Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal Tα1-promoter-driven GFP

The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal Tα1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.     

Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells and mouse central nervous system

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2, by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most Nestin-positive cells as well as beta-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroid plexus. The RANDAM-2 expression commenced at E6.0, and the antigen was distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in mouse central nervous system (CNS).     

Neuronal differentiation of P19 embryonal carcinoma cells in defined media

The P19 embryonal carcinoma cell line is a useful model system for analyzing the factors that regulate neuronal differentiation. In order to analyze the extrinsic factors that are involved in differentiation, it is necessary to carry out experiments in fully defined media. Here we have investigated the neuronal differentiation of P19 cells in two defined media. Cells that are propagated and induced with retinoic acid in standard serum-containing medium are capable of differentiating into neuron-like cells in N2 medium. Dividing fibroblast-like cells also appeared in these cultures. After about 10 days in culture in N2 medium, the great majority of neuron-like cells died. On the other hand, culturing induced cells in N2 medium for 5 days and then switching to a defined medium consisting of Neurobasal medium plus B27 supplement allowed the neuron-like cells to survive for prolonged periods of time. This defined medium thus provides a suitable system for analyzing extrinsic factors that affect the survival and differentiation of P19 neurons. P19 cells induced with retinoic acid and plated in N2 were exposed to bFGF and EGF, which are known to be mitogens for neuronal precursor cells. Both growth factors were mitogenic for a subpopulation of the induced cells. In separate experiments, cells cultured in N2 in the presence of RA were induced to differentiate into neuron-like cells. © 1995 Wiley-Liss, Inc.     

Glial-guided neuronal migration in P19 embryonal carcinoma stem cell aggregates

During development of the nervous system, neuronal precursors that originated in proliferative regions migrate along radial glial fibers to reach their final destination. P19 embryonal carcinoma (EC) stem cells exposed to retinoic acid (RA) differentiate into neurons, glia, and fibroblast-like cells. In this work, we induced P19 aggregates for 4 days with RA and plated them onto tissue culture dishes coated with poly-L-lysine. Several cells migrated out of and/or extended processes from the aggregates after 24 hr. Some cell processes were morphologically similar to radial glial fibers and stained for glial fibrillar acidic protein (GFAP) and nestin. Large numbers of migrating cells showed characteristics similar to those of bipolar migrating neurons and expressed the neuronal marker microtubule-associated protein 2. Furthermore, scanning electron microscopy analysis revealed an intimate association between the radial fibers and the migrating cells. Therefore, the migration of neuron-like cells on radial glia fibers in differentiated P19 aggregates resembled some of the migration models used thus far to study gliophilic neuronal migration. In addition, HPTLC analysis in this system showed the expression of 9-O-acetyl GD3, a ganglioside that has been associated with neuronal migration. Antibody perturbation assays showed that immunoblockage of 9-O-acetyl GD3 arrested neuronal migration in a reversible manner. In summary, we have characterized a new cell culture model for investigation of glial-guided neuronal migration and have shown that 9-O-acetyl GD3 ganglioside has an important role in this phenomenon.     

Glial-derived nexin, a differentially expressed gene during neuronal differentiation, transforms HEK cells into neuron-like cells

Glial-derived nexin (GDN) is a proteinase inhibitor secreted from glial cells and it can enhance neuronal function. However, its expression and function in neuronal differentiation are not, as yet, well-known. In the present study, we analyzed glial-derived nexin gene expression in dissociated neural stem/progenitor cells (NS/PCs) (D0) from the embryonic mouse cerebral cortex, expanded NS/PC cultures (D4 and D10 cultures) and cultured neurons (E15) using a semi-quantitative RT-PCR assay. Our data suggest that mouse GDN, homologue of human GDN, was significantly up-regulated in the expanded NS/PC cultures and cultured neurons. To analyze its function in neuronal differentiation, human GDN cDNA was cloned into bicistronic plasmids containing green fluorescent protein (GFP) and the resulting plasmids were transfected into rodent primary NS/PCs and non-neuronal human embryonic kidney (HEK) cells. Our data suggest that the ectopic expression of human GDN triggered the expression of the neuronal marker TuJ1 in both NS/PCs and HEK cells. We conclude that GDN is up-regulated during neuronal differentiation and plays a role in transforming non-neuronal HEK cells into neuron-like cells.     

In vivo and in vitro neurogenesis in human olfactory epithelium

The birth and differentiation of neurons have been extensively studied in the olfactory epithelium (OE) of rodents but not in humans. The goal of this study was to characterize cellular composition and molecular expression of human OE in vivo and in vitro. In rodent OE, there are horizontal basal cells and globose basal cells that are morphologically and functionally distinct. In human OE, however, there appears to be no morphological distinction among basal cells, with almost all cells having round cell bodies similar to rodent globose basal cells. Unlike the case in rodents, human basal cells, including putative neuronal precursors, express p75NGFR, suggesting a distinctive role for p75NGFR in human OE neurogenesis. Molecular expression of neuronal cells during differentiation in human OE grossly follows that in rodents. However, the topographical organization of immature and mature ORNs in human OE differs from that of rodents, in that immature and mature ORNs in humans are dispersed throughout the OE, whereas rodent counterparts have a highly laminar organization. These observations together suggest that the birth and differentiation of neuronal cells in human OE differ from those in rodents. In OE explant culture, neuronal cells derived from human OE biopsy express markers for immature and mature neurons, grossly recapitulating neuronal differentiation of olfactory neurons in vivo. Furthermore, small numbers of cells are doubly label for bromodeoxyuridine and olfactory marker protein, indicating that neuronal cells born in vitro reach maturity. These data highlight species-related differences in OE development and demonstrate the utility of explant culture for experimental studies of human neuronal development.     

Environmental co-regulation of substance P, somatostatin and neurotransmitter synthesizing enzymes in cultured sympathetic neurons

Mechanisms regulating peptide neurotransmitter metabolism were examined in dissociated cell cultures of the neonatal rat superior cervical ganglion (SCG). The pineal gland, a target of the SCG, produced a soluble factor (PCM) which increased substance P (SP) levels more than 15-fold in sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Elimination of the non-neuronal cells decreased SP to negligible levels and abolished the stimulatory effects of PCM on SP expression. These observations suggest that ganglion non-neuronal cells stimulate sympathetic expression of SP, and that the pineal influences neuronal SP by acting on, or in concert with, ganglion support cells. PCM also influenced other neurotransmitter systems. In the presence of ganglion non-neuronal cells, PCM treatment increased cholineacetyltransferase (CHAC) and decreased tyrosine hydroxylase (TOH) and somatostatin (SO). By contrast, PCM treatment of pure neuronal cultures resulted in negligibleCHAC and SP levels and a doubling of SO with a small increase in TOH. In sympathetic neurons, SP expression may be associated with cholinergic development, whereas SO may be associated with noradrenergic phenotypic expression. Moreover, there is a reciprocal relationship between SP and SS expression by sympathetic neurons analogous toe previously described relationship between noradrenergic and cholinergic expression^17–19.     

In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line

The multipotent embryonal carcinoma (EC) P19 cell line differentiates into neurons, glia and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing grafted P19 cells were studied with intracellular recording and labelling techniques to examine the development of electrophysiological and morphological properties of P19-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned striatum. Cells from 1-week-old grafts had a range of immature electrophysiological characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In contrast, the majority of cells recorded from 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of age, the majority of P19-derived neurons had mature neuronal electrophysiological characteristics including RMP's of −60 mV, Rin's of 100–300 MΩ, and overshooting AP's. Morphologically, P19 derived neurons increase in soma size from 12–15 μ in diameter in 7–14-day-old grafts, to 25–35 μ in diameter in grafts 50–120 days old. Developing neurons exhibited a variety of morphotypes with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the electrophysiology of P19-derived neurons, culminating in mature characteristics closely resembling those of adult rodent hippocampal or cortical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development.     

Effects of nerve growth factor on the survival of primary cultured adult and aged mouse sensory neurons

This study investigated the effects of exogenous nerve growth factor (NGF) on the survival and differentiation in primary culture of sensory neurons isolated from adult (6 months) and aged (2 years) mice. For neurons prepared from adult mice, a concentration effect was evident during a 2 week culture period: Neuronal counts in cultures supplemented with 25 and 50 ng/ml NGF did not differ significantly from those of control cultures without exogenous NGF or those with anti-NGF included in the culture medium, whereas cultures supplemented with either 100 or 200 ng/ml NGF contained higher numbers of neurons throughout the culture period. Cultures prepared from aged mice contained less neurons than those from adult mice, although those supplemented with 100 ng/ml NGF retained higher neuronal numbers than cultures from aged mice which did not receive exogenous NGF. Neuronal diameters were measured to investigate whether specific subpopulations of neurons were more dependent on NGF; the results indicate that neurons of a medium-larger diameter were more prevalent than cells with a smaller diameter following NGF administration. A shape index was calculated for each culture regimen; with longer culture periods a higher proportion of spindle-shaped neurons was observed. © 1993 Wiley-Liss, Inc.     

GABAergic immunoreactivity is predominant in neurons derived from expanded human neural precursor cells in vitro

Neural precursor cells have been previously isolated from the developing human nervous system and their properties studied both in vitro and in transplantation paradigms in vivo. However, their ability to differentiate into neurons of different neurochemical phenotypes remains poorly defined. In this study, the default in vitro neuronal differentiation of hENPs derived from five different regions of the human embryonic brain (cerebral cortex, striatum, cerebellum, ventral mesencephalon, and spinal cord) was studied after varying periods of time in culture. The results were directly compared to those from similarly prepared murine ENPs. hENPs prepared from all five regions showed a significant reduction in the number of neurons generated at each passage, such that by passage 4 only between 5 and 10% of cells spontaneously adopted a neuronal phenotype after differentiation in vitro. A similar observation was obtained with murine ENPs. hENPs prepared from more caudal parts of the developing neuroaxis generated fewer neurons compared to the more rostral regions. The only neuronal phenotype identified in these cultures was GABA, with 15-60% of the neurons immunopositive for this neurotransmitter. Thus there appears to be important differences between hENPs dependent on region of origin and time in vitro under standard culture conditions, forming decreasing numbers of neurons with increasing time in culture and more caudal sites of harvest, and with the major identifiable neurotransmitter being GABA. Such characterisation is important in the process of learning how to manipulate the neuronal phenotype of these cells.     

Regulation of alternative splicing in the amyloid precursor protein (APP) mRNA during neuronal and glial differentiation of P19 embryonal carcinoma cells

Cell-specific alteration in the splicing of exons 7, 8 and 15 of the amyloid precursor protein gene was investigated in differenting P19 EC cells into neuronal and glial cells. Exons 7 and 8 were skipped in the neuronal state and exon 15 was skipped in the glial state. Expression of U2AF, one of the essential factor for splicing in mammalian cells, was down-regulated during cellular differentiation. The skipping of exon 15 was suppressed in the glial cells transfected with U2AF. Thus, a reduction in U2AF is believed to play a crucial role in glial-specific splicing of the APP gene.     

Purification and culture of adult rat dorsal root ganglia neurons

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Culture density regulates both the cholinergic phenotype and the expression of the CNTF receptor in P19 neurons

The P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The cells mature into functional neurons, as determined by their ability to release neurotransmitters in a Ca²⁺- and depolarization-dependent manner. P19 neurons in culture represent a mixed population in terms of their neurotransmitter phenotype. The cholinergic phenotype of these neurons is modulated by culture density. Cholinergic markers, such as the vesicular acetylcholine transporter, acetyl cholinesterase, and choline acetyltransferase, are expressed in about 85% of the cells in sparse cultures and are largely suppressed at high cell densities. In contrast, glutamate release is enhanced in dense P19 neuronal cultures. The factor mediating the density effect is concentrated exclusively on the cell membrane of P19 neurons and not on the nonneuronal cells, which also differentiate from P19 embryonal carcinoma cells. This membrane-associated component retains its functionality, even after membrane fixation. The downregulation of the cholinergic properties in dense cultures is paralleled by a downregulation of the α subunit of the ciliary neurotrophic factor (CNTF) receptor. Thus, it is suggested that the membrane-associated factor, which mediates the density effect, downregulates the cholinergic phenotype by inhibiting the responsiveness of these neurons to CNTF. We further suggest that the P19 cell line can serve as a model system for the study of neurotransmitter phenotype acquisition and plasticity throughout neuronal differentiation.     

Stimulation of non-neuronal cell proliferation in vitro by mitogenic factors present in highly purified sympathetic neurons.

Sympathetic neurons have been demonstrated to contain one or more mitogens which are active on highly purified non-neuronal cells cultured in medium containing an optimal concentration of fetal calf serum. Neurons and homologous non-neuronal cells were separated by a method recently developed in this laboratory. The highly purified neurons were either sonicated or homogenized prior to addition to nonneuronal cultures. The presence of neuronal sonicate (1) greatly stimulated [3H]thymidine incorporation into acid-precipitable macromolecules without altering the soluble [3H]thymidine pool, (2) increased both the fraction of non-neuronal cells which took up [3H]thymidine and the density of labeling as observed by autoradiography, and (3) increased the number of cells present in treated cultures after 40 h. The enhancement of [3H]thymidine incorporation was dose-dependent and did not involve cyclic AMP. Addition of neuronal sonicate also caused marked non-neuronal cell elongation which resulted in the elaboration of very long cell processes. The active factor(s) in the neuronal sonicate were partially heat-labile. Norepinephrine was ruled out as a possible mitogenic factor.     

A comparison of two factors affecting the proliferation of non-neuronal (glial) cells in vitro

Earlier studies have shown that the proliferation of sympathetic non-neuronal cells in vitro can be stimulated either by direct contact with growing neurons or by addition of sonicated neurons of the same type to the culture medium. Several lines of evidence presented herein suggest that intact neurons and neuronal sonicate probably stimulate [³H] thymidine incorporation by disticctly different mechanisms. First, mitogenic factors are present in sonicates of cell types (fibroblasts and non-neuronal cells) which do not stimulate non-neuronal cell proliferation when added asintact cells. Second, neuronal sonicate and intact neurons differ in the types of cells which are responsive no their mitogenic influence. Third, intact neurons do not appear to stimulate non-neuronal cell proliferation by the same mechanism as that of neuronal sonicate.Further similarities between stimulation of non-neuronal cell proliferation in vitro and reactive gliosis in vivo are discussed.     

Enhanced neuronal differentiation in a three-dimensional collagen-hyaluronan matrix

Efficient 3D cell systems for neuronal induction are needed for future use in tissue regeneration. In this study, we have characterized the ability of neural stem/progenitor cells (NS/PC) to survive, proliferate, and differentiate in a collagen type I-hyaluronan scaffold. Embryonic, postnatal, and adult NS/PC were seeded in the present 3D scaffold and cultured in medium containing epidermal growth factor and fibroblast growth factor-2, a condition that stimulates NS/PC proliferation. Progenitor cells from the embryonic brain had the highest proliferation rate, and adult cells the lowest, indicating a difference in mitogenic responsiveness. NS/PC from postnatal stages down-regulated nestin expression more rapidly than both embryonic and adult NS/PC, indicating a faster differentiation process. After 6 days of differentiation in the 3D scaffold, NS/PC from the postnatal brain had generated up to 70% neurons, compared with 14% in 2D. NS/PC from other ages gave rise to approximately the same proportion of neurons in 3D as in 2D (9-26% depending on the source for NS/PC). In the postnatal NS/PC cultures, the majority of betaIII-tubulin-positive cells expressed glutamate, gamma-aminobutyric acid, and synapsin I after 11 days of differentiation, indicating differentiation to mature neurons. Here we report that postnatal NS/PC survive, proliferate, and efficiently form synapsin I-positive neurons in a biocompatible hydrogel.     

Astroglia genesis in vitro: distinct effects of retinoic acid in different phases of neural stem cell differentiaion

In the developing CNS, the manifestation of the macroglial phenotypes is delayed behind the formation of neurons. The “neurons first – glia second” principle seems to be valid for neural tissue differentiation throughout the neuraxis, but the reasons behind are far from clear. In the presented study, the mechanisms of this timing were investigated in vitro, in the course of the neural differentiation of one cell derived NE-4C neuroectodermal stem and P19 embryonic carcinoma cells. The data demonstrated that astrocyte formation coincided in time with the maturation of postmitotic neurons, but the close vicinity of neurons did not initate astrocyte formation before schedule. All-trans retinoic acid, a well-known inducer of neuronal differentiation, on the other hand, blocked effectively the astroglia production if present in defined stages of the in vitro neuroectodermal cell differentiation. According to the data, retinoic acid plays at least a dual role in astrogliogenesis: while it is needed for committing neural progenitors for a future production of astrocytes, it prevents premature astrogliogenesis by inhibiting the differentiation of primed glial progenitors.     

Survival of chick embryo sympathetic neurons in cell culture

Changes in neuronal numbers during the development of the chick embryo paravertebral sympathetic nervous system have been examined using cell culture techniques. Early sympathetic ganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequently there is a large decrease in the number of neurons and an increase in the population of non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-day or older embryos than those from earlier stages of development.