Cross-regulation of Connexin43 and ß-catenin influences differentiation of human neural progenitor cells.

Connexin43 (Cx43) is the most widely and abundantly expressed gap junction (GJ) protein and it is strongly associated with the regulation of cell cycle progression. Emerging roles for Cx43 in cell adhesion and migration during neural differentiation have also been recently recognized, and this has emphasized the involvement of Cx43 in different physiological process beyond its role as a GJ protein. In this study, we explore the function of Cx43 in the differentiation of human neural progenitor cells (hNPCs) using viral vectors that mediate the overexpression or knockdown of the protein. Results showed that in the absence of this protein fetal cortex-derived hNPCs differentiated toward a neuronal phenotype at expenses of a glial phenotype. Furthermore, the silencing of Cx43 did not affect hNPC proliferation rate or numbers of apoptotic cells. The increase in the number of neurons was not recapitulated when GJ intercellular communications were pharmacologically blocked, and this suggested that Cx43 was influencing hNPCs differentiation with a GJ-independent effect. In addition, Cx43 knockdown significantly increased ß-catenin signaling, which has been shown to regulate the transcription of pro-neuronal genes during embryonic neural development. Our results add further support to the hypothesis that Cx43 protein itself regulates key signaling pathways during development and neurogenesis beyond its role as GJ protein.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Clusterin secreted by astrocytes enhances neuronal differentiation from human neural precursor cells.

Neuronal differentiation from expanded human ventral mesencephalic neural precursor cells (NPCs) is very limited. Astrocytes are known to secrete neurotrophic factors, and so in order to enhance neuronal survival from NPCs, we tested the effect of regional astrocyte-conditioned medium (ACM) from the rat cortex, hippocampus and midbrain on this process. Human NPC's were expanded in FGF-2 before differentiation for 1 or 4 weeks in ACM. The results show that ACM from the hippocampus and midbrain increase the number of neurons from expanded human NPCs, an effect that was not observed with cortical ACM. In addition, both hippocampal and midbrain ACM increased the number and length of phosphorylated neurofilaments. MALDI-TOF analysis used to determine differences in media revealed that although all three regional ACMs had cystatin C, α-2 macroglobulin, extracellular matrix glycoprotein and vimentin, only hippocampal and midbrain ACM also contained clusterin, which when immunodepleted from midbrain ACM eliminated the observed effects on neuronal differentiation. Furthermore, clusterin is a highly glycosylated protein that has no effect on cell proliferation but decreases apoptotic nuclei and causes a sustained increase in phosphorylated extracellular signal-regulated kinase, implicating its role in cell survival and differentiation. These findings further reveal differential effects of regional astrocytes on NPC behavior and identify clusterin as an important mediator of NPC-derived neuronal survival and differentiation.

Astrocytes promote neurogenesis from oligodendrocyte precursor cells.

The oligodendrocyte precursor cell (OPC) has until recently been regarded as a lineage-restricted precursor cell. Considerable interest has been generated by reports suggesting that OPCs may possess a wider differentiation potential than previously assumed and thus be considered a multipotential stem cell. This study examined the neuronal differentiation potential of rat, postnatal cortical OPCs in response to extracellular cues in vitro and in vivo. OPCs did not exhibit intrinsic neuronal potential and were restricted to oligodendrocyte lineage potential following treatment with the neural precursor mitogen fibroblast growth factor 2. In contrast, a postnatal hippocampal astrocyte-derived signal(s) is sufficient to induce functional neuronal differentiation of cortical OPCs in vitro in population and single cell studies. Co-treatment with Noggin, a bone morphogenetic protein antagonist, did not attenuate neuronal differentiation. Following transplantation to the adult rat hippocampus, cortical OPCs expressed doublecortin, a neuroblast-associated marker. The present findings show that hippocampal, astrocyte-derived signals can induce the neuronal differentiation of OPCs through a Noggin-independent mechanism.

Improving the survival of human CNS precursor-derived neurons after transplantation.

We have examined the effects of predifferentiation and energy substrate deprivation on long-term expanded human neural precursor cells (HNPCs). The pre-differentiation of HNPC cultures produced large numbers of neurons (>60%) and mature glial cells capable of generating glycogen stores that protected the neuronal population from experimental metabolic stress. When predifferentiated HNPCs were transplanted into intact adult rat hippocampus, fewer cells survived compared to undifferentiated HNPC transplants. This cell death was completely attenuated, however, when predifferentiated HNPC cultures were pretreated to boost glial energy stores and resulted in greatly increased neuronal survival in vivo. The transplanted cells primarily engrafted within the granular layer of the dentate gyrus, where a large proportion of the predifferentiated HNPCs co-expressed neuronal markers whereas most HNPCs outside of the neuronal layer did not, indicating that the predifferentiated cells remained capable of responding to local cues in the adult brain. Undifferentiated HNPCs migrated more widely in the brain after grafting than did the predifferentiated cells, which generally remained within the hippocampus.

Growth factors regulate the survival and fate of cells derived from human neurospheres.

Cells isolated from the embryonic, neonatal, and adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of both multipotent stem cells and more restricted progenitor populations. Neurospheres can also be generated from the embryonic human brain and in some cases have been expanded for extended periods of time in culture. However, the mechanisms controlling the number of neurons generated from human neurospheres are poorly understood. Here we show that maintaining cell-cell contact during the differentiation stage, in combination with growth factor administration, can increase the number of neurons generated under serum-free conditions from 8% to > 60%. Neurotrophic factors 3 and 4 (NT3, NT4) and platelet-derived growth factor (PDGF) were the most potent, and acted by increasing neuronal survival rather than inducing neuronal phenotype. Following differentiation, the neurons could survive dissociation and either replating or transplantation into the adult rat brain. This experimental system provides a practically limitless supply of enriched, non-genetically transformed neurons. These should be useful for both neuroactive drug screening in vitro and possibly cell therapy for neurodegenerative diseases.

Antioxidant strategy to counteract the side effects of antipsychotic therapy: an in vivo study in rats.

The effects of long-term administration of the dopamine D(2) receptor antagonist haloperidol on Parkinsonian symptoms have been shown to persist after cessation of the drug treatment. In order to determine whether the level of tyrosine hydroxylase could be affected by subchronic administration of haloperidol, we examined tyrosine hydroxylase-positive immunoreactive cells in the substantia nigra after blockade of dopaminergic receptors with this antipsychotic. Three weeks of injections with haloperidol (1.5 mg/kg, i.p.) caused a significant decrease in tyrosine hydroxylase-positive cell counts at 24 h (27%), 5 days (21%) and 2 weeks (10%) after the last administration, an effect that was blocked by concurrent administration of the antioxidant, vitamin C. The level of tyrosine hydroxylase returned to baseline after 4 weeks withdrawal, no change being observed at later time-points. Nissl staining demonstrated that no damage to the cell bodies was observed, suggesting that the decrease in tyrosine hydroxylase-positive cells was not due to dopaminergic cell loss. These results demonstrate a depleting action of a short course of haloperidol on nigral tyrosine hydroxylase that outlasts the period of application by 2-4 weeks. Moreover, the current study has shown the effect of the antioxidant vitamin C in protecting haloperidol effects on tyrosine hydroxylase-immunostaining.

Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation.

Worldwideattention is presently focused on proliferating populations of neural precursor cells as an in vitro source of tissue for neural transplantation and brain repair. However, successful neuroreconstruction is contingent upon their capacity to integrate within the host CNS and the absence of tumorigenesis. Here we show that human neural precursor cells express very low levels of telomerase at early passages (less than 20 population doublings), but that this decreases to undetectable levels at later passages. In contrast, rodent neural precursors express high levels of telomerase at both early and late passages. The human neural precursors also have telomeres (approximately 12 kbp) significantly shorter than those of their rodent counterparts (approximately 40 kbp). Human neural precursors were then expanded 100-fold prior to intrastriatal transplantation in a rodent model of Parkinson's disease. To establish the effects of implanted cell number on survival and integration, precursors were transplanted at either 200,000, 1 million, or 2 million cells per animal. Interestingly, the smaller transplants were more likely to extend neuronal fibers and less likely to provoke immune rejection than the largest transplants in this xenograft model. Cellular proliferation continued immediately post-transplantation, but by 20 weeks there were virtually no dividing cells within any of the grafts. In contrast, fiber outgrowth increased gradually over time and often occupied the entire striatum at 20 weeks postgrafting. Transient expression of tyrosine hydroxylase-positive cells within the grafts was found in some animals, but this was not sustained at 20 weeks and had no functional effects. For Parkinson's disease, the principal aim now is to induce the dopaminergic phenotype in these cells prior to transplantation. However, given the relative safety profile for these human cells and their capacity to extend fibers into the adult rodent brain, they may provide the ideal basis for the repair of other lesions of the CNS where extensive axonal outgrowth is required.

Prehospital delay time in acute myocardial infarction: an exploratory study on relation to hospital outcomes and cost.

BACKGROUND: Short prehospital delay is associated with improved outcomes in myocardial infarction, but the impact on cost has not been tested. Shortening delay time could reduce health care expenditures. METHODS AND RESULTS: Two hundred ninety-eight patients were examined with the use of a historic prospective design at 2 hospital sites. A secondary analysis was performed that used patients with confirmed myocardial infarction from the National Register of Myocardial Infarction and direct and indirect costs from the accounting system at the hospitals. Chi-square, Mann Whitney U, and Fisher exact tests were used for comparisons. Delay and 4 sets of variables were regressed on cost with the significant predictors used to construct a final model. The mean age was 71 +/- 14 years old; 62% were men. There were no major differences in demographics, cardiac history, risk factors, and admission characteristics between short and long delayers. Resource utilization and clinical outcomes were similar between the 2 groups; there was no difference in cost. Additional diagnostic procedures (odds ratio 2.92; 95% confidence interval 1.65-5.15) and complications (odds ratio 3.43; 95% confidence interval 2.03-5.82) were significant predictors of cost. Delay was not a predictor of high cost. CONCLUSIONS: Short prehospital delay was not associated with improved clinical outcomes, nor did it predict cost. Explanations include (1) the low utilization of early reperfusion therapy in the short delay group, (2) the study lacked sufficient power to detect a difference in cost between short and long delayers, and (3) the severity of illness could not be adequately measured. This issue warrants further study because of the potential impacts on health care expenditures.

The symptom experience of angina in women.

Angina is the symptom of myocardial ischemia and the most common presentation of women with coronary artery disease. Women have delayed responses to angina and postpone seeking care more than men. Myocardial ischemia is life threatening and timeliness of treatment is critical. Understanding the symptom experience is important to patients and health care providers alike to reduce morbidity and mortality rates. This article reviews current knowledge of the symptom experience to identify gaps in knowledge and provide a basis for future research and interventions. The symptom experience component of the Symptom Management Model is used as an organizing framework. In terms of chest pain perception, biopsychosocially oriented studies are inconsistent. Data suggest that women use different pain descriptors than men and that diabetes, somatic awareness, and hormonal status probably play a role in attenuating or altering anginal pain. In evaluating symptoms, findings suggest that if anginal symptoms were experienced as expected and/or were evaluated as cardiac in origin, response was more appropriate. Even when it is known that the symptoms are related to cardiac disease, women's responses are still delayed because of a need to self-treat to maintain control. If a patient recognizes symptoms to be cardiac in origin, an appropriate evaluation of the urgency of the situation was made more often, and a rapid response was more likely. Therefore, the larger problem may be the accurate perception of the symptom and the recognition that the symptom is cardiac in origin. This article has implications for future research dealing with improved responses.

Mouse epidermal growth factor-responsive neural precursor cells increase the survival and functional capacity of embryonic rat dopamine neurons in vitro.

We have grown expanded populations of epidermal growth factor (EGF)-responsive mouse striatal precursor cells and subsequently co-cultured these with primary E14 rat ventral mesencephalon. The aim of these experiments was to induce dopaminergic (DA) neuronal phenotypes from the murine precursors. While no precursor cell-derived neurons were induced to express tyrosine hydroxylase (TH), there was a dramatic 30-fold increase in the survival of rat-derived TH-positive neurons in the co-cultures. The effect was not explicable solely in terms of total plating density, and was accompanied by a significantly enhanced capacity for [3H]dopamine uptake in the co-cultures compared to rat alone cultures. The present data show that, although primary rat E14 mesencephalic cells are incapable of inducing the development of DA neurons from EGF-responsive mouse neural precursor cells, such precursors will differentiate into cells capable of enhancing the survival and overall functional efficacy of primary embryonic dopamine neurons.

Human neural stem cells: isolation, expansion and transplantation.

Neural stem cells, with the capacity to self renew and produce the major cell types of the brain, exist in the developing and adult rodent central nervous system (CNS). Their exact function and distribution is currently being assessed, but they represent an interesting cell population, which may be used to study factors important for the differentiation of neurons, astrocytes and oligodendrocytes. Recent evidence suggests that neural stem cells may also exist in both the developing and adult human CNS. These cells can be grown in vitro for long periods of time while retaining the potential to differentiate into nervous tissue. Significantly, many neurons can be produced from a limited number of starting cells, raising the possibility of cell replacement therapy for a wide range of neurological disorders. This review summarises this fascinating and growing field of neurobiology, with a particular focus on human tissues.

The development of intracerebral cell-suspension implants is influenced by the grafting medium.

The effect of preparing and grafting embryonic striatal and nigral tissue in four different media was evaluated in vitro and in vivo. The proportion of TH-positive and DARPP-32-positive neurons was determined after 2 days in vitro in standard culture medium following preparation in the different media. The effects were more marked for striatal neurons where DARPP-32 expression in tissue prepared in HBSS was poor compared to other media. TH expression was unaffected by the preparation medium. Striatal grafts derived from tissue prepared and grafted in HBSS were smaller, with fewer DARPP-32 cells, compared to other media. Survival of grafts in combined HBSS and DMEM was very poor. Graft volume and TH cell content was enhanced in tissue prepared in DMEM. These results suggest that preparation protocols optimized for one type of embryonic neuronal population do not necessarily transfer to other neuronal populations.

Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells.

There is increasing evidence that the proteoglycan heparin plays a critical role in the regulation of the activity of FGF-2 by either interacting with its receptor or modifying its stability and functioning. In this study precursor cells were isolated from the rat E14 ventral mesencephalon and cultured as free floating spheres in FGF-2 alone or in combination with heparin or other related proteoglycans, including chondroitin sulfate, keratin sulfate, dermatan sulfate, or hyaluronic acid. Our results show the mitogenic effects of FGF-2 could be potentiated by heparin but not the other four proteoglycans. Sodium chlorate, which blocks the cells ability to sulfate its proteoglycans, was shown to reduce the mitogenic effects of FGF-2 alone to below that of control levels, suggesting that endogenous sulfated molecules are required for the FGF-2 effects on mesencephalic precursors. Cells expanded for 7 days with either FGF-2 or FGF-2 + heparin were plated onto a substrate and allowed to differentiate for a further 7 days in the absence of growth factors. Approximately 6% of the precursors developed into neurons whether grown with or without heparin and none were positive for TH, a marker for dopamine neurons. However, there was a significant decrease in the number of astrocytes developing from cultures grown in FGF-2 + heparin when compared to FGF-2 alone. Interestingly we could not find an EGF responsive cell in the mesencephalon at this embryonic age in the absence or presence of heparin. However, there was a synergistic effect of combining EGF + FGF-2, which could be potentiated by heparin. We conclude that heparin, but not other closely related proteoglycans, is vital for the growth of FGF-2-responsive mesencephalic neural precursors.

A new method for the rapid and long term growth of human neural precursor cells.

A reliable source of human neural tissue would be of immense practical value to both neuroscientists and clinical neural transplantation trials. In this study, human precursor cells were isolated from the developing human cortex and, in the presence of both epidermal and fibroblast growth factor-2, grew in culture as sphere shaped clusters. Using traditional passaging techniques and culture mediums the rate of growth was extremely slow, and only a 12-fold expansion in total cell number could be achieved. However, when intact spheres were sectioned into quarters, rather than mechanically dissociated, cell cell contacts were maintained and cellular trauma minimised which permitted the rapid and continual growth of each individual quarter. Using this method we have achieved a 1.5 million-fold increase in precursor cell number over a period of less than 200 days. Upon differentiation by exposure to a substrate, cells migrated out from the spheres and formed a monolayer of astrocytes and neurons. No oligodendrocytes were found to develop from these human neural precursor cells at late passages when whole spheres were differentiated. This simple and novel culture method allows the rapid expansion of large numbers of non-transformed human neural precursor cells which may be of use in drug discovery, ex vivo gene therapy and clinical neural transplantation.

Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson's disease.

Progenitor cells were isolated from the developing human central nervous system (CNS), induced to divide using a combination of epidermal growth factor and fibroblast growth factor-2, and then transplanted into the striatum of adult rats with unilateral dopaminergic lesions. Large grafts were found at 2 weeks survival which contained many undifferentiated cells, some of which were migrating into the host striatum. However, by 20 weeks survival, only a thin strip of cells remained at the graft core while a large number of migrating astrocytes labeled with a human-specific antibody could be seen throughout the striatum. Fully differentiated graft-derived neurons, also labeled with a human-specific antibody, were seen close to the transplant site in some animals. A number of these neurons expressed tyrosine hydroxylase and were sufficient to partially ameliorate lesion-induced behavioral deficits in two animals. These results show that expanded populations of human CNS progenitor cells maintained in a proliferative state in culture can migrate and differentiate into both neurons and astrocytes following intracerebral grafting. As such these cells may have potential for development as an alternative source of tissue for neural transplantation in degenerative diseases.