Effect of nanostructures on anchoring stem cell-derived neural tissue to artificial surfaces.

OBJECTIVE: Chronic application of brain implants monitoring or modulating neuronal activity are hindered by the foreign body response of the tissue. Topographical modification of implant surfaces may reduce negative tissue response by imitating the structure of the extracellular matrix and therefore affecting the attachment and behavior of neural cells. APPROACH: In our in vitro study, the effect of nanostructuring was investigated on two commercially used neural implant materials: silicon and platinum. The adhesion, survival and arrangement of neural stem cells (NE4C) and microglial cells (BV2) were investigated and compared to nanostructured and flat Si and Pt surfaces using cell viability studies and fluorescent microscopy image analysis. MAIN RESULTS: Our data indicated that neural cells established strong adhesive couplings with each other, instead of binding to the artificial surfaces. SIGNIFICANCE: The phenomena resemble some features of in vivo separation of living tissue from the implanted artificial material, providing an in vitro model for studying immune response.

Deviations in circulating TNFα levels and TNFα production by mononuclear cells in healthy human populations.

OBJECTIVES: Tumor necrosis factor alpha (TNFα) plays a pivotal role in the inflammatory host response. The serum-level of TNFα and the production of TNFα by lympho/monocytes, however, seem to show high individual variations. The goal of the present study was to investigate the variations and inducibility of TNFα-activity in two age-groups of healthy volunteers. METHODS: Sixty elderly, healthy volunteers were studied. These persons were free of malignant diseases, and within three months, they did not have any trauma or inflammatory disease and were not taking any steroids or nonsteroid anti-inflammatory drugs. Thirty young volunteers were also included. Blood samples were taken; lympho/monocytes were separated and cultured with or without endotoxin (LPS) stimulation. Serum and culture supernatant TNFα levels were determined by bioassay using WEHI 164 cells. RESULTS: The results indicated significant individual variations in TNFα levels of healthy volunteers irrespective of age. Subgroups with low, middle, and high serum TNF-levels were distinguished. In about 50% of volunteers with low serum-TNFα activity, LPS stimulation failed to increase the TNFα production by isolated lympho/monocytes. CONCLUSION: Our data suggest a chance to select individuals with enhanced sensitivity for septic complications.

The effect of paternal and maternal history of diabetes mellitus on the development of gestational diabetes mellitus.

BACKGROUND: There is an ongoing debate whether maternal diabetes is a more important risk factor for gestational diabetes (GDM) development than paternal diabetes. AIM: To describe the risk of GDM associated with paternal and maternal diabetes, and to further characterise GDM women with maternal diabetes. SUBJECTS AND METHODS: Case-control study within a population-based GDM screening program in an urban area of Hungary in 2002-2003. All GDM women (no.=133) and an age-matched control group (no.=135) with a mean age of 31 years was evaluated. Blood pressure, anthropometric data, and blood glucose values from a 75 g Oral Glucose Tolerance Test (OGTT) were recorded at 24-28 weeks of gestation. Family history data were by self-report. RESULTS: Known paternal diabetes was not related to GDM risk [odds ratio (OR) 0.83, 95% confidence interval (CI) 0.35-2.00]. Known maternal diabetes (OR 2.90, 95% CI 0.99-8.49) and diabetes in the maternal line (OR 2.83, 95% CI 1.16-6.89) were both related to GDM after adjustment for body mass index (BMI). GDM women with known maternal diabetes had a higher BMI, 31.6 [9.1] kg/m2 median [interquartile range], than GDM women with or without diabetes in the maternal line, 26.1 [4.9] and 26.3 [6.1] kg/m2, respectively, while figures for fasting glucose during OGTT were 5.2 [0.7] vs 4.4 [1.1] vs 4.9 [0.8] mmol/l respectively (all p<0.05). CONCLUSIONS: Maternal history of diabetes and history of diabetes in the maternal line seems to be a stronger predictor of GDM than paternal history.

A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells.

Cell adhesivity is a basic biological principle, which provides mechanisms for construction of multicellular organisms, tissue genesis, migration and individual cell survival. In vivo, the cell adhesive environment is provided by extracellular matrix molecules, neighboring cell surfaces and soluble factors delivered either by tissue cells or by blood circulation. The exact molecular composition of the microenvironment of a cell is not properly understood. The nondefined molecular composition of "native" adhesive components hinders their application when defined culture conditions are necessary, as, for an example, growing human cells for further clinical application. Applying large, substrate-coating molecules as backbones for carrying specific adhesive peptide motifs provides a relatively cheap, reproducible, and chemically defined group of synthetic adhesion molecules. Here, we report on the design, synthesis, and testing of a novel cyclic RGD-containing coating material, which promotes initial attachment, spreading, survival, and proliferation of a number of different cell types. The potent adhesive polypeptide-brush, composed of poly[Lys(DL-Ala(m))] branched chain polypeptide (AK) and multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) pentapeptide prevents anoikis and supports cell attachment in the absence of serum or other biological additives. The defined conditions for cell maintenance make this material a promising candidate for coating artificial cell substrates even for therapeutic applications.

Different behaviour of implanted stem cells in intact and lesioned forebrain cortices.

Cell-replacement therapy promises a useful tool to regenerate compromised brain tissue, but the interaction between grafted cells and host tissues is not well understood. In these studies, the fates of neuroectodermal stem cells were compared in 'healthy' or damaged mouse forebrains. One-cell derived, fluorescent GFP-4C neural stem cells were implanted into normal and cold-lesioned mouse cortices. The fates of implanted cells were followed by histological and immunocytochemical assays for a 55-day postimplantation period. Cells were recultivated from lesioned cortices and characterized by cell cycle parameters, chromosome numbers, immunocytochemical markers and in vitro inducibility. Their intracerebral fates were checked upon re-implanting into 'healthy' mouse brain cortices. GFP-4C cells, giving rise to neurones and astrocytes upon in vitro induction, failed to differentiate in either normal or lesioned cortical tissues. The rate of proliferation and the length of the survival, however, depended on the host environment, markedly. In intact cortices, implanted cells formed compact, isolated aggregates and their survival did not exceed 4 weeks. In compromised cortices, GFP-4C cells survived longer than 8 weeks and repopulated the decayed region. The morphology, viability, immunocytochemical properties, in vitro inducibility and chromosome number of cells recultivated from lesioned cortices were identical to those of the master cells. Long-term survival and repopulating capability were due to signals present in the lesioned, but missing from the intact cortical environment. The results underline the importance of host environment in the fate determination of grafted cells and emphasize the need to understand the 'roles' of recipient tissues for potential cell-replacement methodologies.

Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.

Astroglial cells are essential components of the neurogenic niches within the central nervous system. Emerging evidence suggests that they are among the key regulators of postnatal neurogenesis. Although astrocytes have been demonstrated to possess the potential to instruct stem cells to adopt a neuronal fate, little is known about the nature of the glia-derived instructive signals. Here we propose that all-trans retinoic acid, one of the most powerful morphogenic molecules regulating neuronal cell fate commitment, may be one of the glia-derived factors directing astroglia-induced neurogenesis. According to data obtained from several complementary approaches, we show that cultured astrocytes express the key enzyme mRNAs of retinoic acid biosynthesis and actively produce all-trans retinoic acid. We show that blockage of retinoic acid signaling by the pan-RAR antagonist AGN193109 prevents glia-induced neuron formation by noncommitted stem cells. Therefore, we provide strong in vitro evidence for retinoic acid action in astroglia-induced neuronal differentiation.

Electrophysiological characterization of neural stem/progenitor cells during in vitro differentiation: study with an immortalized neuroectodermal cell line.

Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.

SCL, GATA-2 and Lmo2 expression in neurogenesis.

SCL, Lmo2 and GATA factors form common transcription complexes during hematopoietic differentiation. The overlapping expression of SCL with GATA-2 and GATA-3 in the developing brain indicated that these factors might collaborate also in the course of neural tissue differentiation. The expression pattern of Lmo2 in the developing CNS, however, is not well understood. Here, we show that neural cells in the early embryonic chick mid- and hindbrain express SCL and GATA-2, while Lmo2 is expressed only in vascular elements. The lack of Lmo2 transcripts in neural cells demonstrated that SCL and GATA-2 cannot form common complexes with Lmo2 in the developing brain. In the course of neural tissue genesis, GATA-2 mRNA appeared prior to the SCL transcript. While GATA-2 expression decreased with maturation, SCL expression persisted at a high level also in post-neurogenic periods. The temporal pattern of SCL and GATA-2/3 expression was investigated also in vitro, in the course of induced neurogenesis by NE-4C neural stem cells. While GATA-2 expression increased from the very beginning of differentiation, SCL expression appeared only in more differentiated cells expressing proneural genes. GATA-3 expression, on the other hand, was detected only in advanced stages of the neuronal maturation, which were characterised by the activation of the Math2 neuronal gene. Similarly to the hematopoietic differentiation, GATA-2 expression precedes the activation of both SCL and GATA-3, and may play roles in the activation of the SCL gene in neuronal development. In contrast to hematopoietic differentiation, however, our results failed to demonstrate co-assembling of GATA factors or SCL with Lmo2. While overlapping expression of GATA-2/3 and SCL was detected, Lmo2 activation could not be demonstrated in neural cells in the investigated period of neuronal development.

Fate of cloned embryonic neuroectodermal cells implanted into the adult, newborn and embryonic forebrain.

NE-4C, one-cell derived neuroectodermal stem cells expressing a reporter gene--green fluorescent protein (GFP) or heat-resistant alkaline phosphatase (PLAP)--or prelabeled with bromodeoxyuridine (BrdU) were implanted into the forebrain of adult, new-born and fetal mice and into the mid- and forebrain vesicles of early chick embryos. The fate of implanted cells in the mouse and chick hosts was followed up to 6 and 2 weeks, respectively. Neural differentiation was monitored by detecting the expression of neuron-specific markers and GFAP. NE-4C cells integrated into the early embryonic brain tissue and developed into morphologically differentiated neurons. The same cells produced expanding tumor-like aggregates in the newborn forebrain and were expelled from the adult forebrain parenchyma. In the adult brain, long-term survival and integration of stem cells were revealed only in neurogenic zones. The data suggest that noncommitted, proliferating neuroectodermal progenitors can integrate into the brain tissue at time and site of tissue genesis.

Role of gamma-aminobutyric acid in early neuronal development: studies with an embryonic neuroectodermal stem cell clone.

gamma-Aminobutyric acid (GABA) has been known to function as an autocrine/paracrine signal molecule in addition to its well-known inhibitory neurotransmitter function. Studies on the developing brain and on primary brain cell cultures provided evidence for a variety of GABA functions in periods preceding the formation of synapses. The exact role of GABA in the early neural development, however, is still not well understood. In this study, one-cell-derived NE-4C neuroectodermal stem cells were induced to form neurons and astrocytes in vitro, and the role of GABA was investigated in defined phases of neurogenesis. Noninduced NE-4C cells contained GABA, expressed GABA(A)R alpha subunits, and carried functional GABA(A) ion channels. A moderate cytoplasmic GABA content was detected during the entire period of differentiation. By the time of the formation of differentiated neurons, neuron-like cells with both high and low GABA content were clearly distinguishable. HPLC analysis indicated that NE-4C cells released GABA into their fluid environment during all stages of neuronal development. By using the patch-clamp technique, GABA-evoked currents were recorded during the entire proliferation/differentiation period, whereas a GABA-evoked increase in intracellular Ca(2+) was detected only during the maturation of postmitotic neuronal precursors. Bicuculline blocked both the ion currents and the [Ca(2+)](i) increase in response to GABA. Neuron formation was facilitated by GABA through GABA(A) ion channels during postmitotic differentiation, but not earlier during the phases of cell fate commitment. Although the data clearly demonstrate an early responsiveness to GABA, understanding the significance of GABA influence in early neural cell fate decisions will require further investigation.

Enhancing effect of zinc on astroglial and cerebral endothelial histamine uptake.

We have studied the effect of zinc ion on the uptake of histamine (HA) into cultured astroglial and cerebral endothelial cells and established that Zn(2+) enhances the uptake of the amine dose-dependently and in remarkable extents by increasing the V(max) to about 3-fold (from 3.25 +/- 0.42 to 8.50 +/- 0.97 pmol/mg protein/min in astroglial cells) without altering the K(M) (0.20 +/- 0.03 microM) significantly. The stimulatory effect of zinc ion showed strong sensitivity for VUF 8407, an inhibitory compound of astroglial and cerebral endothelial uptake of HA. In the presence of 20 microM VUF 8407 the zinc-enhanced uptake was reduced by about 50% in both cell types. Binding measurements revealed increased capacities of the zinc-exposed HA binding (B(max)= 0.41 +/- 0.05 increased to 1.21 +/- 0.16 pmol/mg protein in astroglial membranes and B(max) = 0.25 +/- 0.03 enhanced to 1.05 +/- 0.12 pmol/mg protein in cerebral endothelial membranes) but statistically unchanged affinity of the ligand for HA carrier (K(D) values calculated as 35.2 +/- 3.4 nM and 45.1 +/- 3.8 nM for astroglial bindings; whereas 25 +/- 2.1 nM and 30 +/- 2.6 nM for cerebral endothelial bindings of the amine). The compound VUF 8407 reduced the B(max) of zinc-exposed HA binding of astroglial membranes but did not modify the K(D) of the zinc-exposed membrane significantly. The ex vivo experiments confirmed our in vitro findings; an i.c.v. dose of 0.4 micromol/kg ZnSO(4,) 24 hr after the injection, enhanced the uptake of [(3)H]HA into dissociated hypothalamic and cerebellar cells to about 2- and 3-fold, respectively. Present data clearly showed that zinc exposures enhance the astroglial and the cerebral endothelial uptake of HA in vitro and it might be considered that zinc produces similar effects in vivo. Free zinc may participate in the regulation of the extraneuronal HA concentration and this metal ion (endogenous or exogenous) might be favored in the removal of the amine from the interstitial space especially in conditions with relatively high HA.

GABA signalling during development: new data and old questions.

In addition to being the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to play a morphogenetic role in embryonic development. During the last decade, considerable progress has been made in elucidating the molecular mechanisms involved in GABA synthesis and biological action. The present review is an attempt to summarise recent results on the ontogeny of the different components of embryonic GABA signalling with an emphasis on the synthesis of GABA by different molecular forms of glutamic acid decarboxylase (GAD).

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

Cultured astrocytes react to LPS with increased cyclooxygenase activity and phagocytosis.

Phagocytosis and prostaglandin E(2) production were investigated in purified cultures of perinatal rat forebrain astrocytes. Light and electron microscopic data indicated that astrocytes respond to bacterial endotoxin, lipopolysaccharide (LPS) by increased phagocytosis and by activating the cyclooxygenase enzyme-pathway. LPS-inducible phagocytosis of astrocytes was demonstrated by electron microscopic studies on colloidal gold uptake and by photometric determination of fluorescent bead ingestion. The internalisation of fragments of the plasma membrane was shown by histochemical detection of membrane-bound ecto-ATPase activity within intracellular vesicles. Activation of the cyclooxygenase pathway, a characteristic reaction of immune cells under inflammatory conditions, was also detected in astroglial cells upon treatment with LPS. The increased prostaglandin E(2) (PGE(2)) production by astrocytes in response to LPS was reduced by the non-steroid anti-inflammatory drug, indomethacin. Our data indicate that astrocytes display some tissue-protective reactions in response to inflammation inducing factors, even in the absence of peripheral immune cells or central microglia. The role of inducible astrocytic phagocytosis in a non-immune protection-pathway is discussed.

Proliferative and migratory responses of astrocytes to in vitro injury.

An in vitro "scratch-wound" model was used to evoke and investigate some astroglial responses to mechanical injury. The changes in the morphology, locomotion, and proliferation of injured astrocytes were analysed under culture conditions devoid of blood-derived cells responsible for activating the inflammatory cascade. The rate of proliferation was determined by immunocytochemical detection of BrdU-incorporating cells located next to or far from the wound. The motility of individual cells and the mass-advancement of cell-assemblies were monitored by computer controlled video-microscopy both in injured monolayers and in preparations of single cells or aggregates of astrocytes. The large sets of digitalized data allowed a reliable statistical evaluation of changes in cell positions providing a quantitative approach for studies on dynamics of cell locomotion. The results indicated that cultivated astrocytes respond to injury (1) with enhanced nestin immunoreactivity at the expanding processes, (2) with increased mitotic activity exceeding the rate caused by the liberation from contact inhibition, but (3) without specific, injury-induced activation of cell locomotion. Some advantages and drawbacks of "scratch-wound" models of astrocytic responses to mechanical injury are presented and discussed.

Dynamics of cell aggregation during in vitro neurogenesis by immortalized neuroectodermal progenitors.

Early events of in vitro neuronal development were studied by inducing neuron formation in a neuroectodermal cell line, NE-4C/A3, derived from the embryonic forebrain vesicles of p53-deficient mice. Neuronal differentiation was initiated by treating the cells with all-trans retinoic acid (RA). By the second day of RA treatment compact cell aggregates were formed. The first signs of neuronal cell fate decision were revealed inside the aggregates. To elucidate the process of aggregate formation, the dynamics of cell clustering and the migration of individual cells were investigated by a novel computer-controlled videomicroscopic system. Besides real-time observation of cell motility, the system allowed statistical analysis of large sets of data providing quantitative evaluation of cell locomotion during an early, critical phase of RA induced neuron formation. The results showed that chemoattractants did not play a principal role in cell aggregation. Retinoic acid, on the other hand, was found to cause a rapid decrease in the average migratory velocity without changing the randomness of migratory routes. The data indicated that aggregation was facilitated by increased cohesion upon incident collision of randomly encountering cells. The resulting compact cell clusters provided the structural conditions for contact communication apparently needed for the neuronal differentiation of NE-4C/A3 cells.

Calcium current activated by potassium ions in voltage-clamped rat hippocampal pyramidal neurones.

1. Neuronal activity results in local elevation of extracellular K+ concentration ([K+]o). 2. Using the patch-clamp technique in the whole-cell configuration, we investigated whether extracellular K+ activates non-voltage-operated Ca2+ channels in pyramidal cells cultured from rat embryonic hippocampi. 3. K+ (12 mM) reversibly activated a sustained inward current at a holding potential of -100 mV. Membrane conductance and variance of noise were significantly increased by K+. This current could be observed at membrane potentials negative to +60 mV. 4. Inhibitors of inward rectifier K+ channels and hyperpolarization-induced cation current reduced the current only at potentials negative to -50 mV. 5. The K+-induced current was activated in Na+-free but not in Ca2+-free medium, did not depend on cytosolic [Cl-], and was blocked by Cd2+ but not by organic channel inhibitors. 6. Half-maximal activation of the current (at -100 mV) was attained at [K+]o approximately 20 mM. 7. The current is similar to Igl, a K+-induced Ca2+ current described in glomerulosa cells. It was also present in pyramidal cells from prefrontal cortex but not in hippocampal bipolar and glial cells. 8. Activation of K+-induced Ca2+ current may elevate cytoplasmic [Ca2+] at [K+]o levels which are insufficent to activate voltage-dependent Ca2+ channels.