Ibrahim Kassis

Neuroprotection and immunomodulation with mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis.

OBJECTIVE To investigate the therapeutic potential of mesenchymal stromal cells (MSCs) in the chronic model of experimental autoimmune encephalomyelitis (EAE). DESIGN Mesenchymal stromal cells were obtained from the bone marrow of naïve C57BL and green fluorescent protein-transgenic mice and cultured with Eagle minimum essential medium/alpha medium after removal of adhering cells. Following 2 to 3 passages, MSCs were injected intraventricularly or intravenously into mice in which chronic EAE had been induced with myelin oligodendrocyte glycoprotein 35-55 peptide. RESULTS In 8 separate experiments, the intravenously and intraventricularly injected green fluorescent protein-positive MSCs were attracted to the areas of central nervous system inflammation and expressed galactocerebroside, O4, glial fibrillary acidic protein, and beta-tubulin type III. The clinical course of chronic EAE was ameliorated in MSC-treated animals (0% mortality; mean [SE] maximal EAE score, 1.76 [1.01] and 1.8 [0.46] in the intraventricular and intravenous groups, respectively, vs 13% and 21% mortality and 2.80 [0.79] and 3.42 [0.54] mean maximal score in the controls). A strong reduction in central nervous system inflammation, accompanied by significant protection of the axons (86%-95% intact axons vs 45% in the controls) was observed in the animals injected with MSCs (especially following intraventricular administration). Mesenchymal stromal cells injected intravenously were detected in the lymph nodes and exhibited systemic immunomodulatory effects, downregulating proliferation of lymphocytes in response to myelin antigens and mitogens. Mesenchymal stromal cells cultured with fibroblast growth factor and brain-derived neurotrophic factor in vitro acquired neuronal-lineage cell morphology and expressed beta-tubulin type III, nestin glial fibrillary acidic protein, and O4. CONCLUSIONS Our results indicate that stem cells derived from bone marrow may provide a feasible and practical way for neuroprotection, immunomodulation, and possibly remyelination and neuroregeneration in diseases such as multiple sclerosis.

Bone Marrow Mesenchymal Stem Cells: Agents of Immunomodulation and Neuroprotection

Mesenchymal stromal cells (MSC) are part of the bone marrow stem cells repertoire which also includes the main stem cells population of the bone marrow, the hematopoietic stem cells. The main role of MSCs is to support hematopoiesis but they can also give rise to cells of the mesodermal layers. Recently, significant interactions between MSCs and cells from the immune system have been demonstrated: MSCs were found to downregulate T and B lymphocytes, natural killer cells (NK) and antigen presenting cells through various mechanisms, including cell-to cell interaction and soluble factor production. Besides the immunomodulatory effects, MSCs were shown to possess additional stem cells features, such as the self-renewal potential and multipotency. Their debatable transdifferentiation potential to cells of the endo- and exo-dermal layer, including cells of the CNS, may explain in part their reported neuroprotective effects. Studies in vitro and in vivo (in cells cultures and in animal models) have indicated neuroprotective effects. MSCs are believed to promote functional recovery following CNS injury or inflammation, by producing trophic factors that may facilitate the mobilization of endogenous neural stem cells and promote the regeneration or the survival of the affected neurons. These immunomodulatory and neuroprotective features could make MSCs potential candidates for future therapeutic modalities in immune-mediated and neurodegenerative diseases.

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.

The potential use of stem cells in multiple sclerosis: an overview of the preclinical experience.

The reported neurodegeneration process in multiple sclerosis may explain the lack of efficacy of the currently used immunomodulating modalities and the irreversible axonal damage, which results in accumulating disability. Efforts for neuroprotective treatments have not been, so far, successful in clinical studies in other CNS diseases. Therefore, for MS, the use of stem cells may provide a logical solution, since these cells can migrate locally into the areas of white matter lesions (plaques) and have the potential to support local neurogenesis and rebuilding of the affected myelin. This may be achieved both by support of the resident CNS stem cells repertoire and by differentiation of the transplanted cells into neurons and myelin-producing cells (oligodendrocytes). Stem cells were also shown to possess immunomodulating properties, inducing systemic and local suppression of the myelin-targeting autoimmune lymphocytes. Several types of stem cells (embryonic and adult) have been described and extensively studied in animal models of CNS diseases. In this review, we summarize the experience with the use of different types of stem cells in the animal models of MS (EAE) and we describe the advantages and disadvantages of each stem cell type for future clinical applications in MS.

Repeated immunization of mice with phosphorylated-tau peptides causes neuroinflammation ☆

The recent studies of others and of us showing robust efficacy of anti-tangle immunotherapy, directed against phosphorylated (phos)-tau protein, may pave the way to clinical trials of phos-tau immunotherapy in Alzheimers-disease and other tauopathies. At this stage addressing the safety of the phos-tau-immunotherapy is highly needed, particularly since we have previously shown the neurotoxic potential of tau-immunotherapy, specifically of full-length unphosphorylated-tau vaccine under a CNS-proinflammatory milieu [induced by emulsification in complete-Freunds-adjuvant (CFA) and pertussis-toxin (PT)] in young wild-type (WT)-mice. The aim of our current study was to address safety aspects of the phos-tau-immunotherapy in both neurofibrillary-tangle (NFT)-mice as well as in WT-mice, under challenging conditions of repeated immunizations with phos-tau peptides under a CNS-proinflammatory milieu. NFT- and WT-mice were repeatedly immunized (7 injections in adult-, 4 in aged-mice) with phos-tau peptides emulsified in CFA-PT. A paralytic disease was evident in the phos-tau-immunized adult NFT-mice, developing progressively to 26.7% with the number of injections. Interestingly, the WT-mice were even more prone to develop neuroinflammation following phos-tau immunization, affecting 75% of the immunized mice. Aged mice were less prone to neuroinflammatory manifestations. Anti-phos-tau antibodies, detected in the serum of immunized mice, partially correlated with the neuroinflammation in WT-mice. This points that repeated phos-tau immunizations in the frame of a proinflammatory milieu may be encephalitogenic to tangle-mice, and more robustly to WT-mice, indicating that - under certain conditions - the safety of phos-tau immunotherapy is questionable.

T-cell reactivity against AQP4 in neuromyelitis optica

Neuromyelitis optica (NMO) is an idiopathic demyelinating disease of the CNS that can be clearly distinguished from multiple sclerosis (MS) by clinical, neuroradiogic, and pathologic criteria and the presence of the highly specific serum autoantibodies against the water channel aquaporin-4 (AQP4).1 Although studies support a central role of the anti-AQP4 antibodies in the pathogenesis of NMO, their exact involvement in the immunopathogenetic cascade of the disease is still not clear, and T cells seem to be equally crucial for the full development of clinical and histopathologic NMO.

Clinical experience with stem cells and other cell therapies in neurological diseases

To overcome the limited capacity of the CNS for regeneration, the theoretical alternative would be to use stem cells for more effective management of chronic degenerative and inflammatory neurological conditions, and also of acute neuronal damage from injuries or cerebrovascular diseases. Although the adult brain contains small numbers of stem cells in restricted areas, this intrinsic stem cell repertoire is small and does not measurably contribute to functional recovery. Embryonic cells carrying pluripotent and self-renewal properties represent the stem cell prototype, but there are additional somatic stem cells that may be harvested and expanded from various tissues during adult life. Stem cell transplantation is based on the assumption that such cells may have the potential to regenerate or support the survival of the existing, partially damaged cells. This review summarizes the state-of-the-art and the clinical worldwide experience with the use of various types of stem cells in neurological diseases.

Hematopoietic stem cell transplantation in multiple sclerosis

It is widely accepted that the main common pathogenetic pathway in multiple sclerosis (MS) involves an immune-mediated cascade initiated in the peripheral immune system and targeting CNS myelin. Logically, therefore, therapeutic approaches to the disease include modalities aiming at downregulation of the various immune elements that are involved in this immunological cascade. Since the introduction of interferons in 1993, more specific immunoactive drugs have been introduced, but still most of them can, at best, effectively modulate only the early relapsing phases of MS. The more progressed phases of the disease are not efficiently amendable by the existing immunomodulatory drugs. Moreover, localized and compartmentized inflammation in the CNS, which seems to be mostly responsible for the chronic axonal damage and resulting progression of disability, is less affected by the current drugs. A more radical approach to suppress all the inflammation in MS, including that into the CNS, could theoretically be achieved with high-dose immunosuppression using strong cytotoxic medications and resetting of the immune system by hematopoietic stem cell transplantation (HSCT). HSCT, both allogeneic and autologous, has been tried as a novel therapeutic approach in various autoimmune diseases. During the last 15 years several (mostly open) clinical studies evaluated the effect of HSTC on MS patients; the published papers showed that a high proportion of the HSCT-treated MS patients were stabilized, or even improved after the transplantation and have generally indicated a beneficial effect on disease progression. In this review, the rationale of HSCT and the summary of the results of the existing clinical trials are presented. Despite the fact that it is difficult to collectively summarize the results of all the trials, due to lack of uniformity in the conditioning and treatment protocols and of completed controlled studies, these clinical studies have provided a strong ‘proof of concept’ for HSCT in MS and have significantly contributed to our understanding of the advantages and disadvantages of each approach and HSCT protocol.

Characterization of human sporadic ALS biomarkers in the familial ALS transgenic mSOD1G93A mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder of motor neurons. Although most cases of ALS are sporadic (sALS) and of unknown etiology, there are also inherited familial ALS (fALS) cases that share a phenotype similar to sALS pathological and clinical phenotype. In this study, we have identified two new potential genetic ALS biomarkers in human bone marrow mesenchymal stem cells (hMSC) obtained from sALS patients, namely the TDP-43 (TAR DNA-binding protein 43) and SLPI (secretory leukocyte protease inhibitor). Together with the previously discovered ones-CyFIP2 and RbBP9, we investigated whether these four potential ALS biomarkers may be differentially expressed in tissues obtained from mutant SOD1(G93A) transgenic mice, a model that is relevant for at least 20% of the fALS cases. Quantitative real-time PCR analysis of brain, spinal cord and muscle tissues of the mSOD1(G93A) and controls at various time points during the progression of the neurological disease showed differential expression of the four identified biomarkers in correlation with (i) the tissue type, (ii) the stage of the disease and (iii) the gender of the animals, creating thus a novel spatiotemporal molecular signature of ALS. The biomarkers detected in the fALS animal model were homologous to those that were identified in hMSC of our sALS cases. These results support the possibility of a molecular link between sALS and fALS and may indicate common pathogenetic mechanisms involved in both types of ALS. Moreover, these results may pave the path for using the mSOD1(G93A) mouse model and these biomarkers as molecular beacons to evaluate the effects of novel drugs/treatments in ALS.

Rare combination of myasthenia and motor neuronopathy, responsive to Msc-Ntf stem cell therapy

A 75-year-old man was referred to the Hadassah Medical Center with a 6-month history of progressive limb weakness, dysarthria, and cognitive deterioration. His past medical history included prostate hyperplasia, hypothyroidism, diabetes mellitus, cardiac arrhythmias controlled by pacemaker implantation (1997), and hypertension. Autoimmune myasthenia gravis (MG) had been diagnosed 2 years earlier, based on symptoms of fluctuating fatigue, dysarthric speech, eyelid ptosis, and seropositivity for muscle acetylcholine receptor (AChR) binding antibody. MRI did not reveal thymic enlargement, and malignancy markers (and whole-body computed tomography) were negative. Moderate improvement followed treatment, initially with intravenous immune globulin and later with low dose corticosteroids, pyridostigmine, and azathioprine. The patient was evaluated previously at the Mayo Clinic (Rochester, Minnesota) and had been diagnosed with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The diagnosis was based on the cognitive exam and clinical features of progressive diffuse upper and lower motor neuron dysfunction with electromyographic findings (low motor amplitudes, no focal slowing or conduction blocks, no postexercise facilitation, presence of diffuse fibrillation and fasciculation potentials, and large complex motor unit potentials in all limbs) fulfilling the El-Escorial criteria for ALS. MRI and positron emission tomography scans of the brain showed moderate atrophy predominantly affecting the frontal and temporal lobes. Spinal fluid was acellular with slightly elevated total protein (50 mg%). Autoimmune serology revealed antibodies specific for muscle (AChR binding 9.47 nmol/L [normal 0.00–0.02]; AChR modulating 100% loss [normal 0–20%]; striational 15,360 [normal <60]) and thyroglobulin, and antinuclear antibody. On admission, the patient had memory impairment and signs of frontotemporal functional deficits, typical of FTD. Dysarthria rendered speech totally incomprehensible. He had mild eyelid ptosis bilaterally (without extraocular muscle weakness or diplopia) and was markedly quadriparetic (confined to wheelchair) with bilateral foot drop. Distal hand and foot muscles were moderately atrophic, and fasciculations were prominent in all limbs. Deep tendon reflexes were brisk in arms and legs (except hypoactive Achilles reflexes), and an extensor plantar response was evoked bilaterally. Superficial sensation was normal, and vibration sense was reduced slightly distally in the legs. Due to the diagnosis of MG, the patient did not meet inclusion criteria for the Hadassah clinical trial in ALS with autologous enhanced mesenchymal stem cells (MSCNTF, BrainstormVR , Petach Tikva; NCT01051882). The Hadassah Ethics Committee issued a special license for treatment on a compassionate basis. MSC-NTF (prepared from the patient’s bone marrow) were injected intrathecally (1.5 3 10 per kilogram of body weight) and at 24 sites along the biceps and triceps muscles of the right arm (1.5 3 10 per site). The intrathecal and intramuscular administration of the cells were chosen based on previous animal and clinical studies from our groups, which showed good migration of the intrathecally injected cells to the CNS and amelioration of the “dying-back” phenomenon by intramuscularly injected MSC in early stages of ALS in the SOD mouse model (unpublished data and Dadon-Nachum et al.). For the next 2 days the patient had a low-grade fever, headache, and was more confused, but at discharge, these problems had completely subsided. Treatment with azathioprine (125 mg/day) was discontinued 1 month before the injection and readministered 30 days after the treatment. Pyridostigmine (60 mg 3 times daily) and low dose oral prednisolone (10 mg/day) were continued. At 1 month after transplantation, the patient and his family reported significant improvement in cognition, speech, and muscle power. He was able to walk at least 20 meters without any support. The dysarthria improved to the extent he was able to clearly deliver a speech to an audience. ALS Functional Score Scale-Revised (ALSFRS-R, performed at all time points by the same evaluator and confirmed by a second senior examiner) score rose from 36 to 44, and respiratory forced vital capacity (FVC) and cognitive function also improved significantly (Supplementary Table 1, which is available online, and Fig. 1). VC 2013 Wiley Periodicals, Inc.