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The excitatory amino acid transporter EAAC1 is an electrogenic Na+- and K+-gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl− conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3− and Cl− of the conductance mode. [Copyright &y& Elsevier]

In this study, a bioinformatics analysis and luciferase reporter assay revealed that microRNA-141 could silence the expression of lncRNA-HOTAIR by binding to specific sites on lncRNA-HOTAIR. We used superparamagnetic iron oxide nanoparticles (SPIONs) to mediate the high expression of microRNA-141 (SPIONs@miR-141) in human amniotic epithelial stem cells (HuAESCs), which was followed by the induction of the differentiation of HuAESCs into dopaminergic neuron-like cells (iDNLCs). qPCR, western blot, immunofluorescence staining and HPLC all suggested that SPION-mediated overexpression of miR-141 could promote an increased expression of brain-derived neurotrophic factor (BDNF), DAT and 5-TH in HuAESC-derived iDNLCs. The RIP and ChIP assay also showed that overexpression of miR-141 could significantly inhibit the recruitment and binding of lncRNA-HOTAIR to EZH2 on BDNF gene promoter. cDNA microarray analysis revealed that the expression levels of 190 genes were much higher in iDNLCs than in HuAESCs. Finally, a protein interaction network analysis and identification showed that in the iDNLC group with SPIONs@miR-141, factors that interact with BDNF, such as FGF8, SHH, NTRK3 and CREB1, all showed significantly higher expression levels compared with those in the SPIONs@miR-Mut. Therefore, this study confirmed that the highly efficient expression of microRNA-141 mediated by SPIONs could improve the efficiency of HuAESCs differentiation into dopaminergic neuron-like cells., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Parkinson's disease (PD) is a common age-associated neurodegenerative disorder. To date, stem cell transplantation therapy has been developed to replace lost or damaged neural cells in PD patients, in whom dopaminergic neuron cells are lost. Here, we show that CD44+ human amniotic fluid cells (HuAFCs) can be induced to become functional dopaminergic neuronal-like cells in vitro. Furthermore, when CD44+ or CD44- HuAFCs were transplanted into 6-hydroxydopamine (6-OHDA)-treated PD rats, the results indicated that CD44+ HuAFCs expressed multiple dopaminergic neuron cell markers and were ameliorative to behavioral recovery in PD rats after induction both in vitro and in vivo. CD44- HuAFCs did not fully differentiate into dopaminergic neuronal-like cells. When compared with CD44- HuAFCs, CD44+ HuAFCs showed increased activity in regeneration of dopaminergic neuron cell-like cells, increased migration distances, and improvement of animal behavior in the PD rat model. Therefore, CD44+ HuAFCs could be a source of dopaminergic neuronal-like cells with a potential use in cell-replacement therapy for PD.

Pancreatic and duodenal homeobox factor 1 (PDX-1) maintains β-cell function and differentiation via direct regulation of multiple islet cell genes. However, the molecular mechanisms involved in this process remain unknown. Here, we show that PDX-1 plays an important role in the induction of CD44+/CD105+ human amniotic fluid cells (HuAFCs) into functional pancreatic β-cell-like cells in vitro. CD44+/CD105+ HuAFCs were transfected with either siRNA targeting PDX-1 (siRNA-PDX-1) or mock plasmid (siRNA-MOCK). Following induction, siRNA-MOCK-transfected cells differentiated into β-cell-like cells that expressed multiple islet cell markers and produced insulin and C-peptide in a glucose-regulated manner. However, siRNA-PDX-1-transfected cells did not fully differentiate into β-cell-like cells. Further, we observed epigenetic changes at the PDX-1 gene locus in induced CD44(+)/CD105(+) HuAFCs. Therefore, CD44+/CD105+ HuAFCs could be a source of human pancreatic β-cell-like cells with potential uses in cell replacement therapy for diabetes.

The concept of cancer stem cells (CSCs) provides a new paradigm for understanding cancer biology. Here we report how culture conditions affect the characteristics of primary ovarian cancer cells. Cancer cells disaggregated from ovarian serous adenocarcinoma and maintained in serum-free system culture formed sphere cells that exhibited several properties expected for CSCs. These include self-renewal, overexpression of stemness genes as detected by QPCR analysis, greater tumorigenicity and enhanced drug resistance. The serum-free culture system enriched the percentage of CD133+/CD117+ expressing cells in sphere cells as determined by flow cytometric analysis, immunostaining and Western blot analysis. A cDNA microarray showed that there were 2111 genes exhibiting more than a 2-fold difference in expression. Subsequent ontological analysis revealed that a large proportion of the classified genes were related to cell communication, cell-cell adhesion, cellular development and extracellular matrix. We suggest that the sphere cell subpopulation may be a more reliable model than differentiated cells grown in the presence of serum for understanding the biology of primary ovarian cancer.

Gamma-aminobutyric acid (GABA) transporter subtype 1 (GAT1), which transports extracellular GABA into presynaptic neurons, plays an important regulatory role in the function of GABAergic systems. However, the contributions of the GAT1 in regulating mental status are not fully understood. In this paper, we observed the behavioral alterations of GAT1 knockout (GAT1(-/-)) mice using several depression- and anxiety-related models (eg, the forced-swimming test and the tail-suspension test for testing depression-related behaviors; the open-field test, the dark-light exploration test, the emergence test, and the elevated plus maze (EPM) test for anxiety-related behaviors). Here we found that GAT1(-/-) mice showed a lower level of depression- and anxiety-like behaviors in comparison to wild-type mice. Furthermore, GAT1(-/-) mice exhibited measurable insensitivity to selected antidepressants and anxiolytics such as fluoxetine, amitriptyline, buspirone, diazepam, and tiagabine in the tail-suspension test and/or the EPM test. Moreover, the basal level of corticosterone was found to be significantly lower in GAT1(-/-) mice. These results showed that the absence of GAT1 affects mental status through enhancing the GABAergic system, as well as modifying the serotonergic system and the hypothalamic-pituitary-adrenal (HPA) activity in mice.

Unlabelled: We investigated the feasibility of radioiodine therapy targeting hepatoma cells (MH3924A) by tissue-specific expression of the human sodium/iodide symporter (hNIS) gene directed by the murine albumin enhancer and promoter (mAlb)., Methods: The cell-specific transcriptional activity of mAlb was examined by a luciferase assay in several transiently transfected cell lines. MH3924A cells were stably transfected with the recombinant retroviral vector, in which hNIS complementary DNA expression was driven by mAlb and coupled to hygromycin resistance gene using an internal ribosomal entry site (IRES). Functional hNIS expression in hepatoma cells was confirmed by an iodide uptake assay. In imaging studies, the tumor-bearing ACI rats were intravenously injected with (131)I and imaged with a gamma-camera. Biodistribution was studied at 30 min and at 1, 3, 6, and 25 h after injection of (131)I. Toxic effects of (131)I on hepatoma cells were studied in vitro and in vivo., Results: Stably transfected MH3924A cells concentrated (125)I up to 240-fold higher than the wild-type cells. The iodide uptake in stably transfected cells was inhibited by ouabain and sodium perchlorate but increased by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. An in vitro clonogenic assay revealed an 86% decrease in colony number in stably transfected cells after exposure to 3.7 MBq/mL of (131)I and only about 8% in hNIS-negative control cells. Furthermore, the in vivo study showed intense tracer accumulation in hNIS-expressing tumors after administration of (131)I. At 3 h after intraperitoneal injection, the transfected tumors accumulated (131)I 19.2-fold higher than the parental tumors in a biodistribution study. Moreover, administration of a therapeutic dose of (131)I resulted in an inhibition of hNIS-expressing tumor growth, whereas control tumors continued to increase in size., Conclusion: A therapeutic effect of (131)I on hepatoma cells in vitro and in vivo has been demonstrated after tumor-specific iodide uptake induced by mAlb-directed hNIS gene expression. Because a stable transformed cell line has been used in these experiments, the clinical potential of this strategy must be evaluated after in vivo transfection of hepatoma cells.

The aim of this study was to investigate the culture conditions of skin-derived mesenchymal stem cell (sMSCs) and to explore a new cell source for central nervous system cell transplantation. The cells from skins of mice were primarily isolated and cultured in serum-free medium, and they were transferred into serum-containing medium after passaged 2, and the passaged cells were identified by immunocytochemistry and induced to differentiate into multiple lineages. The results indicated that a population of sMSCs could be isolated from skins, they could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. About 60% of sMSCs expressed vimentin and the majorities of these cells expressed fibronectin. They could differentiate into adipocytes, osteogenic cells and fibroblast-like cells, they could differentiate into neurons with a simple protocol, and almost 50-60% of these cells expressed neuron specific enolase (NSE) and neurofilament (NF); and the differentiated neurons showed typical complicated morphology of neurons. In conclusion, skin contains stem cells that are capable of multiple differentiation; they could be cultured in vitro for long time and could maintain their characteristics of stem cells, and they may represent an alternative autologous stem cell source for CNS cell transplantation.

Objective: To investigate the possibility of inducing adipose tissue-derived stromal cells to differentiate into neuron-like cells, and to explore a new cell source for central nervous system transplantation., Methods: beta-mercaptoethanol was adopted to induce the cells to differentiate; undifferentiated cells and differentiated cells were identified with immunocytochemistry., Results: A population of adipose tissue-derived stromal cells were isolated from adult rat adipose tissue; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. beta-mercaptoethanol induced the stem cells to express nestin, characteristic of neuronal precursor stem cells at early stage of differentiation, and at late stage they exhibited a neuronal phenotype, expressing neuron-specific enolase (NSE) and neurofilament(NF); with an optimal differentiation protocol, almost 60%-85% of the cells expressed NSE and NF., Conclusion: The data support the hypothesis that adult adipose tissue contains stem cells capable of differentiating into neurons.

Objectives: To assess the culture and differentiation of neural stem cells in embryonic mice and set up a basis for further research in to neural stem cells., Methods: Embryonic cortices of mice were dissociated and single cell suspensions were achieved by mechanical methods in sterile conditions, and cells were seeded in uncoated plate in N2 medium. The cells were passaged by mechanical methods, frozen and thawed by general procedure. They were identified by immunocytochemical techniques., Results: Neural stem cells from embryonic mice were successfully cultured forming typical neurospheres in suspension. Neurons, astrocytes and oligodendrocytes were differentiated from neural stem cells, with a ratio of 7%, 85% - 90% and 2% - 4% respectively., Conclusions: Neural stem cells, which can be cultured and passaged steadily in vitro and they are the ideal cell sources for cell transplantation and gene therapy.