Ofer Sadan

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.

Bone-marrow-derived mesenchymal stem cell therapy for neurodegenerative diseases.

Background: Stem-cell-based therapy is a promising new approach to handling neurodegenerative diseases. One of the most promising cellular sources is bone-marrow-derived mesenchymal stem cells (MSCs) also termed multipotent stromal cells. MSCs represent an autologous source and are abundant and non-tumorigenic. Additionally, MSCs possess the useful characteristics of homing and chemokine secretion. Objective/methods: Since neurodegenerative diseases have many pathological processes in common, a specific therapeutic agent could potentially ameliorate the symptoms of several distinct neurodegenerative diseases. In this review we demonstrate the wide variety of mechanisms by which MSCs can influence neurodegenerative processes. Results/conclusions: The mechanisms by which transplanted MSCs influence neurodegenerative diseases can be broadly classified as cellular replacement or paracrine secretion, with the latter subdivided into trophic factor secretion or immunomodulation by cytokines. Emerging research suggests that genetic manipulations before transplantation could enhance the therapeutic potential of MSCs. Such manipulation could turn the cells into a ‘Trojan horse’, to deliver specific proteins, or promote reprogramming of the MSCs into the neural lineage. Clinical trials testing MSC-based therapies for familial amyotrophic lateral sclerosis and multiple sclerosis are in progress.

Intravitreal Injections of Neurotrophic Factors Secreting Mesenchymal Stem Cells Are Neuroprotective in Rat Eyes following Optic Nerve Transection

PURPOSE To evaluate the neuroprotective effect of intravitreal injections of neurotrophic factors secreting mesenchymal stem cells (NTF-SCs) on the survival of retinal ganglion cells (RGCs) in rat eyes after optic nerve transection (ONT). METHODS Rat and human bone marrow-derived mesenchymal stem cells (MSCs) were induced to secrete high levels of NTF. The neuroprotective effect from intravitreally injected untreated MSCs or NTF-SCs was compared with that from PBS injections using an ONT model in 146 rats. RGCs were labeled by applying rhodamine dextran to the orbital optic nerve or by injecting Fluorogold into the superior colliculus. Cell- and saline-treated eyes were compared 8 days after ONT. For tracking, MSCs and NTF-SCs were labeled with PKH26 and analyzed at 2 hours and at 10, 17, and 24 days using immunohistochemistry and RT-PCR. RESULTS Mean RGC survival at 8 days after transection increased significantly after intravitreal injections of human NTF-SCs (69% ± 3%) or of untreated human MSCs (66% ± 5%) versus PBS (46% ± 3%; P = 0.0005 and P = 0.03, respectively). In an additional set of experiments, human NTF-SCs versus PBS were significantly neuroprotective, but bone marrow-derived rat NTF-SCs were not (P = 0.001 and P = 0.1, respectively). Immunohistochemistry demonstrated that human-derived MSCs, human NTF-SCs, and rat-derived NTF-SCs survived at least 24 days after intravitreal injection. CONCLUSIONS Bone marrow-derived MSCs can deliver NTFs by intravitreal injection and can be neuroprotective after ONT. This approach might be further studied to deliver NTFs by autotransplantation in glaucomatous eyes.

Migration of Neurotrophic Factors‐Secreting Mesenchymal Stem Cells Toward a Quinolinic Acid Lesion as Viewed by Magnetic Resonance Imaging

Stem cell‐based treatment is a promising frontier for neurodegenerative diseases. We propose a novel protocol for inducing the differentiation of rat mesenchymal stem cells (MSCs) toward neurotrophic factor (NTF)‐secreting cells as a possible neuroprotective agent. One of the major caveats of stem cell transplantation is their fate post‐transplantation. To test the viability of the cells, we tracked the transplanted cells in vivo by magnetic resonance imaging (MRI) scans and validated the results by histology. MSCs went through a two‐step medium‐based differentiation protocol, followed by in vitro characterization using immunocytochemistry and immunoblotting analysis of the cell media. We examined the migratory properties of the cells in the quinolinic acid (QA)‐induced striatal lesion model for Huntingtons disease. The induced cells were labeled and transplanted posterior to the lesion. Rats underwent serial MRI scans to detect cell migration in vivo. On the 19th day, animals were sacrificed, and their brains were removed for immunostaining. Rat MSCs postinduction exhibited both neuronal and astrocyte markers, as well as production and secretion of NTFs. High‐resolution two‐dimensional and three‐dimensional magnetic resonance images revealed that the cells migrated along a distinct route toward the lesion. The in vivo MRI results were validated by the histological study, which demonstrated that phagocytosis had only partially occurred and that MRI could correctly depict the status of the migrating cells. The results show that these cells migrated toward a QA lesion and therefore survived for 19 days post‐transplantation. This gives hope for future research harnessing these cells for treating neurodegenerative diseases.

Mesenchymal stem cells induced to secrete neurotrophic factors attenuate quinolinic acid toxicity: A potential therapy for Huntington's disease

Huntingtons disease (HD) is a hereditary, progressive and ultimately fatal neurodegenerative disorder. Excitotoxicity and reduced availability of neurotrophic factors (NTFs) likely play roles in HD pathogenesis. Recently we developed a protocol that induces adult human bone marrow derived mesenchymal stem cells (MSCs) into becoming NTF secreting cells (NTF(+) cells). Striatal transplantation of such cells represents a promising autologous therapeutic approach whereby NTFs are delivered to damaged areas. Here, the efficacy of NTF(+) cells was evaluated using the quinolinic acid (QA) rat model for excitotoxicity. We show that NTF(+) cells transplanted into rat brains after QA injection survive transplantation (19% after 6 weeks), maintain their NTF secreting phenotype and significantly reduce striatal volume changes associated with QA lesions. Moreover, QA-injected rats treated with NTF(+) cells exhibit improved behavior; namely, perform 80% fewer apomorphine induced rotations than PBS-treated QA-injected rats. Importantly, we found that MSCs derived from HD patients can be induced to become NTF(+) cells and exert efficacious effects similarly to NTF(+) cells derived from healthy donors. To our knowledge, this is the first study to take adult bone marrow derived mesenchymal stem cells from patients with an inherited disease, transplant them into an animal model and evidence therapeutic benefit. Using MRI we demonstrate in vivo that PBS-treated QA-injected striatae exhibit increasing T(2) values over time in lesioned regions, whereas T(2) values decrease in equivalent regions of QA-injected rats treated with NTF(+) cells. We conclude that NTF cellular treatment could serve as a novel therapy for managing HD.

Differentiated mesenchymal stem cells for sciatic nerve injury.

Sciatic nerve injury is common and may cause neurological deficits. Previous studies showed that administration of neurotrophic factors (NTFs), naturally occurring proteins that support the development and survival of neurons, preserved and protected damaged motor neuron in the injured sciatic nerve. We have been successful in converting bone marrow-derived mesenchymal stem cells into astrocyte-like cells that produce and secrete NTFs (NTF+ cells). These cells demonstrate typical astrocyte morphology, express characteristic astrocyte markers and secrete high levels of NTFs. We have already shown that these cells and their conditioned media can protect neurons in culture and in animal models of neurodegenerative diseases. In the current study we examined whether NTF+ cells are capable of rescuing motor neurons in a rat sciatic nerve injury model, where the right hind limb sciatic nerve was crushed. Rats were transplanted with NTF+ cells, MSCs or PBS into the lesion site. In rats injected with the NTF+ cells motor function was markedly preserved. Moreover, NTF+ cells significantly inhibited the degeneration of the neuromuscular junctions and preserved the myelinated motor axons. Our findings suggest that autologous therapeutic approach can alleviate signs of sciatic nerve injury and probably other neurological disorders.

Comparative characterization of bone marrow-derived mesenchymal stromal cells from four different rat strains

BACKGROUND AIMS Bone marrow (BM) multipotent mesenchymal stromal cells (MSC) hold great potential for cell-based regenerative medicine. Because of the growing use of autologous rat MSC transplantation in various rat models, there is a need to establish minimal criteria for rat MSC characterization independent of the specific strain employed in each study. We aimed to compare the phenotypic and functional traits of BM MSC from the four strains of rats commonly used in research: Fisher, Lewis, Sprague-Dawley and Wistar. METHODS Rat MSC were isolated from the BM of the four different rat strains in an identical fashion. Cells were characterized for their cell-surface phenotype in early and late passage. Functional mesenchymal differentiation capacities were examined following adipogenic and osteogenic inductions. Population doubling times were determined across the four strains throughout 10 passages. In vitro proliferation assays of immune cells were conducted following co-culture of spleen cells and MSC of the four different strains. RESULTS We found that rat MSC from different strains exhibited similar cell-surface phenotype. Expansion rates and differentiation capacities of the MSC were also similar across the different strains. Co-culture of rat MSC with spleen cells obtained from rats of a different strain did not induce proliferation of immune cells. CONCLUSIONS Our findings suggest that BM-derived MSC from different strains share similar characteristics, in contrast to the variations previously described in the characterization of mice MSC from different strains.

Mapping apparent eccentricity and residual ensemble anisotropy in the gray matter using angular double-pulsed-field-gradient MRI

Conventional diffusion MRI methods are mostly capable of portraying microarchitectural elements such as fiber orientation in white matter from detection of diffusion anisotropy, which arises from the coherent organization of anisotropic compartments. Double‐pulsed‐field‐gradient MR methods provide a means for obtaining microstructural information such as compartment shape and microscopic anisotropies even in scenarios where macroscopic organization is absent. Here, we apply angular double‐pulsed‐gradient‐spin‐echo MRI in the rat brain both ex vivo and in vivo for the first time. Robust angular dependencies are detected in the brain at long mixing time (tm). In many pixels, the oscillations seem to originate from residual directors in randomly oriented media, i.e., from residual ensemble anisotropy, as corroborated by quantitative simulations. We then developed an analysis scheme that enables one to map of structural indices such as apparent eccentricity (aE) and residual phase (φ) that enables characterization of the rat brain in general, and especially the rat gray matter. We conclude that double‐pulsed‐gradient‐spin‐echo MRI may in principle become important in characterizing gray matter morphological features and pathologies in both basic and applied neurosciences. Magn Reson Med, 2012.

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 novel brain-targeted antioxidant (AD4) attenuates haloperidol-induced abnormal movement in rats: implications for tardive dyskinesia.

Background:Tardive dyskinesia (TD), characterized by abnormal movements, is the major late-onset chronic side effect of antipsychotic treatment found in about 30% of those patients. The association of oxidative stress and the release of free radicals is one of the hallmarks of dopaminergic malfunctions and is one of the leading theories suggested for the pathophysiology of TD. To this day, no brain-targeted antioxidant has been tested as a potential treatment of TD. In light of this assumption, the authors chose a novel, low-molecular weight thiol antioxidant, N-acetyl cysteine amide (AD4), that crosses the blood-brain barrier as a possible treatment of TD. Objective:To examine the protective effects of the novel brain-penetrating antioxidant AD4 on TD experimental models. Methods:The typical vacuous chewing movement occurs in rats following chronic haloperidol injections (1.5 mg/kg/day intraperitoneally for 21 days). This purposeless mouth opening in the vertical plane is similar to TD symptoms in humans. The authors tested rats treated with haloperidol without or with AD4 in the drinking water (1 g/kg orally). Thiobarbituric acid reactive substances and anticarbonyl antibodies were used to measure oxidation of membranes and proteins. Results:Haloperidol increased the vacuous chewing movements to 66.5 ± 7.6 movements/5 minutes compared with 16.4 ± 2.4 movements/5 minutes in untreated rats (P < 0.01). Coadministration of haloperidol and AD4 decreased the vacuous chewing movements level to 42.1 ± 6.7 movements/5 minutes (P < 0.05). Haloperidol also increased the level of lipid peroxidation and protein oxidation in the rat brain, whereas coadministration with AD4 preserved their normal levels. Conclusion:Haloperidol causes behavioral abnormalities associated with oxidative stress in rats, similar to TD. AD4, the brain-targeted potent antioxidant, reduces the cellular oxidation markers and improves the typical clinical behavior. Hence, AD4 is a potential new treatment of antipsychotic-induced TD.