Non-neuronal Cultures Research Articles

Method of obtaining of nonneuronal cultures of astrocytes, derivated from human mesenchymal stem cells

Objective. Elaboration of method for obtaining of nonneuronal cultures of astrocytes, derivatives from human mesenchymal stem cells of various origin. Materials and methods. Mesenchymal stem cells were obtained from human peripheral and umbilical blood in accordance to standard procedure, using the immune-magnet separation method and distributed into three groups, depending on primary material of origin and the cellular differentiation technique applied: the cells, which were obtained from the umbilical blood and if our modified technique of differentiation (CBMSC) used, and the cells, which were obtained from peripheral blood and the differentiation technique used was standard (PBMSC-1) and modified (PBMSC-2) protocols. As primary antibodies standard sets β- III-Tubulin (Sigma, USA), GFAP, Nestin (Abcam) served, while as secondary antibodies - іmmunoglobulins G, conjugated by activators CNTF, BMP2/4 and FGF1 (Sigma, USA) in accordance to instruction. For visualization of the results obtained microscope EVOS FL LIFE TECHNOLOGIES was used with obtaining of view under ×40 objective. Results. From majority of cells from the PBMSC-1 group the answer was not obtained, while from approximately 80% of all stimulated cells of the PBMSC-2 group the answer was obtained, and the both lines content consisted of 20 - 30% of GFAP-positive cells. This makes background to consider, that expression of GFAP only is insufficient to identify the mature and functional astrocytes. Conclusion. Astrocytes from group PBMSC-1 have had answered very rarely on ATP-stimulation, while astrocytes from groups CBMSC and PBMSC-2 demonstrated characteristic answer. Thus, there was demonstrated, that morphologically close astrocytic lines and single cells owes different functional profile.

Stimulation of cyclic GMP production via a nitrosyl factor in sensory neuronal cultures by algesic or inflammatory agents.

Capsaicin stimulates cyclic GMP production via nitric oxide (NO) (or another nitrosyl factor) in dorsal root ganglion (DRG) neurons maintained in culture. The purpose of the present study was to characterize further capsaicin stimulation of cyclic GMP production in DRG cells maintained in culture, investigate other algesic and/or inflammatory agents for effects on cyclic GMP production, and examine cells responsible for NO production and cyclic GMP production. Capsaicin stimulation of cyclic GMP production in DRG cells was dose dependent, receptor mediated, and attenuated by hemoglobin. Prostaglandin E2, substance P, and calcitonin gene-related peptide did not affect basal, capsaicin-stimulated, or bradykinin-stimulated cyclic GMP production. Other inflammatory or algesic agents, including serotonin, histamine, ATP, glutamate, aspartate, and NMDA, did not affect cyclic GMP production. Pretreatment of DRG cells with lipopolysaccharide increased basal cyclic GMP production in neuronal but not in nonneuronal cultures and facilitated stimulation of cyclic GMP production by L-arginine. Capsaicin pretreatment of neuronal DRG cultures, which destroys capsaicin-sensitive (small diameter) afferent neurons, attenuated capsaicin- and bradykinin-stimulated cyclic GMP production but did not affect basal or sodium nitroprusside-stimulated cyclic GMP production. These results indicate that capsaicin elicits production of a nitrosyl factor via capsaicin-sensitive (small diameter) neurons. Capsaicin evoked cyclic GMP production in nonneuronal DRG cultures in the presence but not in the absence of apposed neuronal DRG cultures. Overall, these findings suggest that specific exogenous (or endogenous) substances may stimulate production of a nitrosyl factor(s) by a subset of DRG neurons, and nitrosyl factors produced by these neurons may affect cyclic GMP production in neighboring neuronal or non-neuronal cells.

Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons.

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.

Modulation of neuronal choline acetyltransferase activity by factors derived from cultures of non-neuronal cells from the CNS

We have found that cholinergic neurons in spinal cord-dorsal root ganglion cultures derived from E12–E13 mouse embryos are sensitive, as measured by changes in choline acetyltransferase activity, to factors secreted by non-neuronal cells derived from the same tissue at an identical developmental stage. Conditioned medium was produced by incubating non-neuronal cultures for 4 days in defined medium. The cholinotrophic activity present in the conditioned medium had a molecular weight of greater than 50,000 as determined by ultrafiltration and bound wheat germ lectin and heparin sepharose. Total RNA isolated from the non-neuronal cells, used to produce the conditioned medium, was translated in frog oocytes. Conditioned medium from the injected oocytes was also found to contain cholinotrophic activity. In contrast, the conditioned medium from water-injected oocytes was inactive.The interaction between the cholinotrophic activity in conditioned medium from frog oocytes and known second messengers was also examined. Dibutyryl cyclic AMP produced a concentrationdependent increase in choline acetyltransferase activity. If a maximal effective dose of dibutyryl cyclic AMP was added in conjunction with a maximal effective dose of conditioned medium from oocytes injected with total RNA a nearly additive response was noted. In contrast, the phorbol ester, phorbol 12-myristate 13-acetate, produced a biphasic change in the level of choline acetyltransferase activity; with lower doses stimulating and higher doses inhibiting the enzyme activity. When conditioned medium from oocytes injected with non-neuronal cell RNA was added in conjunction with the phorbol ester a decrease in the physiological response was noted.

Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide.

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.

Iron uptake by glial cells.

Dynamic studies of iron metabolism in brain are generally unavailable despite the fact that a number of neurologic conditions are associated with excessive accumulation of iron in central nervous tissue. Cortical non-neuronal (glial) cultures were prepared from fetal mouse brain. After 13 days the cultures were exposed to radiolabeled iron. Brisk and linear total iron uptake and ferritin iron uptake occurred over 4 hours. When methylamine or ammonium chloride was added, (both known inhibitors of transferrin iron release because of their lysosomotropic properties), total iron uptake was diminished. Further studies indicated that methylamine inhibits glial cell ferritin iron incorporation. Glial cell iron transport is similar to previously reported neuronal cell iron transport (1) but glial cell iron uptake proceeds at a faster rate and is more susceptible to the inhibition of certain lysosomotropic agents. The data reinforces the likelihood that iron uptake by nervous tissues is transferrin-mediated.

Cerebellar astroglial cells in primary culture: Expression of different morphological appearances and different ability to take up [ 3H] d-aspartate and [ 3H]GABA

In non-neuronal cultures of cells dissociated from postnatal rat cerebellum astrocytes, identified by the presence of the marker protein glial fibrillary acidic protein (GFAP), displayed two distinct morphological forms. One class was stellate in shape with radially distributed fine processes, while the other class was more varied in shape being polygonal or elongate. [ 3H]thymidine incorporation experiments revealed that cells of both morphologies were able to incorporate this nucleoside, suggesting the capacity for both cell types to undergo cell division. An autoradiographic study of the uptake of [ 3H] d-aspartate and [ 3H]GABA revealed that whilst the two classes of astrocytes took up the aspartate to apparently the same extent, only the stellate cells were found to be heavily labeled following incubation with [ 3H]GABA. A study of the cultures over a 12-day period showed that there was a disappearance of the stellate astrocytes. The time of disappearance was found to be dependent upon the initial plating density; the stellate morphology was apparent longer in lower density cultures. Time lapse studies suggested that one of the reasons for the disappearance of the stellate cells might be that in fact they underwent a change in shape following certain cell-cell interactions, but cell death also has to be considered as a further possibility. The relationships between the two classes of astroglial cells in these cultures is not yet clear. The possibilities are that they represent two different types of astrocytes, or just one type at different stages of differentiation, or maybe a combination of the two possibilities.

Lactate uptake and release in the presence of glucose by sympathetic ganglia of chicken embryos and by neuronal and nonneuronal cultures prepared from these ganglia.

Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14-15 days old. The chains, typically containing 30-40 micrograms of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6-17 mM glucose, various concentrations of lactate, and either [U-14C]lactate, [1-14C]glucose, or [6-14C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5-6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with Vmax = 360 mumol/g protein/h and Km = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO2. Glucose uptake averaged about 108 mumol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 mumol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO2 and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10-20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.

Conditioned medium from cultures of embryonic neurons contains a high molecular weight factor which induces acetylcholine receptor aggregation on cultured myotubes

The developmental mechanisms involved in the formation of stable arrays of postsynaptic neurotransmitter receptors near sites of neurotransmitter release are essentially unknown. However, several recent studies have shown that cells or tissues of neural origin produce macromolecular factors which induce an increase in the number of acetylcholine (ACh) receptors or the number of receptor aggregates on cultured embryonic myotubes. We have tested primary cultures of embryonic neurons and other cell types for the release of an ACh receptor aggregation factor. Conditioned medium from the cultures was applied to cultures of embryonic rat myotubes for 1 day; ACh receptors on the myotubes were stained with tetramethylrhodamine-labeled alpha-bungarotoxin and ACh receptor aggregation activity, defined as the change in the number of receptor aggregates per myotube, was assayed. Aggregation activity with a molecular weight greater than or equal to 50,000 was released by cultures of neurons from sympathetic ganglia, dorsal root ganglia, spinal cord, and cerebellum. Little or no activity was released by glial or other non-neuronal cultures. Release of aggregation activity by different neuronal cell types varied by up to an order of magnitude; however, this variation was not well correlated with the differences in ACh synthesis. The factor(s) in neuronal cell conditioned medium induced a rearrangement of pre-existing receptors at the cell surface, and its action was not dependent on new protein synthesis. The results of this study are consistent with the idea that one or more receptor aggregation factors secreted by neurons are involved in the organization of neurotransmitter receptors during synapse formation in vivo.

Preparation and partial characterization of highly purified primary cultures of neurons and non-neuronal (glial) cells from embryonic chick cerebral hemispheres and several other regions of the nervous system

Purified cultures of neurons and non-neuronal (glial) cells were prepared from the cerebral hemispheres of 10-day chick embryos by a method previously used for embryonic chick sympathetic ganglia 16. This technique separates these cell types on the basis of both: (1) differences in the adhesiveness of neurons and non-neuronal cells to a collagen substrate; and (2) the capacity of neurons to form homotypic aggregates. Purity of the cerebral non-neuronal cultures was determined to be ≥ 99.5% by microscopic examination, while that of the cerebral neuronal cultures was only 92%. Modification of the technique by periodic redissociation of the neuronal aggregates during cell separation increased the purity of the neuronal cultures to ≥ 97% as determined both by microscopic examination and by measurement of levels of butyrylcholinesterase, an enzyme present in the non-neuronal cells. Highly purified cultures of neurons were also prepared from the optic lobes of 10-day chick embryos (≥ 98%), but attempts to obtain non-neuronal cultures of reasonable density from this tissue were unsuccessful. In addition, highly purified non-neuronal cultures (≥ 99.5%) were prepared from the dorsal root ganglia of 12-day chick embryos, but cultures enriched with dorsal root neurons could only be partially purified (82%). Specific activity of butyrylcholinesterase in cerebral non-neuronal cells was found to vary inversely with the density of non-neuronal cells.

Neuronal stimulation of non-neuronal (glial) cell proliferation: Lack of specificity between different regions of the nervous system

Purified and recombined primary cultures of neurons and non-neuronal (glial) cells were prepared from the cerebral hemispheres of 10-day chick embryos. A̧ddition of cerebral neurons to homologous non-neuronal cultures stimulated incorporation of [ 3H]thymidine by 2.8-fold and increased the frequency of labelling of the non-neuronal cells by 3.5-fold as visualized by autoradiography. In contrast, cerebral neurons did not stimulate the proliferation of either embryonic chick fibroblasts or leptomeningeal cells. Furthermore, addition of either fibroblasts or extra non-neuronal cells did not stimulate non-neuronal cell proliferation. These data demonstrate for the first time that neurons isolated from the central nervous system can selectively stimulate the proliferation of homologous non-neuronal cells. Cell proliferation was also studied in cultures containing both homologous and heterologous combinations of neuronal and non-neuronal cells prepared from several different portions of the nervous system (cerebral hemispheres, optic lobes, sympathetic ganglia, and sensory ganglia). Addition of embryonic neurons stimulated non-neuronal cell proliferation in all cell combinations. Thus, neurons isolated from one region of the nervous system can stimulate the proliferation of non-neuronal cells isolated from other neural regions.

GABA uptake and release in purified neuronal and nonneuronal cultures from chick embryo retina

Uptake and release of γ-aminobutyric acid (GABA) have been studied using glia-free, purified neuronal cultures from 8-day chick embryo retina. At 3 days in vitro 65% of the neurons showed high-affinity GABA uptake. These neurons appeared heavily labeled after incubation in 5 × 10 −8 M [ 3H]GABA, but no labeling was detected when the incubation was carried out at 4°C, or in the absence of Na +ions. Diaminobutyric acid (DABA) also blocked completely the neuronal uptake of GABA, while β-alanine was ineffective at similar concentrations. At 6 days in vitro Na +-and temperature-dependent GABA uptake was present in 50% of the neurons. In addition, in 80% of those neurons the uptake was insensitive to DABA or β-alanine, whereas in the remaining 20% it was blocked by DABA but not by α-alanine. Important developmental changes were also found in the capacity of the neurons to release GABA into the medium. Spontaneous GABA release (i.e. that taking place in regular medium, containing 5 mM K +) was higher at 3 than at 6 days in vitro. However, increasing the K + concentration to 56 mM had minimal effects at 3 days in vitro, but induced a 2 to 3-fold increase in GABA release at 6 days in vitro. This K +-induced release appeared to be Ca 2+-dependent, since it was substantially reduced in the presence of 10 mM Co 2+. Cultures containing a confluent monolayer of nonneuronal flat cells were generated by seeding retinal cell suspensions on poorly adhesive substrate. Retina nonneuronal cells showed, during the first 10 days in vitro, a high-affinity mechanism for GABA uptake which was Na +- and temperature-dependent, and was reduced by 85% by DABA but was practically unaffected by β-alanine. This uptake mechanism seemed to be lost towards the end of the second week in vitro, and could not be detected after 21 days culture.

14C]Glucose metabolism in sympathetic ganglia of chicken embryos and in primary cultures of neurons and of other cells from these ganglia.

Metabolism of [1-14C]glucose and [6-14C]glucose was measured in sympathetic ganglia excised from chicken embryos 12-16 days old and in primary cultures of neurons or nonneurons prepared from these ganglia. Some metabolic rates tended to change with the tissue/medium ratio, so this variable had to be controlled. Less C-6 than C-1 od glucose was put out in CO2 by all three types of preparations, indicating operation of the hexosemonophosphate shunt. The C-6/C-1 ratio was greater for the neuronal cultures and for intact ganglia than for the nonneuronal cultures. The C-6/C-1 ratio for the neurons increased with the amount of tissue added to a given volume of incubation medium, in agreement with previous experiments on embryonic dorsal root ganglia (Larrabee, 1978). Per unit of protein, the output of C-1 of glucose in CO2 was higher in both the neuronal and the nonneural cultures than in intact ganglia, whereas that of C-6 was higher in the neuronal cultures and lower in the nonneuronal ones than in the ganglia. The rates of release in lactate of C-1 and C-6 of glucose were 3-5 times higher from both types of cultures than from intact ganglia. The average rates of incorporation of C-1 and C-6 of glucose into tissue constituents were lower in the cultures than in intact ganglia, significantly so for incorporation of C-6 in the nonneuronal cultures.

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 non-neuronal 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.

Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: A new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates

A new method has been developed for the preparation of essentially pure primary cultures of neurons and non-neuronal cells from 11-day embryonic chick sympathetic ganglia. This method utilizes (1) differences in cell-to-substrate adhesiveness between neurons and non-neuronal cells and (2) the capacity of neurons to form homotypic aggragates. The maximum difference in adhesiveness between neuronal and non-neuronal cells occurred when the ganglia were dissociated with trypsin following collection in a salt solution lacking divalent cations. This difference allowed the preparation of highly purified non-neuronal cultures and 85–90% pure neuronal cultures. Intermittent agitation during the period of cell separation markedly increased the purity of the neuronal cultures by (1) inhibiting neuronal but not non-neuronal cell attachment and (2) facilitating the formation of homotypic neuronal aggregates in the supernatant. Neuronal and non-neuronal cultures prepared under these conditions were more than 99% pure on the basis of both morphological and biochemical analyses. Both cell types exhibited attachment efficiences greater than 95% and have been maintained for several weeks in vitro. Thus, completely isolated neuronal and non-neuronal cultures can be prepared and maintained for prolonged periods in the absence of cells of the other type.

Neuronal stimulation of [ 3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl- 3H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [ 3H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [ 3H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [ 3H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [ 3H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [ 3H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.