Morphological and molecular expression patterns of neural precursor cells derived from human fetal spinal cord in two-, three-dimensional, and organoid culture environments.

Recently, interest in three-dimensional (3D) cell or tissue organoids that may, in vitro, overcome not only the practical problems associated with fetal tissue transplantation, but also provide a potential source for the regeneration of injured spinal cords, has been increasing steadily. In this study, we showed that human neural precursor cells (hNPCs) derived from the fetal spinal cord could be incubated in serum free medium at two dimensional (2D), three dimensional (3D) and tissue organoid-systems. Additionally, we investigated morphological changes over time along with the expression of proteoglycans, collagen, or myelin in 2D, 3D and tissue-like organoids. 2D cells exhibited a spindle-shaped morphology with classic hill and valley growth patterns, while 3D cells grew as clusters of undifferentiated cells and cell sheets (tissue organoids) that gradually rolled up like a carpet without forming a circular cell mass. Immunostaining was performed to demonstrate the expression of TUJ-1, MAP-2, GAD 65/67 and ChAT in 2D cells or tissue-like organoids, which stained positively for them. In addition, we observed the immunoreactivity of HNu, NG2, TUJ-1, and GFAP in tissue-like organoids. The organoid culture system studied in our work may be used as therapeutic agents for spinal cord injury (SCI), and as raw materials needed for development of new medicines to improve human responses and cure diseases.

Dose-Dependent Cytotoxicity of Gold Nanoparticles on Human Neural Progenitor Cells and Rat Brain.

Gold nanoparticles (GNPs) are utilized as diagnostic and therapeutic tools to detect and treat human disease. Researchers in the field of nanotoxicity are working to determine the physicochemical properties of nanoparticles that lead to toxicity in an effort to establish safe design rules. In this study, we performed the MTT and terminal transferase dUTP nick end labeling (TUNEL) assays to verify the cytotoxicity of GNPs on rat brain tissue and human neural progenitor cells (NPCs). As results, we observed that GNPs induced apoptosis in NPCs. NPCs were markedly damaged following the administration of 200 µM and 2 mM GNPs, whereas 2 µM GNPs showed slightly increased damage relative to that of the control. In addition, TUNEL-positive cells were densely distributed at regions surrounding the GNP injection site in the brain 7 days after the GNPs injection. During long-term GNPs exposure, TUNEL-positive cells were rarely observed in the cerebral cortex. In this study, we observed that apoptosis increased in proportion to GNP concentrations in the brain and in cultivated NPCs. These result suggest that large GNPs (<100 nm) are toxic and that the cytotoxicity increased as the concentration of GNPs increased in NPCs or in the brain.

Cytotoxicity of gold nanoparticles in human neural precursor cells and rat cerebral cortex.

Nanoparticles are promising tools for the advancement of drug delivery, medical imaging, and as diagnostic sensor. Medical nanodevices should develop miniaturization, because it would be injected into a human body. Gold nanoparticles (GNPs) with different sizes and shapes have therapeutic potential as a result of their small size, robust nature, excellent biocompatibility and optical properties. However, the application of GNPs as medical nanodevices it is necessary to know the biodegradation, biocompatibility, and development of surface coating which avoid the accumulation of nanoparticles. In this study, we carry out an in vitro toxicity and in vivo gene expression study using two kinds of GNPs. We found that GNPs toxicity is dependent on the dose or size administrated after the injected GNPs into the brain, and small particle size GNPs appeared more nestin expression compared to large particle size at short term implantation. These findings of toxicity of GNPs may play an important role in development of in vivo tools for the safety of GNPs.

Cytotoxicity of Gold Nanorods and Nanowires on Cultivated Neural Precursor Cells.

Given the emergence of nanotherapeutics and nanodiagnostics as key tools in today's medicine, it has become of critical importance to define the interactions of nanomaterials with biological systems. The biomedical applications of nanoparticles (NPs) in chemical sensing, biological imaging, drug delivery, photothermal therapy and cancer treatment have been demonstrated. Gold NPs as new biomedical tools are the focus of research due to their ease of synthesis, chemical stability and unique optical properties. Therefore, there is a need to establish the toxicity, side effects and safety of gold NPs for human applications. To study the in vitro cytotoxicity of gold NPs, we performed MTT assay using two types of gold NPs such as gold nanorods (GNRs) and gold nanowires (GNWs). The percentage cytotoxicity of damaged neural precursor cells (NPCs) that were treated with 100 mg GNRs was 97.5±3.9%; and proportion of damaged NPCs following the administration of the same dose of GNWs was 98.8±0.3%. The cytotoxicity of 10 mg GNRs in NPCs was 54.4±8.3%, whereas it was 98.7±0.6% for the same dose of GNWs. Then, to verify that gold NPs induced apoptotic cell death in NPCs, the LIVE/DEAD Viability/Cytotoxicity assay was performed. We observed that cell death of NPCs increased with an increase in quantity of both types of gold NPs. Cell viability assessed the overall dose-dependent toxicity of NPs in cultured cells. As the results suggest, this study demonstrated that treatment with gold NPs resulted in cellular toxicity in a dose-dependent manner in cultured NPCs.

Exogenous GDNF increases the migration of the neural stem cells with no protection against kainic acid-induced excitotoxic cell death in rats.

Glia cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuron types. In this study, we have evaluated the utility of adenovirus-based vectors (Ad) and hippocampal neural stem cells (NSCs) as genetic tools for the delivery of a therapeutic protein, GDNF, in hippocampus tissues damaged by kainic acid (KA)-induced excitotoxicity. The experimental animals were treated with KA 3 days prior to exposure to Ad-GDNF, NSCs, and NSCs infected with Ad-GDNF (Ad-GDNF-NSCs). Seven days after the treatments with Ad-GDNF, NSCs and Ad-GDNF-NSCs, the effects of the treatments were evaluated. GAD-67 labeled cells originating from the transplanted NSCs were observed at increased levels in the Ad-GDNF-NSCs-treated rats as compared to the NSCs-only rats. In situ apoptosis assays showed that the levels of TUNEL-positive cells were slightly, but not significantly, reduced in the Ad-GDNF and Ad-GDNF-NSCs groups, as compared to the saline and NSCs only groups. GDNF expression by NSCs and Ad-GDNF was upregulated as the consequence of adenoviral gene delivery in the NSCs and Ad-GDNF-treated rats, and the transplanted NSCs were shown to have migrated to the hippocampal regions in Ad-GDNF-NSCs rats to a greater degree than in the NSCs-only rats. Furthermore, in the region in which the NSCs were detected, GDNF and GAD-67 expression were increased. These results indicate that the migration and differentiation of NSCs may be associated with the expression of GDNF. However, cell death consequent to KA administration was not prevented by upregulated GDNF and NSCs transplantation. Collectively, our results indicate that GDNF may exert effects on the migration and differentiation of NSCs, but there are no protective properties with regard to excitotoxically damaged hippocampal tissue.

Cultured cambial meristematic cells as a source of plant natural products.

A plethora of important, chemically diverse natural products are derived from plants. In principle, plant cell culture offers an attractive option for producing many of these compounds. However, it is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. To bypass the dedifferentiation step, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Using a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. This notion was further supported by the morphology of CMCs, their hypersensitivity to γ-irradiation and radiomimetic drugs and their ability to differentiate at high frequency. Suspension culture of CMCs derived from Taxus cuspidata, the source of the key anticancer drug, paclitaxel (Taxol), circumvented obstacles routinely associated with the commercial growth of DDCs. These cells may provide a cost-effective and environmentally friendly platform for sustainable production of a variety of important plant natural products.

Performance analysis of the ARCHITECT anti-cyclic citrullinated peptide antibody in the diagnosis of rheumatoid arthritis.

BACKGROUND: Anti-cyclic citrullinated peptide (CCP) antibody is emerging as an important diagnostic marker for rheumatoid arthritis (RA). We evaluated the analytical and diagnostic performance of the ARCHITECT anti-CCP (Abbott Diagnostics), a new fully automated chemiluminescent microparticle immunoassay. METHODS: Serum samples from 69 patients with RA and 86 non-RA patients were used to evaluate the performance of the ARCHITECT anti-CCP assay, and the results were compared with those of EliA CCP (Phadia). The optimal cut-off value was calculated using receiver operating characteristic (ROC) curve analysis. RESULTS: Within-run and total imprecision (%CV) of the ARCHITECT anti-CCP were <6% and good linearity was observed over the claimed range. The areas under the ROC curves for the ARCHITECT anti-CCP and EliA CCP were 0.90 and 0.89, respectively. The sensitivity and specificity were 76.8% and 95.3% for the ARCHITECT anti-CCP and 78.3% and 95.3% for EliA CCP using the manufacturer's cut-off thresholds. Both assays showed sensitivity of 84.1% and specificity of 94.2% using the optimal cut-off values. CONCLUSIONS: The analytical performance of the ARCHITECT anti-CCP was satisfactory and diagnostic performance was comparable to that of EliA CCP. The use of optimal cut-off thresholds can yield higher sensitivity with minimal loss of specificity.

The identification of proteoglycan, collagen and neuron in precursor cells from human fetal spinal cord.

After spinal cord injury (SCI), a loss of myelinating oligodendrocytes and neurons occurs. The functional recovery of injured spinal cords is the principal objective of SCI repair. Cell transplantation may prove beneficial to help replace lost myelin and spinal cord circuitry. In this study, we demonstrated that neural precursor cells (hNPCs) from human fetal spinal cord express three types of proteoglycan proteins-chondroitin sulfate, keratan sulfate, and cartilage proteoglycan (an extracellular matrix detected in normal spinal cord), and non-proteoglycan matrix collagen. Both proteoglycan and collagen evidenced profound immunoreactivity in double-stained cell clusters. In addition, whether or not hNPCs were capable of differentiating into a variety of cells, including GABAergic and cholinergic neurons, were assessed. The differentiated cells of eight passages grown on a monolayer expressed the human nuclear protein (HNu), the progenitor marker nestin, GAD, ChAT, TJU, and MAP-2. These results indicate that hNPCs may prove to be candidate cells for therapeutic SCI strategies.

Mitochondrial DNA in patients with essential tremor.

Essential tremor (ET) is one of the most common of the movement disorders. However, there has been little agreement in the neurological literature regarding diagnostic criteria for ET. It is not clear to what extent ET is associated with defects of mitochondrial DNA. In this study, we analyzed mitochondrial DNA (mtDNA) from the blood cells of the normal and ET patients using the long and accurate polymerase chain reaction (LA-PCR) and PCR. The large deletions were detected within several regions of mtDNA, but were not detected in the D-loop or CO I regions in ET patients. From our study, it is suggested that ET is a disorder showing a deficiency of mtDNA multicomplexes, and it also appears that mitochondrial dysfunction could be one of the causative factors of ET.

Neuroprotection of adenoviral-vector-mediated GDNF expression against kainic-acid-induced excitotoxicity in the rat hippocampus.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. In the present study, we examined the protective roles of adenoviral-vector-delivered GDNF (Ad-GDNF) in the hippocampus damaged by kainic-acid (KA)-induced excitotoxicity using GAD-67 immunoreactivity, immunoblot analysis, behavioral test, 5-bromo-2-deoxyuridine (BrdU) and TUNEL assay. Ad-GDNF was pre-inoculated into the KA-treated rat hippocampus 7 days before KA injection. Ad-GDNF resulted in the suppression of KA-induced tonic-clonic convulsions. In situ apoptosis assay demonstrated a significant reduction in apoptotic cells in the CA3 and dentate hilus regions of the Ad-GDNF-pre-inoculated rats (Ad-GDNF-KA), compared to the KA rats. Striking reductions in the density of GAD-67 neurons were also observed in the CA3 and dentate hilus regions of the KA rats. On the other hand, the number of GAD-67-positive cells was recovered to the control levels in the Ad-GDNF-KA rats. Immunoblot analysis further confirmed that GAD-67 and Bcl-2 expression increased in the Ad-GDNF-KA rats compared to KA rats. Taken together, these results suggest that Ad-GDNF may serve to control KA-induced hippocampal cell loss and behavioral seizure.

The change of the neuron-glia differentiation rate in human neural precursor cells (HPCs) and Ad-BDNF-/-GDNF-infected HPCs following the administration of a neurotoxin.

Neurotrophic factors promote the survival of various neurons, including peripheral autonomic and sensory neurons, as well as central motor and dopamine neurons, and it is expected that they could function as therapeutic agents for neurodegenerative disease. We examined the changes in the neuron-glia differentiation rate in normal human neural precursor cells (HPCs), Ad-BDNF- and Ad-GDNF-infected HPCs following their treatment with 6-OHDA. We isolated the precursor cells from the human fetal midbrain. To investigate the expression of differentiated cell markers within neurons and glia after 6-OHDA-induced toxicity in HPCs, immunocytochemistry was performed. Our results showed that the treatment with 6-OHDA (100, 200, 300, 400 and 500 microM) for 24 h decreased the viability of the HPCs in vitro. Among the growth factors tested, BDNF and GDNF protected the HPCs against 6-OHDA-induced toxicity. Approximately, 5.8+/-2.2% and 0.5+/-0.1% of the HPCs treated with 6-OHDA were positive for the neuron marker, MAP2, and the oligodendrocyte marker, GalC, respectively, while 13.8+/-3.2% and 1.1+/-0.36% of the Ad-BDNF- or Ad-GDNF-infected HPCs treated with 6-OHDA stained positive for MAP2 and GalC, respectively. These results suggest that cocktail therapy using human precursor cells (HPCs) and certain neurotrophic factors (BDNF, GDNF) provide direct protection against 6-OHDA-induced toxicity and has an effect on the differentiation rate.

Apoptosis and nestin expression in the cortex and cultured astrocytes following 6-OHDA administration.

We used the dopaminergic neurotoxicant, 6-hydroxydopamine (6-OHDA), as a tool to characterize the origins of the astrocytic response to injury. Reactive astrocytes were examined by immunocyto- and histo-chemical visualization of nestin protein in the brain and cultivated cells. Following 6-OHDA (dose-dependent) treatment, the expression of nestin-like immunoreactive cells in the corpus callosum and cerebral cortex was increased compared with that of the control animals, indicating that a significant up-regulation of nestin protein occurred in these regions. In the corpus callosum and cerebral cortex, the majority of the nestin-like immunoreactive cells showed a distribution and pattern similar to those of the glial fibrillary acid protein (GFAP)-immunoreactive cells. Double immunofluorescence measurements showed that 100% of the nestin-like immunoreactive cells expressed GFAP-immunoreactive cells, indicating that these nestin-like immunoreactive cells belong to a reactive population of the astrocytes. In this study, we observed the morphological changes in the astrocytes following 6-OHDA administration, demonstrating that 6-OHDA induced injury leads to a rapid and transient up-regulation of nestin-like immunoreactivity in activated astrocytes.

Tamoxifen-induced cell death and expression of neurotrophic factors in cultured C6 glioma cells.

The effects of ( Z)-2[ p -(1,2-diphenyl-1-butenyl)phenoxy]-N ,N -dimethylamine citrate (tamoxifen) on cell survival and the expression of neurotrophic factors (NTF) were investigated in rat C6 glioma cells (C6). C6 cells do not express the estrogen receptor. Cytotoxic effect was detected from 24 h after the treatment with 10 microM tamoxifen and increased with time in a dose-dependent manner. C6 cells treated with tamoxifen also displayed various morphological types such as elliptical, round and aggregated form. As the treatment time increased, the proliferation of C6 cells was reduced remarkably and most of them became the round or aggregated form. To examine the relationship of the expression of NTF and the cytotoxicity of tamoxifen, the mRNA level of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and basic fibroblast growth factor (bFGF) was measured after 24 h treatment with tamoxifen by RT-PCR. The expression of mRNA of BDNF or GDNF in C6 cells treated with various concentrations of tamoxifen was comparable to controls. The expression of bFGF mRNA was significantly reduced in C6 cells treated with 10 or 15 microM tamoxifen. The results suggest that tamoxifen exerts cytotoxic effect on estrogen receptor-negative C6 cells through the inhibition of the transcription of bFGF.

Identification and functional evidence of GABAergic neurons in parts of the brain of adult zebrafish (Danio rerio).

The distribution of GABA-containing neurons was studied in the brain of the adult zebrafish by Nissl staining and immunohistochemistry. GABA immunoreactivity (GABA-IR) was demonstrated in parts of the brain such as olfactory bulb (OB), telencephalon, tectum stratum, and in the hypothalamus. GABA-IR appeared in the area where Nissl-stained cell bodies were abundant. The internal cellular layer of the OB was most densely stained by Nissl staining, and also showed a high level of GABA-IR. The telencephalon and the hypothalamus revealed a similar pattern to the OB in terms of Nissl staining and GABA-IR. However, the distribution and shape of stained cells of the tectum stratum were distinct from those in other regions: Nissl-stained neurons were ubiquitously present throughout all cellular layers including the stratum griseum centrale, the stratum album centrale (SAC), and the stratum periventriculare (SP). However, GABA-IR was weakly expressed in a limited number of neurons only in the SAC and SP. Whether GABA serves as an inhibitory neurotransmitter was also tested in the isolated telencephalon preparation by using extracellular field potential recordings. The synaptic activity recorded in the posterior dorsal telencephalon in response to the electrical stimulation of the anterior dorsal telencephalon was increased in the presence of the GABAA receptor antagonist, BMI, suggesting an inhibitory role for GABA-immunoreactive neurons in the adult brain of the zebrafish.

Neurotrophic factor in the treatment of Parkinson disease.

OBJECT: Parkinson disease (PD) is a well-known degenerative disease resulting in the depletion of dopamine-producing neurons in the pars compacta of the substantia nigra. Adenoviral vector delivery of neurotrophic factors may provide a potential therapy for PD. The authors examined whether glial cell line-derived neurotrophic factor (GDNF) delivered via adenoviral vector (Ad-GDNF) could promote functional recovery in a rat model of PD. Additionally, they examined whether neural precursor cells (NPCs) provide the therapeutic potential of cultured neural cells for cell regeneration and replacement in PD. METHODS: All animals underwent stereotactic injection of 6-hydroxydopamine into the right substantia nigra. Eight weeks later, the rats were tested for apomorphine-induced rotational asymmetry and evaluation of explanted grafts infected with the complementary DNA for GDNF containing NPCs and NPCs alone. In the NPC cultures of embryonic rat striata, the authors found that basic fibroblast growth factor induced the proliferation of stem cells, which give rise to spheres of undifferentiated cells that generate neurons and glia. CONCLUSIONS: In this study the authors found that the reduction of apomorphine-induced rotation was more prominent in parkinsonian rats that received Ad-GDNF-treated grafts containing NPCs (61%) than in those that received grafts of NPCs alone (16%).

Improved blood compatibility and decreased VSMC proliferation of surface-modified metal grafted with sulfonated PEG or heparin.

Although the technique of coronary stenting has remarkably improved long-term results in recent years, (sub)acute thrombosis and late restenosis still remain problems to be solved. Metallic surfaces were regarded as thrombogenic, due to their positive surface charges, and stenosis resulted from the activation and proliferation of vascular smooth muscle cells (VSMCs). In this study, a unique surface modification method for metallic surfaces was studied using a self-assembled monolayer (SAM) technique. The method included the deposition of thin gold layers, the chemisorption of disulfides containing functional groups, and the subsequent coupling of PEG derivatives or heparin utilizing the functional groups of the disulfides. All the reactions were confirmed by ATR-FTIR and XPS. The surface modified with sulfonated PEG (Au-S-PEG-SO3) or heparinized PEG (Au-S-PEG-Hep) exhibited decreased static contact angles and therefore increased hydrophilicity to a great extent, which resulted from the coupling of PEG and the ionic groups attached. In vitro fibrinogen adsorption and platelet adhesion onto the Au-S-PEG-SO3 or Au-S-PEG-Hep surfaces decreased to a great extent, indicating enhanced blood compatibility. This decreased interaction of the modified surfaces should be attributed to the non-adhesive property of PEG and the synergistic effect of sulfonated PEG. The effect of the surface modification on the adhesion and proliferation of VSMCs was also investigated. The modified Au-S-PEG-SO3 or Au-S-PEG-Hep surfaces also exhibited decreased adhesion of VSMCs, while the deposited gold layer itself was effective. The enhanced blood compatibility and the decreased adhesion of VSMCs on the modified metallic surfaces may help to decrease thrombus formation and suppress restenosis. It would therefore be very useful to apply these modified surfaces to stents for improved functions. A long-term in vivo study using animal models is currently under way.