ResearchPad - nestin Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity]]> Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (≥75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.

<![CDATA[Genetically engineered rat gliomas: PDGF-driven tumor initiation and progression in tv-a transgenic rats recreate key features of human brain cancer]]>

Previously rodent preclinical research in gliomas frequently involved implantation of cell lines such as C6 and 9L into the rat brain. More recently, mouse models have taken over, the genetic manipulability of the mouse allowing the creation of genetically accurate models outweighed the disadvantage of its smaller brain size that limited time allowed for tumor progression. Here we illustrate a method that allows glioma formation in the rat using the replication competent avian-like sarcoma (RCAS) virus / tumor virus receptor-A (tv-a) transgenic system of post-natal cell type-specific gene transfer. The RCAS/tv-a model has emerged as a particularly versatile and accurate modeling technology by enabling spatial, temporal, and cell type-specific control of individual gene transformations and providing de novo formed glial tumors with distinct molecular subtypes mirroring human GBM. Nestin promoter-driven tv-a (Ntv-a) transgenic Sprague-Dawley rat founder lines were created and RCAS PDGFA and p53 shRNA constructs were used to initiate intracranial brain tumor formation. Tumor formation and progression were confirmed and visualized by magnetic resonance imaging (MRI) and spectroscopy. The tumors were analyzed using histopathological and immunofluorescent techniques. All experimental animals developed large, heterogeneous brain tumors that closely resembled human GBM. Median survival was 92 days from tumor initiation and 62 days from the first point of tumor visualization on MRI. Each tumor-bearing animal showed time dependent evidence of malignant progression to high-grade glioma by MRI and neurological examination. Post-mortem tumor analysis demonstrated the presence of several key characteristics of human GBM, including high levels of tumor cell proliferation, pseudopalisading necrosis, microvascular proliferation, invasion of tumor cells into surrounding tissues, peri-tumoral reactive astrogliosis, lymphocyte infiltration, presence of numerous tumor-associated microglia- and bone marrow-derived macrophages, and the formation of stem-like cell niches within the tumor. This transgenic rat model may enable detailed interspecies comparisons of fundamental cancer pathways and clinically relevant experimental imaging procedures and interventions that are limited by the smaller size of the mouse brain.

<![CDATA[Abnormal differentiation of Sandhoff disease model mouse-derived multipotent stem cells toward a neural lineage]]>

In Sandhoff disease (SD), the activity of the lysosomal hydrolytic enzyme, β-hexosaminidase (Hex), is lost due to a Hexb gene defect, which results in the abnormal accumulation of the substrate, GM2 ganglioside (GM2), in neuronal cells, causing neuronal loss, microglial activation, and astrogliosis. We established induced pluripotent stem cells from the cells of SD mice (SD-iPSCs). In the present study, we investigated the occurrence of abnormal differentiation and development of a neural lineage in the asymptomatic phase of SD in vitro using SD mouse fetus-derived neural stem cells (NSCs) and SD-iPSCs. It was assumed that the number of SD mouse fetal brain-derived NSCs was reduced and differentiation was promoted, resulting in the inhibition of differentiation into neurons and enhancement of differentiation into astrocytes. The number of SD-iPSC-derived NSCs was also reduced, suggesting that the differentiation of NSCs was promoted, resulting in the inhibition of differentiation into neurons and enhancement of that into astrocytes. This abnormal differentiation of SD-iPSCs toward a neural lineage was reduced by the glucosylceramide synthase inhibitor, miglustat. Furthermore, abnormal differentiation toward a neural lineage was reduced in SD-iPSCs with Hexb gene transfection. Therefore, differentiation ability along the time axis appears to be altered in SD mice in which the differentiation ability of NSCs is promoted and differentiation into neurons is completed earlier, while the timing of differentiation into astrocytes is accelerated. These results clarified that the abnormal differentiation of SD-iPSCs toward a neural lineage in vitro was shown to reflect the pathology of SD.

<![CDATA[Changes in Expression of Aquaporin-4 and Aquaporin-9 in Optic Nerve after Crushing in Rats]]>

The purpose of this study was to determine the temporal and spatial changes in the expression of AQP4 and AQP9 in the optic nerve after it is crushed. The left optic nerves of rats were either crushed (crushed group) or sham operated (sham group), and they were excised before, and at 1, 2, 4, 7, and 14 days later. Four optic nerves were pooled for each time point in both groups. The expression of AQP4 and AQP9 was determined by western blot analyses. Immunohistochemistry was used to determine the spatial expression of AQP4, AQP9, and GFAP in the optic nerve. Optic nerve edema was determined by measuring the water content in the optic nerve. The barrier function of the optic nerve vessels was determined by the extravasated Evans blue dye on days 7 and 14. The results showed that the expression of AQP4 was increased on day 1 but the level was significantly lower than that in the sham group on days 4 and 7 (P<0.05). In contrast, the expression of AQP9 gradually increased, and the level was significantly higher than that in the sham group on days 7 and 14 (P<0.05, Tukey-Kramer). The down-regulation of AQP4 was associated with crush-induced optic nerve edema, and the water content of the nerve was significantly increased by 4.3% in the crushed optic nerve from that of the untouched fellow nerve on day 7. The expression of AQP4 and GFAP was reduced at the crushed site where AQP4-negative and AQP9-positive astrocytes were present. The barrier function was impaired at the crushed site on days 7 and 14, restrictedly where AQP4-negative and AQP9-positive astrocytes were present. The presence of AQP9-positive astrocytes at the crushed site may counteract the metabolic damage but this change did not fully compensate for the barrier function defect.

<![CDATA[Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells]]>

Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response.

<![CDATA[Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy]]>

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L,10 mg/kg/day), tempol (T,100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well as tubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR.

<![CDATA[Prognostic significance of nestin expression in patients with resected non-small cell lung cancer treated with platinum-based adjuvant chemotherapy; relationship between nestin expression and epithelial to mesenchymal transition related markers]]>


Although adjuvant platinum-based chemotherapy (AC) has been shown to improve survival of patients with completely resected stage II and stage IIIA non-small cell lung cancer (NSCLC), its effect is limited. Nestin is a class VI intermediate filament protein expressed in neural stem cells and several cancer cells including NSCLC. In the present study, we aimed to determine its prognostic significance concerning survival in NSCLC patients receiving AC.


Nestin expression in cancer cells was immunohistochemically studied in 90 patients with completely resected stage II and stage IIIA NSCLC treated with AC and its association with clinicopathologic parameters, including ABCG2, E-cadherin, and vimentin expression, was evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of nestin expression on survival.


Nestin expression was observed in 28 of the 90 (31.1%) NSCLCs. Clinicopathologically, nestin expression was associated with loss of E-cadherin expression (P = 0.006) and vimentin positive expression (P < 0.001). In survival analysis, nestin expression was significantly associated with a poorer prognosis (P = 0.028). Multivariable analysis confirmed that nestin expression is an independent prognostic indicator in NSCLC patients receiving AC (HR = 2.56; 95% CI, 1.23–5.30, P = 0.01).


The present study reveals that nestin expression is a prognostic indicator of a poorer survival probability in NSCLC patients receiving AC, although its prognostic significance still requires confirmation with larger patient populations.

<![CDATA[Clusters of amniotic fluid cells and their associated early neuroepithelial markers in experimental myelomeningocele: Correlation with astrogliosis]]>

Myelomeningocele (MMC) is the most common and severe disabling type of spina bifida resulting in the exposure of vulnerable spinal cord to the hostile intrauterine environment. In this study, we sought to examine the cellular content of fetal amniotic fluid (AF) in MMC and explore a correlation between these cells and pathological development of MMC. MMC was induced in fetal rats by exposing pregnant mothers to all-trans retinoic acid and AF samples were collected before term. Cells were isolated from AF samples and morphologically and phenotypically characterized in short-term cultures. In addition, the spinal cord injury in MMC fetuses was assessed by immunohistochemical examination of astrogliosis. We identified a population of cells from the AF of MMC fetuses (MMC-AF) that formed adherent clusters of tightly packed cells, which were absent from the AF of normal control fetuses (norm-AF). MMC-AF clusters contained cells co-expressing adherens junction associated proteins (ZO-1), N-cadherin and F-actin at sites of cell-cell contacts. In addition, they expressed markers of early neuroepithelial cells such as SOX-1 and Pax-6 along with other stem/progenitor cell markers such as SOX-2 and nestin. Subpopulations of cells in MMC-AF clusters also expressed more advanced differentiation markers such as doublecortin and GFAP. We found that the appearance of cluster forming cells in cultures from MMC-AF correlated with activation of astrogliosis associated with the spinal cord injury in MMC fetuses. In summary, we identified a neuroepithelial cell population in the AF of MMC fetuses that formed adherent clusters in culture and we characterized cellular markers of these cells. Our data suggests that the phase of the disease is a crucial factor in the emergence of these cells into the AF and that these cells may provide a new and important platform for studying the progression of MMC and development of improved strategies for the repair and diagnosis of MMC prenatally.

<![CDATA[Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells]]>

Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.

<![CDATA[Nestin expression is upregulated in the fibrotic rat heart and is localized in collagen-expressing mesenchymal cells and interstitial CD31(+)- cells]]>

Renal and lung fibrosis was characterized by the accumulation of collagen-immunoreactive mesenchymal cells expressing the intermediate filament protein nestin. The present study tested the hypothesis that nestin expression was increased in the hypertrophied/fibrotic left ventricle of suprarenal abdominal aorta constricted adult male Sprague-Dawley rats and induced in ventricular fibroblasts by pro-fibrotic peptide growth factors. Nestin protein levels were upregulated in the pressure-overloaded left ventricle and expression positively correlated with the rise of mean arterial pressure. In sham and pressure-overloaded hearts, nestin immunoreactivity was detected in collagen type I(+)-and CD31(+)-cells identified in the interstitium and perivascular region whereas staining was absent in smooth muscle α-actin(+)-cells. A significantly greater number of collagen type I(+)-cells co-expressing nestin was identified in the left ventricle of pressure-overloaded rats. Moreover, an accumulation of nestin(+)-cells lacking collagen, CD31 and smooth muscle α-actin staining was selectively observed at the adventitial region of predominantly large calibre blood vessels in the hypertrophied/fibrotic left ventricle. Angiotensin II and TGF-β1 stimulation of ventricular fibroblasts increased nestin protein levels via phosphatidylinositol 3-kinase- and protein kinase C/SMAD3-dependent pathways, respectively. CD31/eNOS(+)-rat cardiac microvascular endothelial cells synthesized/secreted collagen type I, expressed prolyl 4-hydroxylase and TGF-β1 induced nestin expression. The selective accumulation of adventitial nestin(+)-cells highlighted a novel feature of large vessel remodelling in the pressure-overloaded heart and increased appearance of collagen type I/nestin(+)-cells may reflect an activated phenotype of ventricular fibroblasts. CD31/collagen/nestin(+)-interstitial cells could represent displaced endothelial cells displaying an unmasked mesenchymal phenotype, albeit contribution to the reactive fibrotic response of the pressure-overloaded heart remains unknown.

<![CDATA[Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells]]>

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs.

<![CDATA[Comparative study of the neural differentiation capacity of mesenchymal stromal cells from different tissue sources: An approach for their use in neural regeneration therapies]]>

Mesenchymal stem cells (MSCs) can trans/differentiate to neural precursors and/or mature neurons and promote neuroprotection and neurogenesis. The above could greatly benefit neurodegenerative disorders as well as in the treatment of post-traumatic and hereditary diseases of the central nervous system (CNS). In order to attain an ideal source of adult MSCs for the treatment of CNS diseases, adipose tissue, bone marrow, skin and umbilical cord derived MSCs were isolated and studied to explore differences with regard to neural differentiation capacity. In this study, we demonstrated that MSCs from several tissues can differentiate into neuron-like cells and differentially express progenitors and mature neural markers. Adipose tissue MSCs exhibited significantly higher expression of neural markers and had a faster proliferation rate. Our results suggest that adipose tissue MSCs are the best candidates for the use in neurological diseases.

<![CDATA[Expression and Prognostic Value of Oct-4 in Astrocytic Brain Tumors]]>


Glioblastomas are the most frequent type of malignant primary brain tumor with a median overall survival less than 15 months. Therapy resistance of glioblastomas has been attributed to the presence of tumor initiating stem-like cells (TSCs). TSC-related markers have therefore been suggested to have promising potentials as prognostic markers in gliomas.

Methodology/Principal Findings

The aim of the present study was to investigate the expression and prognostic impact of the TSC-related marker Oct-4 in astrocytic brain tumors of increasing grade. In total 114 grade II, III and IV astrocytic brain tumors were immunohistochemically stained for Oct-4, and the fraction and intensity of Oct-4 positive cells were determined by morphometric analysis of full tumor sections. Oct-4 was expressed in all tumors, and the Oct-4 positive cell fraction increased with tumor grade (p = 0.045). There was no association between survival and Oct-4 positive cell fraction, neither when combining all tumor grades nor in analysis of individual grades. Oct-4 intensity was not associated with grade, but taking IDH1 status into account we found a tendency for high Oct-4 intensity to be associated with poor prognosis in anaplastic astrocytomas. Double immunofluorescence stainings showed co-localization in the perivascular niches of Oct-4 and two other TSC markers CD133 and nestin in glioblastomas. In some areas Oct-4 was expressed independently of CD133 and nestin.


In conclusion, high Oct-4 fraction was associated with tumor malignancy, but seemed to be without independent prognostic influence in glioblastomas. Identification of a potential prognostic value in anaplastic astrocytomas requires additional studies using larger patient cohorts.

<![CDATA[Molecular Analysis of Stromal Cells-Induced Neural Differentiation of Mouse Embryonic Stem Cells]]>

Deriving specific neural cells from embryonic stem cells (ESCs) is a promising approach for cell replacement therapies of neurodegenerative diseases. When co-cultured with certain stromal cells, mouse ESCs (mESCs) differentiate efficiently to neural cells. In this study, a comprehensive gene and protein expression analysis of differentiating mESCs is performed over a two-week culture period to track temporal progression of cells from a pluripotent state to specific terminally-differentiated neural cells such as neurons, astrocytes, and oligodendrocytes. Expression levels of 26 genes consisting of marker genes for pluripotency, neural progenitors, and specific neuronal, astroglial, and oligodendrocytic cells are tracked using real time q-PCR. The time-course gene expression analysis of differentiating mESCs is combined with the hierarchal clustering and functional principal component analysis (FPCA) to elucidate the evolution of specific neural cells from mESCs at a molecular level. These statistical analyses identify three major gene clusters representing distinct phases of transition of stem cells from a pluripotent state to a terminally-differentiated neuronal or glial state. Temporal protein expression studies using immunohistochemistry demonstrate the generation of neural stem/progenitor cells and specific neural lineages and show a close agreement with the gene expression profiles of selected markers. Importantly, parallel gene and protein expression analysis elucidates long-term stability of certain proteins compared to those with a quick turnover. Describing the molecular regulation of neural cells commitment of mESCs due to stromal signaling will help identify major promoters of differentiation into specific cell types for use in cell replacement therapy applications.

<![CDATA[MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation]]>

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral “sponge” to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.