on primary neural tissue (Reynolds and Weiss, 1992, Science 255:1707-1710; Kilpatrick and Bartlett, 1993, Neuron 10:255-265; Ray et al., 1993, Proc. Natl. Acad. Sci. USA 90:3602-3606) suggesting that bFGF and EGF responsiveness may be fundamental properties of neural progenitors.     

FGF2 promotes neuronal differentiation in explant cultures of adult and embryonic mouse olfactory epithelium

Neurogenesis in the adult olfactory epithelium is highly regulated in vivo. Little is known of the molecular signals which control this process, although contact with the olfactory bulb or with astrocytes has been implicated. Explants of mouse olfactory epithelium were grown in the presence or absence of several peptide growth factors. Basic fibroblast growth factor (FGF2) stimulated differentiation of sensory neurons in adult and embryonic olfactory epithelium. Other growth factors tested were ineffective. FGF2-stimulated neurons were born in vitro and expressed neurofilament, neural cell adhesion molecule, and beta-tubulin. The cells also expressed olfactory marker protein, a marker for mature olfactory sensory neurons in vivo. These bipolar neurons did not express glial fibrillary acidic protein or low-affinity nerve growth factor receptor. These results indicate that neither astrocytes nor olfactory bulb are necessary for differentiation of olfactory sensory neurons in vitro.     

In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: inducing effects of brain-derived neurotrophic factor

Neural stem cells proliferate in vitro and form neurospheres in the presence of epidermal growth factor (EGF), and are capable of differentiating into both neurons and glia when exposed to a substrate. We hypothesize that specific neurotrophic factors induce differentiation of stem cells from different central nervous system (CNS) regions into particular fates. We investigated differentiation of stem cells from the postnatal mouse hippocampus in culture using the following trophic factors (20 ng/mL): brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF). Without trophic factors, 32% of stem cells differentiated into neurons by 4 days in vitro (DIV), decreasing to 10% by 14 DIV. Addition of BDNF (starting at either day 0 or day 3) significantly increased neuron survival (31-43% by 14 DIV) and differentiation. Morphologically, many well-differentiated neurons resembled hippocampal pyramidal neurons. 5'-Bromodeoxyuridine labeling demonstrated that the pyramidal-like neurons originated from stem cells which had proliferated in EGF-containing cultures. However, similar application of NT-3 and GDNF did not exert such a differentiating effect. Addition of BDNF to stem cells from the postnatal cerebellum, midbrain, and striatum did not induce these neuronal phenotypes, though similar application to cortical stem cells yielded pyramidal-like neurons. Thus, BDNF supports survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons. The presence of pyramidal neurons in BDNF-treated hippocampal and cortical stem cell cultures, but not in striatal, cerebellar, and midbrain stem cell cultures, suggests that stem cells from different CNS regions differentiate into region-specific phenotypic neurons when stimulated with an appropriate neurotrophic factor.     

A new human breast cancer cell line, KPL-1 secretes tumour-associated antigens and grows rapidly in female athymic nude mice

We recently established a new human breast cell line, designated KPL-1, which was derived from the malignant effusion of a patient with breast cancer. This cell line is highly tumorigenic and grows rapidly in female nude mice. Cytogenetic analysis indicated its human origin and revealed a hypertriploid modal number of chromosomes. Electron microscopic examination suggested that the KPL-1 cells are of epithelial origin. Immunohistochemical studies revealed that the cells express cytokeratin, carcinoembryonic antigen and CA 15-3. They also possess a large number of oestrogen receptors but not progesterone receptors. Interestingly, KPL-1 cells seem to grow oestrogen independently in vitro. No amplification of c-erbB-2, c-myc, H-ras and N-ras genes was detected. KPL-1 cells secrete a large amount of tissue polypeptide antigen (TPA). Although the secretion of CA 15-3 seemed to be constant throughout all cell growth phases, TPA secretion increased during the exponential growth phase and decreased during the plateau phase. Serum TPA levels significantly correlated with the volume of KPL-1 tumours transplanted into nude mice. These data suggest that this KPL-1 cell line may be useful for studying oestrogen-independent growth and the kinetics of tumour-associated antigens in vivo as well as in vitro.     

Identification of apoptotic changes in osteocytes in normal and pathological human bone

MS-430 is a novel synthetic pyrimidine derivative that stimulates regeneration of the nerve as a promoter for various growth factors such as epidermal growth factor (EGF) and nerve growth factor, and differentiation of astrocytes. The effects of MS-430 on the liver were tested using hepatocytes and stellate cells in primary culture isolated from rats. MS-430 enhanced EGF-induced DNA synthesis in hepatocytes while it alone failed to increase the basal DNA synthesis. Albumin mRNA expression in the cells and its amount in the medium were not changed by addition of EGF or MS-430 alone or both. Basic fibroblast growth factor (bFGF) increased DNA and but not collagen synthesis by hepatic stellate cells. Addition of MS-430 inhibited DNA synthesis by hepatic stellate cells at either presence or absence of bFGF, and collagen synthesis at the presence of bFGF. However, MS-430 had no effects on basal or bFGF-stimulated TGFbeta mRNA expression in the cells. These results suggest that MS-430 stimulated proliferation of hepatocytes as a comitogen for EGF without affecting albumin synthesis, and suppressed proliferation of activated hepatic stellate cells and their collagen synthesis without affecting TGFbeta expression.     

Stem cells in the central nervous system

In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results focus attention on how information in external stimuli is translated into the number and types of differentiated cells in the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding of the origin and stability of cell type in the central nervous system.     

Distinct actions of interleukin-9 and interleukin-4 on a hematopoietic stem cell line, EMLC1

EMLC1 is a hematopoietic stem cell line that depends on stem cell factor (SCF) for growth and generates lymphoid, erythroid and myeloid progenitors in the presence of different cytokines. We have studied signaling events leading to cell proliferation and differentiation of EMLC1 mediated by interleukin (IL)-4 and IL-9. It was found that IL-9 enhances SCF-induced cell proliferation and promotes erythropoietin (EPO)-dependent erythroid differentiation of EMLC1 cells. However, IL-9 alone cannot support the growth of this cell line. In contrast, IL-4 by itself is sufficient to promote the growth of EMLC1 cells, even in the absence of SCF. Antiphosphotyrosine immunoblots of total cell lysates demonstrated that IL-4 and IL-9 induce tyrosine phosphorylation of different cellular substrates. Both IL-4 and IL-9 stimulated tyrosine phosphorylation of SHP-2, whereas the 90-kD tyrosine phosphorylated protein induced by IL-9 stimulation is Stat3. We have also shown that IL-4 is much more potent than IL-9 in inducing the expression of primary response gene c-myc. It was further determined that c-myc antisense oligodeoxynucleotide blocked IL-4 supported cell growth. Taken together, these results indicate that IL-4 may serve as a growth-promoting factor for hematopoietic stem cells, and IL-9 enhances both growth and erythroid differentiation of primitive hematopoietic progenitors. The results also suggest that differences in tyrosine phosphorylation induced by IL-4 and IL-9 may in part determine their distinct biological functions.     

Thermal unfolding of small proteins with SH3 domain folding pattern

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34 degrees C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39 degrees C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39 degrees C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39 degrees C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal-glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons.     

A simple neonatal mock circulation enabling pulsatility and different hemodynamical states for neonatal ECMO research: application to assess the effect of a centrifugal pump operated neonatal ECMO system on the circulation

In the developing forebrain, mounting evidence suggests that neural stem cell proliferation and differentiation is regulated by growth factors. In vitro in the presence of serum, stem cell proliferation is predominantly mediated by fibroblast growth factor-2 (FGF-2) whereas neuronal differentiation can be triggered by FGF-1 in association with a specific heparan sulphate proteoglycan. On the other hand, astrocyte differentiation in vivo and in vitro appears to be dependent on signalling through the leukaemia inhibitory factor receptor (LIFR). The evidence suggests that in the absence of LIFR signalling, the stem cell population is present at approximately the same frequency and can generate neurons but is blocked from producing astrocytes that express glial fibrillary acidic protein (GFAP) or have trophic functions. The block can be overcome by other growth factors such as BMP-2/4 or interferon-gamma, providing further evidence that the inhibition to astrocyte development does not result from loss of a precursor population. Signalling through the LIFR, in addition to stimulating astrocyte differentiation, may also inhibit neuronal differentiation, which may explain why this receptor is expressed at the earliest stages of neurogenesis. Another signalling system which also exerts its influence on neurogenesis through active inhibition is Delta-Notch. We show in vitro that at high cell densities which impede neuronal production by FGF-1, lowering the levels of expression of the receptor Notch by antisense oligonucleotide results in a significant increase in neuronal production. Thus, stem cell differentiation appears to be dependent on the outcome of interactions between a number of signalling pathways, some which promote specific lineages and some which inhibit.