Yossef S. Levy

Protective Effects of Neurotrophic Factor–Secreting Cells in a 6-OHDA Rat Model of Parkinson Disease

Stem cell-based therapy is a promising treatment for neurodegenerative diseases. In our laboratory, a novel protocol has been developed to induce bone marrow-derived mesenchymal stem cells (MSC) into neurotrophic factors- secreting cells (NTF-SC), thus combining stem cell-based therapy with the NTF-based neuroprotection. These cells produce and secrete factors such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor. Conditioned medium of the NTF-SC that was applied to a neuroblastoma cell line (SH-SY5Y) 1 h before exposure to the neurotoxin 6-hydroxydopamine (6-OHDA) demonstrated marked protection. An efficacy study was conducted on the 6-OHDA-induced lesion, a rat model of Parkinsons disease. The cells, either MSC or NTF-SC, were transplanted on the day of 6-OHDA administration and amphetamine-induced rotations were measured as a primary behavior index. We demonstrated that when transplanted posterior to the 6-OHDA lesion, the NTF-SC ameliorated amphetamine-induced rotations by 45%. HPLC analysis demonstrated that 6-OHDA induced dopamine depletion to a level of 21% compared to the untreated striatum. NTF-SC inhibited dopamine depletion to a level of 72% of the contralateral striatum. Moreover, an MRI study conducted with iron-labeled cells, followed by histological verification, revealed that the engrafted cells migrated toward the lesion. In a histological assessment, we found that the cells induced regeneration in the damaged striatal dopaminergic nerve terminal network. We therefore conclude that the induced MSC have a therapeutic potential for neurodegenerative processes and diseases, both by the NTFs secretion and by the migratory trait toward the diseased tissue.

Regenerative effect of neural-induced human mesenchymal stromal cells in rat models of Parkinson's disease

BACKGROUND Human bone marrow multipotent mesenchymal stromal cells (hMSC), because of their capacity of multipotency, may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hMSC to replace the midbrain dopamine neurons selectively lost in Parkinsons disease. METHODS Cells were isolated and characterized, then induced to differentiate toward the neural lineage. In vitro analysis of neural differentiation was achieved using various methods to evaluate the expression of neural and dopaminergic genes and proteins. Neural-induced cells were then transplanted into the striata of hemi-Parkinsonian rats; animals were tested for rotational behavior and, after killing, immunohistochemistry was performed. RESULTS Following differentiation, cells displayed neuronal morphology and were found to express neural genes and proteins. Furthermore, some of the cells exhibited gene and protein profiles typical of dopaminergic precursors. Finally, transplantation of neural-induced cells into the striatum of hemi-Parkinsonian rats resulted in improvement of their behavioral deficits, as determined by apomorphine-induced rotational behavior. The transplanted induced cells proved to be of superior benefit compared with the transplantation of naive hMSC. Immunohistochemical analysis of grafted brains revealed that abundant induced cells survived the grafts and some displayed dopaminergic traits. DISCUSSION Our results demonstrate that induced neural hMSC may serve as a new cell source for the treatment of neurodegenerative diseases and have potential for broad application. These results encourage further developments of the possible use of hMSC in the treatment of Parkinsons disease.

Safety and Clinical Effects of Mesenchymal Stem Cells Secreting Neurotrophic Factor Transplantation in Patients With Amyotrophic Lateral Sclerosis: Results of Phase 1/2 and 2a Clinical Trials

IMPORTANCE Preclinical studies have shown that neurotrophic growth factors (NTFs) extend the survival of motor neurons in amyotrophic lateral sclerosis (ALS) and that the combined delivery of these neurotrophic factors has a strong synergistic effect. We have developed a culture-based method for inducing mesenchymal stem cells (MSCs) to secrete neurotrophic factors. These MSC-NTF cells have been shown to be protective in several animal models of neurodegenerative diseases. OBJECTIVE To determine the safety and possible clinical efficacy of autologous MSC-NTF cells transplantation in patients with ALS. DESIGN, SETTING, AND PARTICIPANTS In these open-label proof-of-concept studies, patients with ALS were enrolled between June 2011 and October 2014 at the Hadassah Medical Center in Jerusalem, Israel. All patients were followed up for 3 months before transplantation and 6 months after transplantation. In the phase 1/2 part of the trial, 6 patients with early-stage ALS were injected intramuscularly (IM) and 6 patients with more advanced disease were transplanted intrathecally (IT). In the second stage, a phase 2a dose-escalating study, 14 patients with early-stage ALS received a combined IM and IT transplantation of autologous MSC-NTF cells. INTERVENTIONS Patients were administered a single dose of MSC-NTF cells. MAIN OUTCOMES AND MEASURES The primary end points of the studies were safety and tolerability of this cell therapy. Secondary end points included the effects of the treatment on various clinical parameters, such as the ALS Functional Rating Scale-Revised score and the respiratory function. RESULTS Among the 12 patients in the phase 1/2 trial and the 14 patients in the phase 2a trial aged 20 and 75 years, the treatment was found to be safe and well tolerated over the study follow-up period. Most of the adverse effects were mild and transient, not including any treatment-related serious adverse event. The rate of progression of the forced vital capacity and of the ALS Functional Rating Scale-Revised score in the IT (or IT+IM)-treated patients was reduced (from -5.1% to -1.2%/month percentage predicted forced vital capacity, P < .04 and from -1.2 to 0.6 ALS Functional Rating Scale-Revised points/month, P = .052) during the 6 months following MSC-NTF cell transplantation vs the pretreatment period. Of these patients, 13 (87%) were defined as responders to either ALS Functional Rating Scale-Revised or forced vital capacity, having at least 25% improvement at 6 months after treatment in the slope of progression. CONCLUSIONS AND RELEVANCE The results suggest that IT and IM administration of MSC-NTF cells in patients with ALS is safe and provide indications of possible clinical benefits, to be confirmed in upcoming clinical trials. TRIAL REGISTRATION clinicaltrials.gov Identifiers: NCT01051882 and NCT01777646.

Induction of Neuron-Specific Enolase Promoter and Neuronal Markers in Differentiated Mouse Bone Marrow Stromal Cells

Mesenchymal stem cells in the adult bone marrow are differentiated to connective tissue, muscle, bone, cartilage, and fat cells. Recent studies in cultures, animal models, and humans demonstrated the plasticity of these cells and their capacity to express neuronal markers. However, questions were raised as to whether the neuronal phenotypes reflect transient changes or even fusion with neurons. In this study, we induced the differentiation of mouse stromal cells to neuron-like cells and observed the activation of the tissue-specific promoter of neuron-specific enolase (NSE). We used transgenic (Tg) mice that carry the antiapoptotic human bcl-2 gene, expressed only in neurons under the NSE promoter. Some previous studies have indicated that the transgene induces neuroprotection in various animal models of neurodegenerative diseases. We found that following induction, the mouse stromal cells demonstrate neuronal phenotype and express the neuronal marker, NeuN (neural nuclei protein). However, most of the stromal cells derived from the Tg mice, but not the wild type, also expressed human Bcl-2, as indicated by immunocytochemistry. Furthermore, these induced neuron-like cells were more resistant to cell death induced by dopamine. In conclusion, our experimental models showed that stromal cells might be induced to neuronal phenotypes and activate neuronal-specific promoters. Moreover, neurons targeted over expression of the human bcl-2 gene and provided high resistance against such apoptotic insults. This novel strategy reveals a new horizon in the improvement of gene therapy, based on stem cell transplantation in neurodegenerative diseases.

Embryonic and adult stem cells as a source for cell therapy in Parkinson’s disease

The rationale behind the use of cells as therapeutic modalities for neurodegenerative diseases in general, and in Parkinson’s disease (PD) in particular, is that they will improve patient’s functioning by replacing the damaged cell population. It is reasoned that these cells will survive, grow neurites, establish functional synapses, integrate best and durably with the host tissue mainly in the striatum, renew the impaired wiring, and lead to meaningful clinical improvement. To increase the generation of dopamine, researchers have already transplanted non-neuronal cells, without any genetic manipulation or after introduction of genes such as tyrosine hydroxylase, in animal models of PD. Because these cells were not of neuronal origin, they developed without control, did not integrate well into the brain parenchyma, and their survival rates were low. Clinical experiments using cell transplantation as a therapy for PD have been conducted since the 1980s. Most of these experiments used fetal dopaminergic cells originating in the ventral mesencephalic tissue obtained from fetuses. Although it was shown that the transplanted cells survived and some patients benefited from this treatment, others suffered from severe dyskinesia, probably caused by the graft’s excessive and uncontrolled production and release of dopamine. It is now recognized that cell-replacement strategy will be effective in PD only if the transplanted cells have the same abilities, such as dopamine synthesis and control release, reuptake, and metabolizing dopamine, as the original dopaminergic neurons. Recent studies on embryonic and adult stem cells have demonstrated that cells are able to both self-renew and produce differentiated tissues, including dopaminergic neurons. These new methods offer real hope for tissue replacement in a wide range of diseases, especially PD. In this review we summarize the evidence of dopaminergic neuron generation from embryonic and adult stem cells, and discuss their application for cell therapy in PD.

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.

Hemin-induced Apoptosis in PC12 and Neuroblastoma Cells: Implications for Local Neuronal Death Associated with Intracerebral Hemorrhage

The exact pathogenesis of neuronal death following bleeding in brain parenchyma is still unknown. Hemoglobin (Hb) toxicity has been postulated to be one of the underlying mechanisms. The purpose of this study was to examine the possible contribution to neurotoxicity of each of the Hb compounds and to characterize the death pathway. Pheochromocytoma (PC12) and neuroblastoma (SH-SY5Y) cell lines were exposed to Hb, globin, hemin, protoporphyrin IX, and iron for 1.5–24 h. We found that Hb and hemin are highly toxic (LD50 of 8 and 20 μmol/l, respectively) as compared to globin that was not toxic. In addition, protoporphyrin IX and iron, compounds of hemin, were less toxic than hemin itself (LD50 of 962 and 2070 μmol/l, respectively). We also demonstrated that non-specific protein digestion with proteinase-K, markedly increased Hb toxicity. Hemin-treated cells caused a typical apoptotic cell-death pattern as indicated by DNA fragmentation, caspase activation and reduction in the mitochondrial membrane potential. Treatment with the antioxicant N-acetyl-l-cysteine or iron chelator, deferoxamine, diminished hemin-induced cell death, indicating a role of oxidative stress in this deleterious process. Thus, therapeutic strategies, based on antioxidant, iron chelator and anti-apoptotic agents may be effective in counteracting Hb neurotoxicity.

Stem Cells as a Source for Cell Replacement in Parkinson’s Disease

rodegenerative disease of the basal ganglia (BG), consisting of a remarkable diversity of neuroactive substances, organized into functional subsystems. Pathologically, it is characterized by continuous dopaminergic cell loss in the nigrostriatal and other dopaminergic systems that are found outside the extrapyramidal system, and its main classic triad of signs involves resting tremor, rigidity, and bradykinesia. The disease affects about 1% of the population more than 50 years of age. Current treatment regimes for PD consist primarily of pharmacologic supplementation of the dopaminergic loss with dopamine (DA) agonist and L-3-4-dihydroxyphenylalanine (L-DOPA, levodopa), a precursor of DA. Levodopa that can readily cross the blood-brain barrier is the most effective agent controlling the symptoms of PD. Most PD patients have a good initial response to levodopa, but, after a few years, become subject to adverse effects, which include dyskinesia, fluctuations of efficacy (onoff effect), freezing, mental changes, and loss of efficacy. Functional replacement of specific neuronal populations through transplantation of neural tissue represents an attractive therapeutic strategy for treating neurodegenerative disorders such as PD. Given that most neurodegenerative diseases affect the neuronal populations of specific neurochemical phenotypes, an ideal source material for transplantation would be a reproducible cell that could be instructed to assume the desired neuronal phenotype upon differentiation. The strategy of cell replacement therapy seeks to replace the loss in synaptic signaling caused by neuronal degeneration. In late 1970s, Bjorklund and collaborators demonstrated that the transplantation of embryonic DA neural tissue, obtained from the fetal ventral mesencephalon, could reverse the symptoms of DA depletion in the unilateral 6-hydroxydopamine (6-OHDA)-treated rat model of PD.1,2 Encouraged by these findings in animal models, Lindvall and Hagell3 launched a clinical program in 1984–1985 to attempt transplantation of embryonic neural tissues into the brains of PD patients. Clinical trials with transplantation of human embryonic mesencephalic tissue into the caudate and putamen (striatum) of PD patients were initiated in 1987, and about 350 patients have since undergone transplantation.3 These clinical tests showed that grafts of fetal ventral mesencephalon successfully survive and reduce motor symptoms.4–10 Although transplantation is a promising treatment for PD, it requires as many as 5–10 fetal brains for only one PD patient, thus causing ethical and practical problems and limiting its clinical application. The mammalian adult brain is a very plastic system that is capable of incorporating transplanted stem cells into functional neurotransmission. In recent years, the questionable benefit and safety of this procedure has been raised, as a control study pointed to the high risk of adverse signs such as tardive dyskenesis.11–13 The challenge of cell replacement in PD is huge and efforts to find the best cell source 7