Dimitrios Karussis

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.

Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis

Transplanted neural precursor cells remyelinate efficiently acutely demyelinated focal lesions. However, the clinical value of cell transplantation in a chronic, multifocal disease like multiple sclerosis will depend on the ability of transplanted cells to migrate to the multiple disease foci in the brain. Here, we expanded newborn rat neural precursor cells in spheres and transplanted them intracerebroventricularly or intrathecally in rats. The cells were labeled by the nuclear fluorescent dye Hoechst or by incubation with BrdU to enable their identification at 2 days and 2 weeks after transplantation, respectively. Spheres consisted of PSA‐NCAM+, nestin+, NG2− undifferentiated precursor cells that differentiated in vitro into astrocytes, oligodendrocytes, and neurons. Spheres that were transplanted into intact rats remained mostly in the ventricles or in the spinal subarachnoid space. Following transplantation at peak of experimental autoimmune encephalomyelitis, cells migrated into the brain or spinal cord parenchyma, exclusively into inflamed white matter but not into adjacent gray matter regions. After 2 weeks, many transplanted cells had migrated into distant white matter tracts and acquired specific markers of the astroglial and oligodendroglial lineages. Thus, the inflammatory process may attract targeted migration of transplanted precursor cells into the brain parenchyma. GLIA 41:73–80, 2003.

Intraventricular transplantation of neural precursor cell spheres attenuates acute experimental allergic encephalomyelitis

Brain transplantation of neural precursor cells (NPCs) has been proposed to enhance CNS regeneration. As the pathogenesis of most acute CNS diseases involves an inflammatory component, we studied whether NPC transplantation affects brain inflammation. Newborn rat multipotential NPCs were transplanted intraventriculary into acute experimental allergic encephalomyelitis (EAE) rats, a model for disseminated brain inflammation. Cells migrated into inflamed white matter and differentiated into glial cells. NPC transplantation attenuated the clinical severity of EAE and the brain inflammation, indicated by reduction in perivascular infiltrates and decreased expression of ICAM-1 and LFA-1. NPCs inhibited basal proliferation and proliferative responses to Concavalin-A and to MOG peptide of EAE rat-derived lymphocytes in vitro. Purified astrocytes inhibited lymphocyte proliferation in vitro, but did not migrate into EAE brains in vivo, and did not reduce EAE severity or brain inflammation. Thus, transplanted NPCs attenuate acute EAE via an anti-inflammatory mechanism which depends on cell ability to migrate into inflamed brain tissue.

The therapeutic potential of mesenchymal stem cell transplantation as a treatment for multiple sclerosis: consensus report of the International MSCT Study Group

Current therapies for multiple sclerosis effectively reduce inflammation, but do little in terms of repair to the damaged central nervous system. Cell-based therapies may provide a new strategy for bolstering regeneration and repair through neuro-axonal protection or remyelination. Mesenchymal stem cells modulate pathological responses in experimental autoimmune encephalitis, alleviating disease, but also stimulate repair of the central nervous system through the release of soluble factors. Autologous and allogeneic mesenchymal stem cells have been safely administered to individuals with hemato-oncological diseases and in a limited number of patients with multiple sclerosis. It is therefore reasonable to move mesenchymal stem cells transplantation into properly controlled human studies to explore their potential as a treatment for multiple sclerosis. Since it is likely that the first such studies will probably involve only small numbers of patients in a few centers, we formed an international panel comprising multiple sclerosis neurology and stem cell experts, as well as immunologists. The aims were to derive a consensus on the utilization of mesenchymal stem cells for the treatment of multiple sclerosis, along with protocols for the culture of the cells and the treatment of patients. This article reviews the consensus derived from our group on the rationale for mesenchymal stem cell transplantation, the methodology for generating mesenchymal stem cells and the first treatment protocol for multiple sclerosis patients.

Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis.

Stem cell transplantation was introduced as a mean of cell replacement therapy, but the mechanism by which it confers clinical improvement in experimental models of neurological diseases is not clear. Here, we transplanted neural precursor cells (NPCs) into the ventricles of mice at day 6 after induction of chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Transplanted cells migrated into white matter tracts and attenuated the clinical course of disease. NPC transplantation down-regulated the inflammatory brain process at the acute phase of disease, as indicated by a reduction in the number of perivascular infiltrates and of brain CD3+ T cells, an increase in the number and proportion of regulatory T cells and a reduction in the expression of ICAM-1 and LFA-1 in the brain. Demyelination and acute axonal injury in this model are considered to result mainly from the acute inflammatory process and correlate well with the chronic neurological residua. In consequence to inhibition of brain inflammation, precursor cell transplantation attenuated the primary demyelinating process and reduced the acute axonal injury. As a result, the size of demyelinated areas and extent of chronic axonal pathology were reduced in the transplanted brains. We suggest that the beneficial effect of transplanted NPCs in chronic EAE is mediated, in part, by decreasing brain inflammation and reducing tissue injury.

Cognitive dysfunction following thalamic stroke: a study of 16 cases and review of the literature

The thalamus is a relay center for afferent sensory pathways that regulates and transmits peripheral stimulation to various representative areas of the cortex. Aphasia, neglect and anosognosia were also reported to occur after thalamic lesions, in the absence of cortical pathology. However, considerable controversy exists as to the pathogenetic mechanisms, and incidence of cognitive abnormalities following thalamic lesions. We present a series of sixteen consecutive stroke patients with thalamic stroke (n=12) or hemorrhage (n=4), admitted to a university based neurology department. Dysphasia was observed in seven of eight patients with left thalamic strokes (five in the territory of the tuberothalamic artery, two inferior-lateral thalamic lesions and one in the area supplied by the anterior choroidal artery). Neglect and anosognosia appeared in five of eight patients with right side thalamic insults (two each in the territories of the tuberothalamic and thalamogeniculate arteries and one in the area supplied by the posterior choroidal artery). These findings reconfirm those found in previous studies and suggest that the thalamus is part of an integral neuronal network concerned with cognitive functions.

The potential use of adult stem cells for the treatment of multiple sclerosis and other neurodegenerative disorders.

No specific treatment exists for patients with multiple sclerosis (MS) who fail to respond to conventional immunosuppressive and immunomodulating modalities. Furthermore, no method is available for regeneration of existing defect in the central nervous system (CNS). The ultimate goals of MS treatment, similarly to other autoimmune diseases, are twofold: first, to eliminate self-reactive lymphocytes and to prevent de novo development of self-reactivity by induction of self-tolerance. Second, attempting regeneration and repair of existing damage. In the case of MS, there is a need to stop the ongoing process of inflammation against the CNS by self-reactive lymphocytes thus facilitating spontaneous re-myelinization while in parallel attempt to recover existing neurological deficits caused by the autoimmune process resulting in demyelinization. Cell therapy stands out as the most rationale approach for neurological regeneration. In the absence of clinically applicable approaches involving the use of embryonic stem cells, we are investigating the feasibility and efficacy of enriched autologous mesenchymal stromal cells (MSC) injected intrathecally and intravenously to induce in situ immunomodulation and neuroprotection and possibly facilitate repair of the CNS in patients with MS and other neurodegenerative disorders. Our preclinical results suggest that bone marrow cells may provide a source of stem cells with a potential for migration into inflamed CNS and differentiate into cells expressing neuronal and glial cell markers. Based on the preclinical data, we are currently evaluating the safety of a similar therapeutic approach in a small group of patients with MS and other neurodegenerative diseases.

Guidelines for autologous blood and marrow stem cell transplantation in multiple sclerosis: a consensus report written on behalf of the European Group for Blood and Marrow Transplantation and the European Charcot Foundation

Abstract Recent reports suggest the possible beneficial effects of haemopoietic stem cell transplantation (HSCT) in autoimmune diseases such as multiple sclerosis (MS). The definition of the risk/benefit ratio for such a treatment is perceived as a major issue for the neurological community worldwide. The First Consensus Conference on Bone Marrow Transplantation in Patients with Multiple Sclerosis was held in Milan, Italy on 21 February 1998. Participants from 16 European, North American, and South American countries discussed the guidelines form performing HSCT in MS. This conference was organized in order to : (a) define criteria for patient selection; (b) define transplantation procedures to maximize efficacy of the treatment and minimize its toxicity; (c) standardize patient outcome evaluation; and (d) establish an international working group to evaluate the efficacy and safety of HSCT in MS and to study the immunological changes related to HSCT in MS patients. During the meeting in Milan agreement was reached on: (a) the preparation and distribution of a consensus report on HSCT in MS and (b) the design of an open trial for an initial assessment of the safety and efficacy of HSCT in MS. The consensus reached during the meeting and the design of the clinical trial are summarized in this contribution.

Chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE): treatment and induction of tolerance, with high dose cyclophosphamide followed by syngeneic bone marrow transplantation

We examined the effect of acute immunosuppression with high dose cyclophosphamide (CY), followed by syngeneic T-cell-depleted bone marrow transplantation (SBMT) on chronic-relapsing autoimmune encephalomyelitis (CR-EAE) induced in SJL/J mice by immunization with mouse spinal cord homogenate (MSCH) in adjuvant. Treatment of mice on day 9 post immunization, before the appearance of clinical signs of the disease, delayed the onset of paralysis, but did not affect its clinical course. Treatment on day 2-3 after the first clinical signs led to complete regression of the disease. During a period of 3 months, only one of the 15 mice treated after the the onset of CR-EAE relapsed, as compared to a total of 21 relapses in the 15 untreated animals. A rechallenge with MSCH in adjuvant on day 78 after immunization induced a severe relapse in all untreated mice, with 78% mortality; in contrast, only 25% of mice treated with CY and SBMT relapsed when similarly rechallenged. Lymphocytes from mice treated with CY and SBMT showed reduced in vitro proliferative responses to myelin basic protein (GMBP) and PPD, even after the rechallenge with MSCH. Our results show that high dose CY for elimination of immunocompetent lymphocytes, followed by SBMT rescue, suppresses CR-EAE and induces tolerance to the immunizing antigens. These results may encourage attempts to apply a similar therapeutic principle in life-threatening human neurological autoimmune diseases.

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.