技术资料

Studies have shown that neural stem/progenitor cell (NPC) transplantation is beneficial in experimental autoimmune encephalomyelitis (EAE),an established animal model of multiple sclerosis (MS). It is unclear whether NPCs have the ability to integrate into the host CNS to replace lost cells or if their main mechanism of action is via bystander immunomodulation. Understanding the mechanisms by which NPCs exert their beneficial effects as well as exploring methods to increase post-transplantation survival and differentiation is critical to advancing this treatment strategy. Using the EAE model and Fas-deficient (lpr) NPCs,we investigated the effects of altering the Fas system in NPC transplantation therapy. We show that transplantation of NPCs into EAE mice ameliorates clinical symptoms with greater efficacy than sham treatments regardless of cell type (wt or lpr). NPC transplantation via retro-orbital injections significantly decreased inflammatory infiltrates at the acute time point,with a similar trend at the chronic time point. Both wt and lpr NPCs injected into mice with EAE were able to home to sites of CNS inflammation in the periventricular brain and lumbar spinal cord. Both wt and lpr NPCs have the same capacity for inducing apoptosis of Th1 and Th17 cells,and minimal numbers of NPCs entered the CNS. These cells did not express terminal differentiation markers,suggesting that NPCs exert their effects mainly via bystander peripheral immunomodulation. View Publication

Plp1 gene expression occurs very early in development,well before the onset of myelination,creating a conundrum with regard to the function of myelin proteolipid protein (PLP),one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity,we found that,at very early time points,PLP-EGFP was expressed in Sox2+ undifferentiated precursors in the spinal cord ventricular zone (VZ),as well as in the progenitors of both neuronal and glial lineages. As development progressed,most PLP-EGFP-expressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord,and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP,normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However,the orientation and length of OPC processes during migration was abnormal in Plp1-null mice,suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth. View Publication

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study,we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42),indicating an increase in transient mPTP opening. Subsequently,the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42,during which time global cellular reactive oxygen species increased,mitochondrial membrane potential decreased,cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism,then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ. View Publication

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor (EPOR). The presence of EPO and its receptor in the CNS suggests a different function for EPO other than erythropoiesis. The purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation of neonatal spinal cord-derived neural progenitor cells. The effect of EPO on cell cycle progression was also examined,as well as the signaling cascades involved in this process. Our results showed that EPOR was present in the neural progenitor cells and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated neural progenitor cells indicated a reduced percentage of cells in G0/G1 phase,whereas the cell proliferation index (S phase plus G2/M phase) was increased. EPO also increased the proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells. With respect to the cell cycle signaling,we examined the cyclin-dependent kinases D1,D2 and E,and cyclin-dependent kinase inhibitors,p21cip1,p27kip1 and p57kip2. No significant differences were observed in the expression of these transcripts after EPO administration. Interestingly,the anti-apoptotic factors,mcl-1 and bcl-2 were significantly increased twofold. Moreover,these specific effects of EPO were eliminated by incubation of the progenitor cells with anti-EPO neutralizing antibody. Those observations suggested that EPO may play a role in normal spinal cord development by regulating cell proliferation and apoptosis. View Publication

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling,immunocytochemistry,electron microscopy,and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones,loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats,the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless,they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition,thereby opening the avenue for stem cell therapy. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication