178 research outputs found
Short-Term Withdrawal of Mitogens Prior to Plating Increases Neuronal Differentiation of Human Neural Precursor Cells
Background: Human neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.Principal Findings: We demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. in this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.Conclusions: These results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Institutos do Milenio de Bioengenharia TecidualUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Ciencias Biomed, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilFAPESP: fellowCNPq: fellowWeb of Scienc
The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells
<p>Abstract</p> <p>Background</p> <p>Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.</p> <p>Findings</p> <p>Here, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1<sub>Lai-Halo</sub>), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.</p> <p>Conclusions</p> <p>Use of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.</p
Marine-based multiproxy reconstruction of Atlantic multidecadal variability
Atlantic multidecadal variability (AMV) is known to impact climate globally, and knowledge about the persistence of AMV is important for understanding past and future climate variability, as well as modeling and assessing climate impacts. The short observational data do not significantly resolve multidecadal variability, but recent paleoproxy reconstructions show multidecadal variability in North Atlantic temperature prior to the instrumental record. However, most of these reconstructions are land-based, not necessarily representing sea surface temperature. Proxy records are also subject to dating errors and microenvironmental effects. We extend the record of AMV 90 years past the instrumental record using principle component analysis of five marine-based proxy records to identify the leading mode of variability. The first principal component is consistent with the observed AMV, and multidecadal variability seems to persist prior to the instrumental record. Thus, we demonstrate that reconstructions of past Atlantic low-frequency variability can be improved by combining marine-based proxies
Is the Presence of Microalbuminuria a Relevant Marker of Kidney Disease?
Levels of urinary albumin excretion that are below the usual limit of detection by qualitative testing, but are above normal levels (microalbuminuria; MA), can be readily identified by simple measures, such as the urinary albumin to creatinine ratio in untimed urine samples. Such measurements, particularly when combined with assessment of estimated glomerular filtration rate (eGFR), have utility as biomarkers for enhanced risk of all-cause mortality, cardiovascular events, progressive chronic kidney disease, and end-stage renal disease in diabetic and nondiabetic subjects. However, it is controversial whether “isolated” MA (MA in the absence of a clear reduction in eGFR, urine sediment abnormalities, or structural renal disease) should be regarded as kidney disease. Such MA could also be regarded as a manifestation of a diffuse endothelial (microvascular) injury and thereby collateral kidney damage. This article reviews the current evidence concerning MA as a marker of kidney disease or kidney damage
GDNF Secreting Human Neural Progenitor Cells Protect Dying Motor Neurons, but Not Their Projection to Muscle, in a Rat Model of Familial ALS
Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of large motor neurons in the brain and spinal cord. Growth factors such as glial cell line derived neurotrophic factor (GDNF) are known to protect motor neurons from damage in a range of models. However, penetrance through the blood brain barrier and delivery to the spinal cord remains a serious challenge. Although there may be a primary dysfunction in the motor neuron itself, there is also increasing evidence that excitotoxicity due to glial dysfunction plays a crucial role in disease progression. Clearly it would be of great interest if wild type glial cells could ameliorate motor neuron loss in these models, perhaps in combination with the release of growth factors such as GDNF.Human neural progenitor cells can be expanded in culture for long periods and survive transplantation into the adult rodent central nervous system, in some cases making large numbers of GFAP positive astrocytes. They can also be genetically modified to release GDNF (hNPC(GDNF)) and thus act as long-term 'mini pumps' in specific regions of the rodent and primate brain. In the current study we genetically modified human neural stem cells to release GDNF and transplanted them into the spinal cord of rats over-expressing mutant SOD1 (SOD1(G93A)). Following unilateral transplantation into the spinal cord of SOD1(G93A) rats there was robust cellular migration into degenerating areas, efficient delivery of GDNF and remarkable preservation of motor neurons at early and end stages of the disease within chimeric regions. The progenitors retained immature markers, and those not secreting GDNF had no effect on motor neuron survival. Interestingly, this robust motor neuron survival was not accompanied by continued innervation of muscle end plates and thus resulted in no improvement in ipsilateral limb use.The potential to maintain dying motor neurons by delivering GDNF using neural progenitor cells represents a novel and powerful treatment strategy for ALS. While this approach represents a unique way to prevent motor neuron loss, our data also suggest that additional strategies may also be required for maintenance of neuromuscular connections and full functional recovery. However, simply maintaining motor neurons in patients would be the first step of a therapeutic advance for this devastating and incurable disease, while future strategies focus on the maintenance of the neuromuscular junction
Biphasic Electrical Currents Stimulation Promotes both Proliferation and Differentiation of Fetal Neural Stem Cells
The use of non-chemical methods to differentiate stem cells has attracted
researchers from multiple disciplines, including the engineering and the
biomedical fields. No doubt, growth factor based methods are still the most
dominant of achieving some level of proliferation and differentiation control -
however, chemical based methods are still limited by the quality, source, and
amount of the utilized reagents. Well-defined non-chemical methods to
differentiate stem cells allow stem cell scientists to control stem cell biology
by precisely administering the pre-defined parameters, whether they are
structural cues, substrate stiffness, or in the form of current flow. We have
developed a culture system that allows normal stem cell growth and the option of
applying continuous and defined levels of electric current to alter the cell
biology of growing cells. This biphasic current stimulator chip employing ITO
electrodes generates both positive and negative currents in the same culture
chamber without affecting surface chemistry. We found that biphasic electrical
currents (BECs) significantly increased the proliferation of fetal neural stem
cells (NSCs). Furthermore, BECs also promoted the differentiation of fetal NSCs
into neuronal cells, as assessed using immunocytochemistry. Our results clearly
show that BECs promote both the proliferation and neuronal differentiation of
fetal NSCs. It may apply to the development of strategies that employ NSCs in
the treatment of various neurodegenerative diseases, such as Alzheimer's
and Parkinson's diseases
Biphasic Electrical Currents Stimulation Promotes both Proliferation and Differentiation of Fetal Neural Stem Cells
The use of non-chemical methods to differentiate stem cells has attracted
researchers from multiple disciplines, including the engineering and the
biomedical fields. No doubt, growth factor based methods are still the most
dominant of achieving some level of proliferation and differentiation control -
however, chemical based methods are still limited by the quality, source, and
amount of the utilized reagents. Well-defined non-chemical methods to
differentiate stem cells allow stem cell scientists to control stem cell biology
by precisely administering the pre-defined parameters, whether they are
structural cues, substrate stiffness, or in the form of current flow. We have
developed a culture system that allows normal stem cell growth and the option of
applying continuous and defined levels of electric current to alter the cell
biology of growing cells. This biphasic current stimulator chip employing ITO
electrodes generates both positive and negative currents in the same culture
chamber without affecting surface chemistry. We found that biphasic electrical
currents (BECs) significantly increased the proliferation of fetal neural stem
cells (NSCs). Furthermore, BECs also promoted the differentiation of fetal NSCs
into neuronal cells, as assessed using immunocytochemistry. Our results clearly
show that BECs promote both the proliferation and neuronal differentiation of
fetal NSCs. It may apply to the development of strategies that employ NSCs in
the treatment of various neurodegenerative diseases, such as Alzheimer's
and Parkinson's diseases
Neural progenitor cells from an adult patient with fragile X syndrome
BACKGROUND: Currently, there is no adequate animal model to study the detailed molecular biochemistry of fragile X syndrome, the leading heritable form of mental impairment. In this study, we sought to establish the use of immature neural cells derived from adult tissues as a novel model of fragile X syndrome that could be used to more fully understand the pathology of this neurogenetic disease. METHODS: By modifying published methods for the harvest of neural progenitor cells from the post-mortem human brain, neural cells were successfully harvested and grown from post-mortem brain tissue of a 25-year-old adult male with fragile X syndrome, and from brain tissue of a patient with no neurological disease. RESULTS: The cultured fragile X cells displayed many of the characteristics of neural progenitor cells, including nestin and CD133 expression, as well as the biochemical hallmarks of fragile X syndrome, including CGG repeat expansion and a lack of FMRP expression. CONCLUSION: The successful production of neural cells from an individual with fragile X syndrome opens a new avenue for the scientific study of the molecular basis of this disorder, as well as an approach for studying the efficacy of new therapeutic agents
Neural Stem/Progenitor Cells from the Adult Human Spinal Cord Are Multipotent and Self-Renewing and Differentiate after Transplantation
Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia
Bioinformatic analyses identifies novel protein-coding pharmacogenomic markers associated with paclitaxel sensitivity in NCI60 cancer cell lines
<p>Abstract</p> <p>Background</p> <p>Paclitaxel is a microtubule-stabilizing drug that has been commonly used in treating cancer. Due to genetic heterogeneity within patient populations, therapeutic response rates often vary. Here we used the NCI60 panel to identify SNPs associated with paclitaxel sensitivity. Using the panel's GI50 response data available from Developmental Therapeutics Program, cell lines were categorized as either sensitive or resistant. PLINK software was used to perform a genome-wide association analysis of the cellular response to paclitaxel with the panel's SNP-genotype data on the Affymetrix 125 k SNP array. FastSNP software helped predict each SNP's potential impact on their gene product. mRNA expression differences between sensitive and resistant cell lines was examined using data from BioGPS. Using Haploview software, we investigated for haplotypes that were more strongly associated with the cellular response to paclitaxel. Ingenuity Pathway Analysis software helped us understand how our identified genes may alter the cellular response to paclitaxel.</p> <p>Results</p> <p>43 SNPs were found significantly associated (FDR < 0.005) with paclitaxel response, with 10 belonging to protein-coding genes (<it>CFTR</it>, <it>ROBO1</it>, <it>PTPRD</it>, <it>BTBD12</it>, <it>DCT</it>, <it>SNTG1</it>, <it>SGCD</it>, <it>LPHN2</it>, <it>GRIK1</it>, <it>ZNF607</it>). SNPs in <it>GRIK1</it>, <it>DCT</it>, <it>SGCD </it>and <it>CFTR </it>were predicted to be intronic enhancers, altering gene expression, while SNPs in <it>ZNF607 </it>and <it>BTBD12 </it>cause conservative missense mutations. mRNA expression analysis supported these findings as <it>GRIK1</it>, <it>DCT</it>, <it>SNTG1</it>, <it>SGCD </it>and <it>CFTR </it>showed significantly (p < 0.05) increased expression among sensitive cell lines. Haplotypes found in <it>GRIK1, SGCD, ROBO1, LPHN2</it>, and <it>PTPRD </it>were more strongly associated with response than their individual SNPs.</p> <p>Conclusions</p> <p>Our study has taken advantage of available genotypic data and its integration with drug response data obtained from the NCI60 panel. We identified 10 SNPs located within protein-coding genes that were not previously shown to be associated with paclitaxel response. As only five genes showed differential mRNA expression, the remainder would not have been detected solely based on expression data. The identified haplotypes highlight the role of utilizing SNP combinations within genomic loci of interest to improve the risk determination associated with drug response. These genetic variants represent promising biomarkers for predicting paclitaxel response and may play a significant role in the cellular response to paclitaxel.</p
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