Study the effect of mesenchymal stem cells on isolated cortical neurons before and after NMDA treatment
ABSTRACT: Primary cortical neurons were isolated from E15 mice and after 5 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium and then untreated or treated for a further 24 h with NMDA. Neuron gene expression was profiled and compared between the four different conditions (neurons, neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to activation of endogenous CNS repair processes and immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of MSC in simplified MSC-neuron co-culture systems and in mice using models of glutamate excitotoxicity. MSC protected primary cortical neurons against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC cm), but not control NIH3T3 cells, was sufficient for this effect. MSC cm neuroprotection in mouse cortical neurons was reduced by neutralizing antibodies to bFGF and associated with altered gene expression in neurons towards an immature phenotype as well as reduced neuronal Grin1, Grin2a and Grin2b mRNA levels in response to NMDA stimulation. Further, MSC cm neuroprotection in rat retinal ganglion cells was associated with absence of glutamate-induced calcium influx. Adoptive transfer of EGFP+MSC in a mouse kainic acid seizure model reduced CA3 neuron damage and hippocampal astrocytosis and resulted in the increased expression of neuronal genes that are upregulated by MSC cm, Bmi1, Ddx4, Ezh1, in the hippocampus. These results show that MSC mediate direct neuroprotection against glutamate excitotoxicity by secreting bFGF, reducing glutamate receptor expression and function and altering neuron gene expression towards an immature pattern, and provide evidence for a link between the therapeutic effects of MSC and the activation of endogenous repair processes following CNS injury. In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm and/or NMDA were investigated using a cDNA spotted macroarray filter. Four samples were analysed in duplicate: neurons alone (untreated), neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA.
Project description:Primary cortical neurons were isolated from E15 mice and after 6 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium. Neuron gene expression was profiled and compared between the two different conditions (neurons and neurons+MSC cm) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to the activation of endogenous CNS protection and repair processes as well as immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of mouse MSC in rodent MSC-neuron co-cultures and mice using models of glutamate excitotoxicity. A 24 hr pre-culture of mouse primary cortical neurons with MSC protected them against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC CM) was sufficient for this effect. Protection by MSC CM was associated with reduced mRNA levels of genes encoding NMDA receptor subunits, and increased levels for genes associated with non-neuronal and stem cell types, as shown by RT-PCR and cDNA microarray analyses. Changes in gene expression were not associated with alterations in cell lineage representation within the cultures. Further, MSC CM-mediated neuroprotection in rat retinal ganglion cells was associated with reduced glutamate-induced calcium influx. The adoptive transfer of EGFP+MSC in a mouse kainic acid epilepsy model also provided neuroprotection against glutamate excitotoxicity in vivo, as shown by reduced neuron damage and glial cell activation in the hippocampus. These results show that MSC mediate direct neuroprotection by reducing neuronal sensitivity to glutamate receptor ligands and altering gene expression, and suggest a link between the therapeutic effects of MSC and the activation of cell plasticity in the damaged CNS. Overall design: In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm were investigated using a cDNA spotted macroarray filter. Two samples were analyzed in duplicate: neurons alone (untreated) and neurons+MSC cm.
Project description:Gene regulation in mammals involves a complex interplay between promoter and distal regulatory elements that function in concert to drive precise spatio-temporal gene expression programs. However, the dynamics of distal gene regulatory elements and its function in transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here we use a combinatorial analysis of genomewide datasets for chromatin accessibility (FAIRE-Seq) and enhancer mark H3K27ac to reveal a highly dynamic nature of chromatin accessibility during neurogenesis that gets restricted to certain genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further reveal that the distal open regions serve as target sites of distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. A prolonged NMDA-driven neural activity results in epigenetic reprogramming at a large number of distal regulatory elements as well as dramatic reorganization of super-enhancers that in turn mediate critical transcriptional responses. Taken together, these findings reveal dynamics of distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate transcriptome underlying neuronal development and function. FAIRE-Seq and H3K27ac profiles for three stages on neuronal differentation viz. neuronal progenitors, day 1 neurons and day 10 neurons, were generated to understand the dynamics of accessible and ehancer chromatin landscape. Along with this we also generated RNASeq and H3K27ac profiles for day 10 neurons upon control and NMDA treatment.
Project description:Hydrogen sulfide (H2S), present in abundance in the mammalian brain, has recently been demonstrated to induce a dose- and time-dependent apoptotic-necrotic continuum in murine primary cortical neurons, which was successfully attenuated upon application of N-methyl-D-aspartate (NMDA) receptor antagonist. The current study focused on gaining an insight into the molecular mechanisms of H2S–mediated neuronal death pertaining to NMDA receptors activation through global gene expression comparisons. A total of 24 RNA samples were analyzed. There are 2 treatment conditions, namely 200uM sodium hydrosulfide and 200uM N-methyl-D-asparate. 3 replicates were collected for each of the selected time-points (5h, 15h and 24h), in addition to 6 replicates of shared vehicle control. The supplementary file 'GSE16035_non-normalized_data.txt' contains non-normalized data for Samples GSM401312-GSM401335.
Project description:To increase our understanding of the mechanisms that underlie neuroprotection following glutamate receptor-induced excitotoxicity, we treated primary CD-1 mouse cortical neurons with NMDA in the presence of absence of neuroprotective compounds. The results indicate a convergence at the level of whole genome expression, despite the divergent chemistries and purported mechanisms of action. While more arrays were performed than published, only the extreme arrays were used in subsequent analyses. Overall design: CD-1 Mouse cortical neurons were exposed to NMDA, in the presence or absence of neuroprotective molecules, followed by a recovery period (16hr) and total RNA extraction.
Project description:Mesenchymal stem cells (MSC) resemble a multipotent adult stem cell population capable of differentiation into a number of different mesodermal cell types including adipodytes, osteoblasts, chondroblasts. Although still in debate there is some evidence, that these cells can also differentiate into cells of non-mesodermal germal layers including hepatocytes, myocytes, cardiomyocytes or neurons. Analysis of these cells in the course of differentiation makes them an intriguing model for the examination of stemness. More importantly the differentiation capacity of MSC raises high hopes for clinical applications. In this study we have isolated MSC from bone marrow under two different culture conditions, from adipose tissue and from umbilical cord blood. The genome wide gene expression profile of these different human MSC was compared with reference RNA of non-multipotent human fibroblasts HS68. The aim of this study was to elucidate common molecular characteristics of MSC as well as differences in their expression profile. These results might help for a better understanding of MSC and contribute to a reliable quality control that will be necessary for clinical applications.
Project description:Synaptic activity drives changes in gene expression to promote long-lasting adaptations of neuronal structure and function. One example of such an adaptive response is the buildup of acquired neuroprotection, a synaptic activity- and gene transcription-mediated increase in the resistance of neurons against harmful conditions. A hallmark of acquired neuroprotection is the stabilization of mitochondrial structure and function. We therefore re-examined previously identified sets of synaptic activity-regulated genes to identify genes that are directly linked to mitochondrial function. In mouse and rat primary hippocampal cultures synaptic activity caused an upregulation of glycolytic genes and a concomitant downregulation of genes required for oxidative phosphorylation, mitochondrial biogenesis and maintenance. Changes in metabolic gene expression were induced by action potential bursting, but not by glutamate bath application activating extrasynaptic NMDA receptors. The specific pattern of gene expression changes suggested that synaptic activity promotes a shift of neuronal energy metabolism from oxidative phosphorylation toward aerobic glycolysis, also known as Warburg effect. The ability of neurons to upregulate glycolysis has, however, been debated. We therefore used FACS sorting to show that, in mixed neuron glia co-cultures, activity-dependent regulation of metabolic gene expression occurred in neurons. Changes in gene expression were accompanied by changes in the phosphorylation-dependent regulation of the key metabolic enzyme, pyruvate dehydrogenase. Finally, increased synaptic activity caused an increase in the ratio of L-lactate production to oxygen consumption in primary hippocampal cultures. Based on these data we suggest the existence of a synaptic activity-mediated neuronal Warburg effect that may promote mitochondrial homeostasis and neuroprotection. Overall design: We compared the mRNA expression profile of primary hippocampal neurons after 4h of basal synaptic activity vs. 4h of action potential bursting. Two independent experiments with independent cell preparations were performed.
Project description:This experiment comprises RNA-seq data used to study evolutionary differences between humans and mice in neuronal activity-dependent transcriptional responses. Activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons were compared with those induced in developing primary- or stem cell-derived mouse cortical neurons 4 hours after KCl-induced membrane depolarisation. Activity-dependent transcriptional responses were also measured in aneuploid mouse neurons carrying human chromosome 21, allowing study of the regulation of Hsa21 genes, plus their mouse orthologs, side-by-side in the same cellular environment of a mouse primary neuron.
Project description:We have employed whole genome microarray expression profiling to identify genes that characterize different populations of human postnatal stem cells and reflect their potency MSC, Mab and MAPC cultures were established from at least three different donors and their transcriptome was assayed using the Agilent 4x44k whole human genome microarray; furthermore, the H9 ESC line from U of Wisconsin and MSC from Lonza were added for comparison MSC = Mesenchymal Stem Cells, MAPC= Multipotent Adult Progenitor Cells, Mab= Mesoangioblasts, ESC= Embryonic Stem Cells
Project description:Mesenchymal stem cells (MSC) are found in many adult tissues. It is generally accepted that this type of stem cell forms different types of progenitor cells, osteocytes, chondrocytes, and adipocytes. There have been also reports about these cells differentiating into cardiac myocytes and neurons. We found primary MSC enriched at the surface of spongious bone. Therefore cellular and environmentally-induced aging of MSC and their response to oxygen tension have been determined. Low oxygen levels appeared to be instructive on the stemness of MSC allowing proliferation upon stimulation while being protective with respect to differentiation and moreover low oxygen provided a milieu for extended cellular life span. Our data are furthermore indicative for cellular senescence eventually taking place in aged MSC, which however appears to complete more rapidly under mild oxidative stress.
Project description:Identification of transcriptional profiles stimulated by the complement protein C1q in rat immature neurons associated with the C1q-dependent neuroprotection observed in vitro. Use of the unbiased whole genome microarray approach to identify genes regulated by C1q. Immature neurons grown in vitro for 3 days were stimulated with 10 nM C1q for 3h.