Memantine Attenuates Cognitive and Emotional Dysfunction in Mice with Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy (SAE) is the most common complication of sepsis, with increased morbidity and mortality. To date, there has still been no established pharmacological therapy. Memantine, as an NMDA (N-methyl-d-aspartate) receptor antagonist, exhibited neuroprotective effects against cognitive and emotional dysfunction in many disorders. We performed cecal ligation and puncture (CLP) inducing sepsis as the ideal animal model of SAE. CLP-induced septic mice were given a memantine treatment through intragastric administration. The novel object recognition test indicated that memantine significantly improved cognitive dysfunction in septic mice. The open field test revealed that the anxiety-like behaviors and locomotion ability of septic mice were relieved by memantine. The pole test further confirmed the protective effects of memantine against immobility. Memantine significantly inhibited the excessive glutamate production and improved impaired neurogenesis on first and seventh day after sepsis, accompanying with reducing proinflammatory cytokines production (tumor necrosis factor alpha (TNF-α), interleukin (IL)-1beta (IL-1ß), and IL-10) and microglia activation in the brain of SAE. In addition, memantine treatment also reducing sepsis-induced brain blood barrier disruption via inhibiting the expression of metalloproteinase-9 (MMP-9). In conclusion, memantine exerted neuro-protective effects against cognitive and emotional defects, which might be considered as a promising therapy for SAE.

Mesenchymal stem cells from human umbilical cord regulate the expression of major histocompatibility complex in human neural stem cells and their lineages.

hNSCs (human neural stem cells) derived from embryonic tissue and aborted fetal brains are considered to be the most promising candidates for neurodegenerative and other CNS(central nervous system) diseases. However, the most common problem, which limited successful use of these allogeneic hNSC therapy, is immune rejection. Mesenchymal stem cells (MSCs) from human umbilical cord (hUC-MSCs) are receiving increasing attention for their immune-modulatory properties. In the current studies, we firstly investigated the immunogenecity of hNSCs as well as their lineages in cultures with the presence or absence of interferon gamma (IFNγ), a pro-inflammatory factors. Our data revealed that the majority of hNSCs and astrocytes expressed MHCI (major histocompatibility complex class I) while neurons hardly expressed MHCI (<5%) in the absence of IFNγ. In addition, neither hNSCs nor neurons expressed MHCII while a subpopulation (about 18 %) of astrocytes expressed MHCII without IFNγ stimulation. However, the addition of IFNγ in cultures significantly increased the expressions of MHCII on hNSCs and astrocytes. However, IFNγ did not affect the expression of MHCI on hNSCs and astrocytes. We then investigated whether hUC-MSCs had the capacity of regulating the immunogenecity of hNSCs as well as their lineages in a co-culture system. We found that hUC-MSCs did not affect the expression of MHCI on hNSCs and their lineages, however, these cells were able to significantly inhibit the IFNγ-induced up-regulation of MHCII on hNSCs and astrocytes (p < 0.001). Thus, our results suggest that hUC-MSCs may serve as potentially useful modulators to reduce the immunogenicity of allogeneic hNSCs in clinical application.

Therapeutic mechanism of human neural stem cell-derived extracellular vesicles against hypoxia-reperfusion injury in vitro.

AIMS: This study aimed to explore that the human neural stem cell derived extracellular vesicles (hNSC-EVs) have therapeutic effect on neuronal hypoxia-reperfusion (H/R) injured neurons in vitro by mediating the nuclear translocation of NF-E2-related factor 2 (Nrf2) to regulate the expression of downstream oxidative kinases. MAIN METHODS: The neuroprotective effects of hNSC-EVs were evaluated in an in vitro neuronal H/R model. Three parameters of hNSC-EVs, structure, phenotype and particle size, were characterized. At the cellular level, a human neuron cerebral ischemic reperfusion (CIR) injury model was constructed. Cell viability, apoptosis, and the amount of reactive oxygen species (ROS) were detected using real-time cell analysis (RTCA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and dichloro-dihydro-fluorescein diacetate (DCFH-DA), respectively. The neuronal axonal elongation was assessed by Opera Phenix™ screening system. The angiogenesis of human umbilical vein endothelial cells (HUVECs) was evaluated by co-culturing HUVECs with hNSC-EVs in Matrigel. The expression of apoptosis and oxidative stress-related proteins in cells and the nuclear transfer of Nrf2 following hypoxia-reperfusion (H/R) was verified by Western-blotting. KEY FINDINGS: We found that the hNSC-EVs can promote the survival of post-H/R injury neurons, inhibit neuronal apoptosis, and enhance nuclear transfer of Nrf2, in response to oxidative stress. We also found the hNSC-EVs can promote the elongation of neuronal axons and the angiogenesis of HUVECs. SIGNIFICANCE: At present, there is no effective therapy for CIR injury. We suggest that the hNSC-EVs could be considered a new strategy to achieve nerve repair for the treatment of neurological diseases, especially stroke.

Co-Transplantation of Human Umbilical Cord Mesenchymal Stem Cells and Human Neural Stem Cells Improves the Outcome in Rats with Spinal Cord Injury.

Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) are promising graft materials for cell therapies in spinal cord injury (SCI) models. Previous studies have demonstrated that MSCs can regulate the microenvironment of NSCs and promote their survival rate. Furthermore, several studies indicate that MSCs can reduce stem cell transplantation-linked tumor formation. To our knowledge, no previous studies have determined whether co-transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and human neural stem cells (hNSCs) could improve the outcome in rats with SCI. Therefore, we investigated whether the transplantation of hUC-MSCs combined with hNSCs through an intramedullary injection can improve the outcome of rats with SCI, and explored the underlying mechanisms. In this study, a moderate spinal cord contusion model was established in adult female Wistar rats using an NYU impactor. In total, 108 spinal cord-injured rats were randomly selected and divided into the following five groups: 1) hUC-MSCs group, 2) hNSCs group, 3) hUC-MSCs+hNSCs group, 4) PBS (control) group, and 5) a Sham group. Basso, Beattie and Bresnahan (BBB) behavioral test scores were used to evaluate the motor function of all animals before and after the SCI weekly through the 8th week. Two weeks after transplantation, some rats were sacrificed, immunofluorescence and immunohistochemistry were performed to evaluate the survival and differentiation of the transplanted stem cells, and brain-derived neurotrophic factor (BDNF) was detected by ELISA in the injured spinal cords. At the end of the experiment, we evaluated the remaining myelin sheath and anterior horn neurons in the injured spinal cords using Luxol Fast Blue (LFB) staining. Our results demonstrated that the surviving stem cells in the hUC-MSCs+hNSCs group were significantly increased compared with those in the hUC-MSCs alone and the hNSCs alone groups 2 weeks post-transplantation. Furthermore, the results of the BBB scores and the remaining myelin sheath evaluated via LFB staining in the injured spinal cords demonstrated that the most significantly improved outcome occurred in the hUC-MSCs+hNSCs group. The hUC-MSCs alone and the hNSCs alone groups also had a better outcome compared with that of the PBS-treated group. In conclusion, the present study demonstrates that local intramedullary subacute transplantation of hUC-MSCs, hNSCs, or hUC-MSCs+hNSCs significantly improves the outcome in an in vivo moderate contusion SCI model, and that co-transplantation of hUC-MSCs and hNSCs displayed the best outcome in our experiment.

Mesenchymal stem cells from bone marrow attenuated the chronic morphine-induced cAMP accumulation in vitro.

Even though opioid tolerance is both a common and a major challenge in medicine, treatment with opioids is currently the primary method used to treat acute and chronic pain. The cAMP accumulation induced by chronic morphine is regarded as one of the molecular mechanisms leading to its tolerance and dependence characteristics. In the present study, we differentiated SH-SY5Y cells into neuron-like cells by retinoic acid (RA), pretreated these cells with morphine, and tested their cAMP levels under different conditions, including co-culture with bone marrow-derived human mesenchymal stem cells from bone marrow (hMSCs-BM) at various hMSCs-BM/SH-SY5Y ratios (1:5, 1:25, and 1:125), by direct cell-to-cell contact or without cell-to-cell contact, and by conditioned medium (CM) from hMSCs-BM. We found that chronic treatment with 10 µM morphine led to cAMP upregulation in those RA-differentiated SH-SY5Y cells while the morphine induced-cAMP accumulation was significantly attenuated by co-culturing with hMSCs-BM by direct cell-to-cell contact at a lower cell ratio (1:25) and a higher cell ratio (1:5). However, at neither the low or higher cell ratios could hMSCs-BM inhibit morphine-induced cAMP accumulation in RA-differentiated SH-SY5Y cells without cell-to-cell contact. In summary, hMSCs-BM can successfully inhibit morphine-induced cAMP up-regulation in RA-differentiated SH-SY5Y cells by cell-to-cell contact at a higher ratio, suggesting that hMSCs-BM may serve as valuable therapeutics to minimize the risk of drug abuse and addiction in the treatment of morphine tolerance and dependence.

Neural Stem Cell-Conditioned Medium Ameliorated Cerebral Ischemia-Reperfusion Injury in Rats.

INTRODUCTION: Our previous study suggested that NSC-CM (neural stem cell-conditioned medium) inhibited cell apoptosis in vitro. In addition, many studies have shown that neurotrophic factors and microparticles secreted into a conditioned medium by NSCs had neuroprotective effects. Thus, we hypothesized that NSC-CM had the capacity of protecting against cerebral I/R injury. METHODS: Adult male Sprague-Dawley rats receiving middle cerebral artery occlusion surgery as an animal model of cerebral I/R injury were randomly assigned to two groups: the control group and NSC-CM-treated group. 1.5 ml NSC-CM or PBS (phosphate buffer saline) was administrated slowly by tail vein at 3 h, 24 h, and 48 h after ischemia onset. RESULTS: NSC-CM significantly ameliorated neurological defects and reduced cerebral infarct volume, accompanied by preserved mitochondrial ultrastructure. In addition, we also found that NSC-CM significantly inhibited cell apoptosis in the ischemic hemisphere via improving the expression of Bcl-2 (B-cell lymphoma-2). CONCLUSION: NSC-CM might be an alternative and effective therapeutic intervention for ischemic stroke.

Umbilical cord-derived mesenchymal stem cells reversed the suppressive deficiency of T regulatory cells from peripheral blood of patients with multiple sclerosis in a co-culture - a preliminary study.

The immunoregulatory function of T regulatory cells (Tregs) is impaired in multiple sclerosis (MS). Recent studies have shown that umbilical cord-derived mesenchymal stem cells (UC-MSCs) exert regulatory effect on the functions of immune cells. Thus, we investigated whether UC-MSCs could improve the impaired function of Tregs from MS patients. Co-cultures of UC-MSCs with PBMCs of MS patients were performed for 3 days. Flow cytometry was used to determine the frequency of Tregs. A cell proliferation assay was used to evaluate the suppressive capacity of Tregs. ELISA was conducted for cytokine analysis in the co-cultures. Our results showed that UC-MSCs significantly increased the frequency of CD4+CD25+CD127low/- Tregs in resting CD4+ T cells (p<0.01) from MS, accompanied by the significantly augmented production of cytokine prostaglandin E2, transforming growth factor (-ß1, and interleukin-10, along with a reduced interferon-γ production in these co-cultures (p<0.05 - 0.01). More importantly, UC-MSC-primed Tregs of MS patients significantly inhibited the proliferation of PHA-stimulated autologous and allogeneic CD4+CD25- T effector cells (Teffs) from MS patients and healthy individuals compared to non-UC-MSC-primed (naïve) Tregs from the same MS patients (p<0.01). Furthermore, no remarkable differences in suppressing the proliferation of PHA-stimulated CD4+CD25- Teffs was observed in UC-MSC-primed Tregs from MS patients and naïve Tregs from healthy subjects. The impaired suppressive function of Tregs from MS can be completely reversed in a co-culture by UC-MSC modulation. This report is the first to demonstrate that functional defects of Tregs in MS can be repaired in vitro using a simple UC-MSC priming approach.

Mesenchymal Stem Cells Reversed Morphine Tolerance and Opioid-induced Hyperalgesia.

More than 240 million opioid prescriptions are dispensed annually to treat pain in the US. The use of opioids is commonly associated with opioid tolerance (OT) and opioid-induced hyperalgesia (OIH), which limit efficacy and compromise safety. The dearth of effective way to prevent or treat OT and OIH is a major medical challenge. We hypothesized that mesenchymal stem cells (MSCs) attenuate OT and OIH in rats and mice based on the understanding that MSCs possess remarkable anti-inflammatory properties and that both OT and chronic pain are associated with neuroinflammation in the spinal cord. We found that the development of OT and OIH was effectively prevented by either intravenous or intrathecal MSC transplantation (MSC-TP), which was performed before morphine treatment. Remarkably, established OT and OIH were significantly reversed by either intravenous or intrathecal MSCs when cells were transplanted after repeated morphine injections. The animals did not show any abnormality in vital organs or functions. Immunohistochemistry revealed that the treatments significantly reduced activation level of microglia and astrocytes in the spinal cord. We have thus demonstrated that MSC-TP promises to be a potentially safe and effective way to prevent and reverse two of the major problems of opioid therapy.

Dopaminergic Neuronal Differentiation from the Forebrain-Derived Human Neural Stem Cells Induced in Cultures by Using a Combination of BMP-7 and Pramipexole with Growth Factors.

Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.

Human umbilical cord-derived mesenchymal stem cells suppress proliferation of PHA-activated lymphocytes in vitro by inducing CD4(+)CD25(high)CD45RA(+) regulatory T cell production and modulating cytokine secretion.

Bone marrow-derived mesenchymal stem cells (MSCs) are promising candidate cells for therapeutic application in autoimmune diseases due to their immunomodulatory properties. Unused human umbilical cords (UC) offer an abundant and noninvasive source of MSCs without ethical issues and are emerging as a valuable alternative to bone marrow tissue for producing MSCs. We thus investigated the immunomodulation effect of umbilical cord-derived MSCs (UC-MSCs) on human peripheral blood mononuclear cells (PBMCs), T cells in particular, in a co-culture system. We found that UC-MSCs efficiently suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated PBMCs (p<0.01). Kinetic analysis revealed that UC-MSCs primarily inhibited the division of generation 3 (G3) and 4 (G4) of PBMCs. In addition, UC-MSCs augmented the expression of CD127(+) and CD45RA(+) but reduced the expression of CD25(+) in PBMCs stimulated by PHA (p<0.05). Furthermore, UC-MSCs inhibited PHA-resulted increase in the frequency of CD4(+)CD25(+)CD127(low/-) Tregs significantly (p<0.01) but augmented PHA-resulted increase in the frequency of CD4(+)CD25(high)CD45RA(+) Tregs to about three times in PBMCs. The levels of anti-inflammatory cytokines, PEG2, TGF-ß, and IL-10 were greatly up-regulated, accompanied by a significant down-regulation of pro-inflammatory IFN-γ in the co-culture (p<0.01). Our results showed that UC-MSCs are able to suppress mitogen-induced PBMC activation and proliferation in vitro by altering T lymphocyte phenotypes, increasing the frequency of CD4(+)CD25(high)CD45RA(+) Tregs, and modulating the associated cytokine production. Further studies are warranted to investigate the therapeutic potential of UC-MSCs in immunologically-diseased conditions.

Intraventricular administration of endoneuraminidase-N facilitates ectopic migration of subventricular zone-derived neural progenitor cells into 6-OHDA lesioned striatum of mice.

Polysialic acid (PSA), a carbohydrate polymer associated with the neural cell adhesion molecule (NCAM), plays an important role in the migration, differentiation and maturation of neuroblasts. Endoneuraminidase-N (Endo-N) can specifically cleave PSA from NCAM. The objective of the present study was to examine: the effect of Endo-N on characteristics of subventricular zone (SVZ)-derived neural progenitor cells (NPCs) in vitro; whether intraventricular administration of Endo-N could increase ectopic migration of SVZ-derived NPCs into 6-hydroxydopamine (6-OHDA)-lesioned striatum, and whether migrated NPCs could differentiate into neuronal and glial cells. In in vitro study, Endo-N was found to inhibit the migration of NPCs, and to enhance the differentiation of NPCs. In in vivo study, mice sequentially received injections of 6-OHDA into the right striatum, Endo-N into the right lateral ventricle, and bromodeoxyuridine (BrdU) intraperitoneally. The data showed that intraventricular injections of Endo-N disorganized the normal structure of the rostral migratory stream (RMS), and drastically increased the number of BrdU-immunoreactive (IR) cells in 6-OHDA-lesioned striatum. In addition, a number of BrdU-IR cells were double labeled for doublecortin (DCX), NeuN or glial fibrillary acidic protein (GFAP). The results suggest that interruption of neuroblast chain pathway with Endo-N facilitates ectopic migration of SVZ-derived NPCs into the lesioned striatum, and migrated NPCs can differentiate into neurons and astrocytes.

A new method to effectively and rapidly generate neurons from SH-SY5Y cells.

It is well known that neurons differentiated from SH-SY5Y cells can serve as cell models for neuroscience research; i.e., neurotoxicity and tolerance to morphine in vitro. To differentiate SH-SY5Y cells into neurons, RA (retinoic acid) is commonly used to produce the inductive effect. However, the percentage of neuronal cells produced from SH-SY5Y cells is low, either from the use of RA treatment alone or from the combined application of RA and other chemicals. In the current study, we used CM-hNSCs (conditioned medium of human neural stem cells) as the combinational inducer with RA to prompt neuronal differentiation of SH-SY5Y cells. We found that neuronal differentiation was improved and that neurons were greatly increased in the differentiated SH-SY5Y cells using a combined treatment of CM-hNSCs and RA compared to RA treatment alone. The neuronal percentage was higher than 80% (about 88%) on the 3rd day and about 91% on the 7th day examined after a combined treatment with CM-hNSCs and RA. Cell maturation and neurite growth of these neuronal cells were also improved. In addition, the use of CM-hNSCs inhibited the apoptosis of RA-treated SH-SY5Y cells in culture. We are the first to report the use of CM-hNSCs in combination with RA to induce neuronal differentiation of RA-treated SH-SY5Y cells. Our method can rapidly and effectively promote the neuronal production of SH-SY5Y cells in culture conditions.

Inducible Lentivirus-Mediated Expression of the Oct4 Gene Affects Multilineage Differentiation of Adult Human Bone Marrow-Derived Mesenchymal Stem Cells.

The octamer-binding transcription factor 4 (Oct4) gene plays an important role in maintaining the undifferentiated state of embryonic stem cells (ESCs) and reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs). In the present study, we transduced human bone marrow-derived mesenchymal stem cells (hMSCs) using tetracycline-on (Tet-On) lentiviruses carrying human Oct4 to examine the effects of regulated expression of human Oct4 on the proliferation and differentiation of hMSCs. hMSCs were efficiently transduced by Tet-On lentiviruses to express regulated levels of human Oct4 with doxycycline (Dox), as examined by immunofluorescent staining, flow cytometry, and quantitative real time-PCR (qRT-PCR) assays. Ectopic expression of Oct4 in transduced hMSCs increased the ability of colony formation. Continued expression of Oct4 further enhanced adipogenic differentiation of hMSCs, and transient expression of Oct4 sufficiently enhanced osteogenic differentiation of hMSCs. qRT-PCR analysis showed that ectopic expression of Oct4 in transduced hMSCs temporally increased the expression of Sox2 and c-Myc. Interestingly, ectopic expression of Oct4 reduced neuronal differentiation of hMSCs when incubated under neuronal differentiation conditions. Our results suggest that ectopic expression of human Oct4 leads to temporal changes in multilineage differentiation of hMSCs and may inhibit neuronal differentiation of hMSCs.

Pre-immunization with an intramuscular injection of AAV9-human erythropoietin vectors reduces the vector-mediated transduction following re-administration in rat brain.

We have recently demonstrated that adeno-associated virus serotype 9 (AAV9)-mediated human erythropoietin (hEPO) gene delivery into the brain protects dopaminergic (DA) neurons in the substantia nigra in a rat model of Parkinson's disease. In the present study, we examined whether pre-exposure to AAV9-hEPO vectors with an intramuscular or intrastriatal injection would reduce AAV9-mediated hEPO transduction in rat brain. We first characterized transgene expression and immune responses against AAV9-hEPO vectors in rat striatum at 4 days, 3 weeks and 6 months, and with doses ranging from 10(11) to 10(13) viral genomes. To sensitize immune system, rats received an injection of AAV9-hEPO into either the muscle or the left striatum, and then sequentially an injection of AAV9-hEPO into the right striatum 3 weeks later. We observed that transgene expression exhibited in a time course and dose dependent manner, and inflammatory and immune responses displayed in a time course manner. Intramuscular, but not intrastriatal injections of AAV9-hEPO resulted in reduced levels of hEPO transduction and increased levels of the major histocompatibility complex (MHC) class I and class II antigen expression in the striatum following AAV9-hEPO re-administration. There were infiltration of the cluster of differentiation 4 (CD4)-and CD8-lymphacytes, and accumulation of activated microglial cells and astrocytes in the virally injected striatum. In addition, the sera from the rats with intramuscular injections of AAV9-hEPO contained greater levels of antibodies against both AAV9 capsid protein and hEPO protein than the other treatment groups. hEPO gene expression was negatively correlated with the levels of circulating antibodies against AAV9 capsid protein. Intramuscular and intrastriatal re-administration of AAV9-hEPO led to increased numbers of red blood cells in peripheral blood. Our results suggest that pre-immunization with an intramuscular injection can lead to the reduction of transgene expression in the striatal re-administration.

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo.

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

Membrane properties of neuron-like cells generated from adult human bone-marrow-derived mesenchymal stem cells.

Adult mesenchymal stem cells (MeSCs) isolated from human bone marrow are capable of generating neural stem cell (NSC)-like cells that can be subsequently differentiated into cells expressing molecular markers for neurons. Here we report that these neuron-like cells had functional properties similar to those of brain-derived neurons. Whole-cell patch-clamp recordings and calcium imaging experiments were performed on neuron-like cells differentiated from bone-marrow-derived NSC-like cells. The neuron-like cells were subjected to current pulses to determine if they were capable of generating depolarization-induced action potentials. We found that nearly all of the cells with neuron-like morphology exhibited active membrane properties in response to the depolarizing pulses. The most common response was a single spike-like event with an overshoot and brief afterhyperpolarization. Cells that did not generate overshooting spike-like events usually displayed rectifying current-voltage relationships. The prevalence of these active membrane properties in response to the depolarizing current pulses suggested that the human MeSCs (hMeSCs) were capable of converting to a neural lineage under defined culture conditions. The spike-like events were blocked by the voltage-gated sodium channel inhibitor lidocaine, but unaffected by another sodium channel inhibitor tetrodotoxin (TTX). In calcium imaging experiments, the neuron-like cells responded to potassium chloride depolarization and l-glutamate application with increases in the cytoplasmic calcium levels. Thus, the neuron-like cells appeared to express TTX-resistant voltage-gated sodium channels, voltage-gated calcium channels, and functional l-glutamate receptors. These results demonstrate that hMeSCs were capable of generating cells with characteristics typical of functional neurons that may prove useful for neuroreplacement therapies.

Phenotypic characteristics of hybrid cells produced by cell fusion of porcine adrenal chromaffin cells with human mesenchymal stem cells: a preliminary study.

BACKGROUND AND PURPOSE: Transplantation of adrenal chromaffin cells (CCs) that release endogenous opioid peptides and catecholamines produces significant antinociceptive effects in patients with terminal cancer pain. In clinical practice, however, obtaining a sufficient number of chromaffin cells may not be possible because of the limited availability of human adrenal tissue. Recent works have shown that fusion of bone marrow-derived cells with differentiated cells can occur spontaneously in vivo and acquire the phenotype of the recipient cells. In this study, we investigated the possibility of producing chromaffin-like cells by fusing human bone marrow-derived mesenchymal stem cells (MeSCs) with post-mitotic porcine CCs in vitro with the application of polyethylene glycol (PEG). METHODS AND RESULTS: Before cell-to-cell fusion was initiated, MeSCs and CCs were labeled by fluorescence dyes DiO (green) and Dil (red), respectively. The hybrid cells generated by PEG-mediated fusion expressed a DiO and Dil double-staining with estimated fusion efficiency at approximately 40% of the total cell population. Further immunocytochemical examination for tyrosine hydroxylase and methionine enkephalin (markers for CCs) demonstrated positive immuno-reactivity in these hybrid cells 2 weeks post-fusion. More interestingly, some of the hybrid cells showed bromodeoxyuridine (BrdU)-positive immunostaining in the nuclei. DISCUSSION: Our results show that these hybrid cells fused by CCs and MeSCs express some characteristics of the CC phenotype. A subpopulation of these hybrid cells are dividing cells with positive BrdU immunostaining, suggesting that a novel cellular production could be developed by a "reprogramming" mechanism through the application of targeted cell fusion strategies.

Genetically engineered human mesenchymal stem cells produce met-enkephalin at augmented higher levels in vitro.

We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patient's bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.