Human mesenchymal stromal cells (hMSCs) were injected into the hippocampus of adult mice 1 day after transient global ischemia. The hMSCs both improved neurologic function and markedly decreased neuronal cell death of the hippocampus. Microarray assays indicated that ischemia up-regulated 586 mouse genes. The hMSCs persisted for <7 days, but they down-regulated >10% of the ischemia-induced genes, most of which were involved in inflammatory and immune responses. The hMSCs also upregulated three mouse genes, including the neuroprotective gene Ym1 that is expressed by activated microglia/macrophages. In addition, the transcriptomes of the hMSC changed with upregulation of 170 human genes and down-regulation of 54 human genes. Protein assays of the hippocampus demonstrated increased expression in microglia/macrophages of Ym1, the cell survival factor insulin-like growth factor 1, galectin-3, cytokines reflective of a type 2 T cell immune bias, and the major histocompatibility complex II. The observed beneficial effects of hMSCs were largely explained by their modulation of inflammatory and immune responses, apparently by alternative activation of microglia and/or macrophages.inflammation ͉ mesenchymal stromal cells ͉ microglia ͉ mesenchymal stem cells O bservations in rodent and primate models suggest that a potential therapy for ischemia of the central nervous system is the administration of the adult stem/progenitor cells from bone marrow referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) (1-3). Administration of MSCs also produced beneficial effects in animal models for neurodegenerative diseases, such as Parkinson's disease, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis (2-5). MSCs initially attracted interest for their ability to differentiate into multiple cellular phenotypes in culture and in vivo (1-7). However, recent observations indicate that only small numbers of the cells engraft into most injured tissues, and they disappear quickly (2-5, 8-10). When human MSCs (hMSCs) were injected into the dentate gyrus (DG) of the hippocampus in adult immunodeficient (ID) mice, most of the cells disappeared within 1 week, but they enhanced proliferation, migration, and neural differentiation of the endogenous neural stem cells (8). These and related observations have focused attention on the paracrine effects of MSCs (2, 3, 11). However, it has not been established whether the beneficial effects of MSCs in ischemic models of brain injury are explained by enhanced neurogenesis (8) or by neuroprotection.Experiments here were performed in a mouse model of global ischemia to assess the neuroprotective effects of hMSCs. Administration of hMSCs 1 day after transient common carotid artery occlusion (tCCAO) improved neurologic function and decreased the delayed neuronal cell death of the hippocampus. Surveys with microarrays indicated that the hMSCs decreased expression of many of the mouse genes that were induced by ischemia and that were involved in inflamma...

The ɛ4 allele of the human apolipoprotein E gene (APOE) is a well-proven genetic risk factor for the late onset form of Alzheimer's disease (AD). However, the biological mechanisms through which the ɛ4 allele contributes to disease pathophysiology are incompletely understood. The three common alleles of APOE, ɛ2, ɛ3 and ɛ4, are defined by two single nucleotide polymorphisms (SNPs) that reside in the coding region of exon 4, which overlaps with a well-defined CpG island (CGI). Both SNPs change not only the protein codon but also the quantity of CpG dinucleotides, primary sites for DNA methylation. Thus, we hypothesize that the presence of an ɛ4 allele changes the DNA methylation landscape of the APOE CGI and that such epigenetic alteration contributes to AD susceptibility. To explore the relationship between APOE genotype, AD risk, and DNA methylation of the APOE CGI, we applied bisulfite pyrosequencing and evaluated methylation profiles of postmortem brain from 15 AD and 10 control subjects. We observed a tissue-specific decrease in DNA methylation with AD and identified two AD-specific differentially methylated regions (DMRs), which were also associated with APOE genotype. We further demonstrated that one DMR was completely un-methylated in a sub-population of genomes, possibly due to a subset of brain cells carrying deviated APOE methylation profiles. These data suggest that the APOE CGI is differentially methylated in AD brain in a tissue- and APOE-genotype-specific manner. Such epigenetic alteration might contribute to neural cell dysfunction in AD brain.

The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ε2/ε3/ε4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ε4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ε2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ε2/ε3/ε4 allele-carrying 3'-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ε2/ε3/ε4 allele-specific manner. These findings implicate a novel functional role for a 3'-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.

Traumatic brain injury (TBI) causes multiple long-term defects including a loss of working memory that is frequently incapacitating. Administrations of mesenchymal stem/stromal cells (MSCs) previously produced beneficial effects in models of TBI as well as other disease models. In several models, the beneficial effects were explained by the MSCs being activated to express TSG-6, a multifunctional protein that modulates inflammation. In a mouse model of TBI, we found the initial mild phase of the inflammatory response persisted for at least 24 hour and was followed by secondary severe response that peaked at 3 days. Intravenous human MSCs or TSG-6 during initial mild phase decreased neutrophil extravasation, expression of matrix metalloproteinase 9 by endothelial cells and neutrophils, and the subsequent blood brain barrier leakage in secondary phase. Administration of TSG-6 also decreased the lesion size at 2 weeks. Importantly, the acute administration of TSG-6 within 24 hour of TBI was followed 6 to 10 weeks later by improvements in memory, depressive-like behavior and the number of newly born-neurons. The data suggested that acute administration of TSG-6 may be an effective therapy for decreasing some of the long-term consequences of TBI.

Multipotent stem=progenitor cells from bone marrow stroma (mesenchymal stromal cells or MSCs) were previously shown to enhance proliferation and differentiation of neural stem cells (NSCs) in vivo, but the molecular basis of the effect was not defined. Here coculturing human MSCs (hMSCs) with rat NSCs (rNSCs) was found to stimulate astrocyte and oligodendrocyte differentiation of the rNSCs. To survey the signaling pathways involved, RNA from the cocultures was analyzed by species-specific microarrays. In the hMSCs, there was an upregulation of transcripts for several secreted factors linked to differentiation: bone morphogenetic protein 1 (BMP1), hepatocyte growth factor (HGF), and transforming growth factor isoforms (TGFb1 and TGFb3). In both the hMSCs and the rNSCs, there was an upregulation of transcripts for Notch signaling. The role of TGFb1 was verified by the demonstration that hMSCs in coculture increased secretion of TGFb1, the rNSCs expressed the receptor, and an inhibitor of TGFb signaling blocked differentiation. The role of Notch signaling was verified by the demonstration that in the cocultures hMSCs expressed a Notch ligand at sites of cell contact with rNSCs, and the rNSCs expressed the receptor, Notch 1. Increased Notch signaling in both cell types was then demonstrated by assays of transcript expression and by a reporter construct for downstream targets of Notch signaling. The results demonstrated that glial differentiation of the rNSCs in the cocultures was driven by increased secretion of soluble factors such as TGFb1 by the hMSCs and probably through increased cell contact signaling between the hMSCs and rNSCs through the Notch pathway.

Mesenchymal stem/progenitor cells (MSCs) improve functional outcome in a number of disease models through suppression of inflammation. However, their effects on neuroinflammation are unknown. In this study, we show that MSCs suppress endotoxin-induced glial activation in organotypic hippocampal slice cultures (OHSCs). Lipopolysaccharide-stimulated OHSCs activated MSCs to increase the expression of cyclo-oxygenase-2 and produce prostaglandin E2. MSC-derived prostaglandin E2, then suppressed proinflammatory cytokine production by the OHSCs. Together, the results suggest the potential anti-inflammatory mechanism of MSCs in models of disease and support earlier observations that MSCs may offer a therapy for neuroinflammation produced by trauma or disease.

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Abstract:The apolipoprotein E (APOE) gene is robustly linked with numerous physiological conditions, including healthy aging, altered cardiovascular fitness, and cognitive function. These connections have been established primarily by phenotype-genotype association studies using APOE's three common genetic variants (ε2, ε3, and ε4). These variants encode for the three apoE protein isoforms (E2, E3, and E4), which have slightly different structures and, consequently, distinct functions in lipid metabolism. However, the differential lipid binding and transferring properties of these isoforms cannot fully explain the association of APOE with such a wide range of physiological phenotypes. One potential explanation for APOE's pleiotropic roles may lie in its unique epigenetic properties. In this article, we present a brief review of the APOE gene and protein, its disease associations, and epigenetic components, with a focus on DNA methylation. We close with a discussion of the prospective epigenetic implications of APOE in disease.