Tali Ben-Zur

Induction of Human Mesenchymal Stem Cells into Dopamine-Producing Cells with Different Differentiation Protocols

Several reports have shown that human mesenchymal stem cells (MSCs) are capable of differentiating outside the mesenchymal lineage. We sought to induce MSCs to differentiate into dopamine-producing cells for potential use in autologous transplantation in patients with Parkinsons disease (PD). Following cell culture with various combinations of differentiation agents under serum-free defined conditions, different levels of up-regulation were observed in the protein expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Further analysis of selected differentiation protocols revealed that the induced cells displayed a neuron-like morphology and expressed markers suggesting neuronal differentiation. In addition, there was an increase in Nurr 1, the dopaminergic transcription factor gene, concomitant with a decrease gamma-aminobutyric acid (GABA)ergic marker expression, suggesting a specific dopaminergic direction. Moreover, the induced cells secreted dopamine in response to depolarization. These results demonstrate the great therapeutic potential of human MSCs in PD.

Wnt Signaling Enhances Neurogenesis and Improves Neurological Function after Focal Ischemic Injury

Stroke potently stimulates cell proliferation in the subventricular zone of the lateral ventricles with subsequent neuroblast migration to the injured striatum and cortex. However, most of the cells do not survive and mature. Extracellular Wnt proteins promote adult neurogenesis in the neurogenic niches. The aim of the study was to examine the efficacy of Wnt signaling on neurogenesis and functional outcome after focal ischemic injury. Lentivirus expressing Wnt3a-HA (LV-Wnt3a-HA) or GFP (LV-GFP) was injected into the striatum or subventricular zone of mice. Five days later, focal ischemic injury was induced by injection of the vasoconstrictor endothelin-1 into the striatum of the same hemisphere. Treatment with LV-Wnt3a-HA into the striatum significantly enhanced functional recovery after ischemic injury and increased the number of BrdU-positive cells that differentiated into mature neurons in the ischemic striatum by day 28. Treatment with LV-Wnt3a-HA into the subventricular zone significantly enhanced functional recovery from the second day after injury and increased the number of immature neurons in the striatum and subventricular zone. This was accompanied by reduced dissemination of the neuronal injury. Our data indicate that Wnt signaling appears to contribute to functional recovery after ischemic injury by increasing neurogenesis or neuronal survival in the striatum.

Dopaminergic differentiation of human mesenchymal stem cells--utilization of bioassay for tyrosine hydroxylase expression.

Parkinsons disease (PD) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra. In PD, the best therapeutic modalities cannot halt the degeneration. The selective hallmark pathology and the lack of effective treatment make PD an appropriate candidate for cell replacement therapy. Adult autologous bone-marrow-derived mesenchymal stem cells (MSCs) have been investigated as candidates for cell replacement strategies. Several laboratories, including ours, have induced MSCs into neuron-like cells demonstrating a variety of neuronal markers including dopaminergic characteristics, such as the expression of tyrosine hydroxylase (TH). This project aimed to induce MSCs into mature dopamine secreting cells and to generate a bioassay to evaluate the induction. For that purpose, we created a reporter vector containing a promoter of TH, the rate-limiting enzyme in the dopamine synthesis and red fluorescent protein DsRed2. Transfection of human neuroblastoma, dopamine synthesizing, SH-SY5Y cells confirmed the reliability of the constructed reporter plasmid. Following dopaminergic differentiation of the transfected human MSCs cells, TH expressing cells were identified and quantified using flow cytometry. Further study revealed that not only did the differentiated cells activate TH promoter but they also expressed TH protein and secreted dopamine. The reported results indicate that MSCs may be primed in vitro towards a dopaminergic fate offering the promise of innovative therapy for currently incurable human disorders, including PD.

Knocking Out DJ-1 Attenuates Astrocytes Neuroprotection Against 6-Hydroxydopamine Toxicity

Astrocytes are the most abundant glial cell type in the brain. Impairment in astrocyte functions can critically influence neuronal survival and leads to neurodegeneration. Parkinson’s disease (PD) is a common neurodegenerative disorder, characterized by motor dysfunction that results from progressive neuronal loss. Astrocytic dysfunction was demonstrated in human samples and in experimental models of PD. Mutations in DJ-1 (PARK7) leading to loss of functional protein cause familial PD and enhance sensitivity to oxidative insults. Recently, an increase in DJ-1’s expression was found in reactive astrocytes in various neurodegenerative disorders. Here we show that lack of DJ-1 attenuates astrocytes’ ability to support neuronal cells, thereby leading to accelerated neuronal damage. DJ-1 knockout mice demonstrated increased vulnerability in vivo to 6-hydroxydopamine (6-OHDA) hemiparkinsonian PD model. Astrocytes isolated from DJ-1 knockout mice showed an inferior ability to protect human neuroblastoma cells against 6-OHDA insult both by co-culture and through their conditioned media, as compared to wild-type astrocytes. DJ-1 knockout astrocytes showed blunted ability to increase the expression of cellular protective mechanisms against oxidative stress mediated via Nrf-2 and HO-1 in response to exposure to 6-OHDA. These experiments demonstrated that lack of DJ-1 impairs astrocyte-mediated neuroprotection.

Altered astrocytic response to activation in SOD1(G93A) mice and its implications on amyotrophic lateral sclerosis pathogenesis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, rapidly progressive, neurodegenerative disease caused by motor neuron degeneration. Despite extensive efforts, the underlying cause of ALS and the path of neurodegeneration remain elusive. Astrocyte activation occurs in response to central nervous system (CNS) insult and is considered a double edged sword in many pathological conditions. We propose that reduced glutamatergic and trophic response of astrocytes to activation may, over time, lead to accumulative CNS damage, thus facilitating neurodegeneration. We found that astrocytes derived from the SOD1G93A ALS mouse model exhibit a reduced glutamatergic and trophic response to specific activations compared to their wild‐type counterparts. Wild‐type astrocytes exhibited a robust response when activated with lipopolysaccharide (LPS), G5 or treated with ceftriaxone in many parameters evaluated. These parameters include increased expression of GLT‐1 and GLAST the two major astrocytic glutamate transporters, accompanied by a marked increase in the astrocytic glutamate clearance and up‐regulation of neurtrophic factor expression. However, not only do un‐treated SOD1G93A astrocytes take up glutamate less efficiently, but in response to activation they show no further increase in any of the glutamatergic parameters evaluated. Furthermore, activation of wild‐type astrocytes, but not SOD1G93A astrocytes, improved their ability to protect the motor neuron cell line NSC‐34 from glutamate induced excitotoxicity. Our data indicates that altered astrocyte activation may well be pivotal to the pathogenesis of ALS.

Placental mesenchymal stromal cells induced into neurotrophic factor-producing cells protect neuronal cells from hypoxia and oxidative stress.

BACKGROUND AIMS Mesenchymal stromal cells (MSC) may be useful in a range of clinical applications. The placenta has been suggested as an abundant, ethically acceptable, less immunogenic and easily accessible source of MSC. The aim of this study was to evaluate the capacity of induced placental MSC to differentiate into neurotrophic factor-producing cells (NTF) and their protective effect on neuronal cells. METHODS MSC were isolated from placentas and characterized by fluorescence-activated cell sorting (FACS). The cells underwent an induction protocol to differentiate them into NTF. Analysis of the cellular differentiation was done using polymerase chain reactions (PCR), immunocytochemical staining and enzyme-linked immunosorbent assays (ELISA). Conditioned media from placental MSC (PL-MSC) and differentiated MSC (PL-DIFF) were collected and examined for their ability to protect neural cells. RESULTS The immunocytochemical studies showed that the cells displayed typical MSC membrane markers. The cells differentiated into osteoblasts and adipocytes. PCR and immunohistology staining demonstrated that the induced cells expressed typical astrocytes markers and neurotrophic factors. Vascular endothelial growth factor (VEGF) levels were higher in the conditioned media from PL-DIFF compared with PL-MSC, as indicated by ELISA. Both PL-DIFF and PL-MSC conditioned media markedly protected neural cells from oxidative stress induced by H(2)O(2) and 6-hydroxydopamine. PL-DIFF conditioned medium had a superior effect on neuronal cell survival. Anti-VEGF antibodies (Bevacizumab) reduced the protective effect of the conditioned media from differentiated and undifferentiated MSC. CONCLUSIONS This study has demonstrated a neuroprotective effect of MSC of placental origin subjected to an induction differentiation protocol. These data offer the prospect of using placenta as a source for stem cell-based therapies.

Intracerebral adult stem cells transplantation increases brain-derived neurotrophic factor levels and protects against phencyclidine-induced social deficit in mice

Stem cell-based regenerative therapy is considered a promising cellular therapeutic approach for the patients with incurable brain diseases. Mesenchymal stem cells (MSCs) represent an attractive cell source for regenerative medicine strategies for the treatment of the diseased brain. Previous studies have shown that these cells improve behavioral deficits in animal models of neurological disorders such as Parkinsons and Huntingtons diseases. In the current study, we examined the capability of intracerebral human MSCs transplantation (medial pre-frontal cortex) to prevent the social impairment displayed by mice after withdrawal from daily phencyclidine (PCP) administration (10 mg kg−1 daily for 14 days). Our results show that MSCs transplantation significantly prevented the PCP-induced social deficit, as assessed by the social preference test. In contrast, the PCP-induced social impairment was not modified by daily clozapine treatment. Tissue analysis revealed that the human MSCs survived in the mouse brain throughout the course of the experiment (23 days). Significantly increased cortical brain-derived neurotrophic factor levels were observed in the MSCs-treated group as compared with sham-operated controls. Furthermore, western blot analysis revealed that the ratio of phosphorylated Akt to Akt was significantly elevated in the MSCs-treated mice compared with the sham controls. Our results demonstrate that intracerebral transplantation of MSCs is beneficial in attenuating the social deficits induced by sub-chronic PCP administration. We suggest a novel therapeutic approach for the treatment of schizophrenia-like negative symptoms in animal models of the disorder.

A DJ-1 Based Peptide Attenuates Dopaminergic Degeneration in Mice Models of Parkinson's Disease via Enhancing Nrf2

Drugs currently used for treating Parkinsons disease patients provide symptomatic relief without altering the neurodegenerative process. Our aim was to examine the possibility of using DJ-1 (PARK7), as a novel therapeutic target for Parkinsons disease. We designed a short peptide, named ND-13. This peptide consists of a 13 amino acids segment of the DJ-1-protein attached to 7 amino acids derived from TAT, a cell penetrating protein. We examined the effects of ND-13 using in vitro and in vivo experimental models of Parkinsons disease. We demonstrated that ND-13 protects cultured cells against oxidative and neurotoxic insults, reduced reactive oxygen species accumulation, activated the protective erythroid-2 related factor 2 system and increased cell survival. ND-13 robustly attenuated dopaminergic system dysfunction and in improved the behavioral outcome in the 6-hydroxydopamine mouse model of Parkinsons disease, both in wild type and in DJ-1 knockout mice. Moreover, ND-13 restored dopamine content in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model. These findings validate DJ-1 as a promising therapeutic target in Parkinsons disease and identify a novel peptide with clinical potential, which may be significant for a broader range of neurological diseases, possibly with an important impact for the neurosciences.

Astrocyte-Like Cells Derived From Human Oral Mucosa Stem Cells Provide Neuroprotection In Vitro and In Vivo

Human oral mucosa stem cells (hOMSC) are a recently described neural crest‐derived stem cell population. Therapeutic quantities of potent hOMSC can be generated from small biopsies obtained by minimally invasive procedures. Our objective was to evaluate the potential of hOMSC to differentiate into astrocyte‐like cells and provide peripheral neuroprotection. We induced hOMSC differentiation into cells showing an astrocyte‐like morphology that expressed characteristic astrocyte markers as glial fibrillary acidic protein, S100β, and the excitatory amino acid transporter 1 and secreted neurotrophic factors (NTF) such as brain‐derived neurotrophic factor, vascular endothelial growth factor, glial cell line‐derived neurotrophic factor, and insulin‐like growth factor 1. Conditioned medium of the induced cells rescued motor neurons from hypoxia or oxidative stress in vitro, suggesting a neuroprotective effect mediated by soluble factors. Given the neuronal support (NS) ability of the cells, the differentiated cells were termed hOMSC‐NS. Rats subjected to sciatic nerve injury and transplanted with hOMSC‐NS showed improved motor function after transplantation. At the graft site we found the transplanted cells, increased levels of NTF, and a significant preservation of functional neuromuscular junctions, as evidenced by colocalization of α‐bungarotoxin and synaptophysin. Our findings show for the first time that hOMSC‐NS generated from oral mucosa exhibit neuroprotective effects in vitro and in vivo and point to their future therapeutic use in neural disorders.

Mesenchymal stem cells protect from sub-chronic phencyclidine insult in vivo and counteract changes in astrocyte gene expression in vitro

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine strategies in brain diseases. Experimental studies have shown that repeated administration of phencyclidine (PCP) leads to schizophrenia-like behavioral changes in mice. The aim of the present study was to explore the effectiveness of MSC transplantation into the hippocampus in attenuating PCP-induced social behavior deficits. PCP was administered subcutaneously to C57bl mice (10mg/kg daily) for 2 weeks. On the first day of PCP administration, adult human MSCs were transplanted into the hippocampus. A week after the last PCP dose, the mice underwent social preference testing. MSC transplantation was associated with a significant reduction in the adverse social behavior induced by PCP. Immunohistochemical analysis revealed that the stem cells survived in the mouse brain, and hippocampal Western blot analysis revealed a statistical trend towards a decrease in cleaved caspase 3 protein levels in the stem cell treated group. Upon in vitro co-culture of astrocytes and MSCs, the MSCs, in the presence of PCP, positively regulated astrocyte expression of genes involved in glutamate metabolism and antioxidant defenses. These findings suggest that MSC transplantation into the hippocampus may serve as a novel neuroprotective tool for the treatment of the PCP-induced schizophrenia-like social endophenotype. The mechanism underlying the beneficial behavioral effect may involve modulation of host astrocyte functioning, including glutamate processing and antioxidant capacity.