Regenerative Effect of Adipose Derived Mesenchymal Stem Cells on Ganglion Cells in the Hypoxic Organotypic Retina Culture.

Background and Objectives: To examine whether ischemic retinal ganglion cells (RGCs) will be salvaged from cell death by human adipose-derived mesenchymal stem cells (ADSCs) in an organotypic retina model. Methods and Results: Deprived of arterial oxygen supply, whole mice retinas were cultured as an ex vivo organotypic cultures on an insert membrane in a 24-well plate. The therapeutic potential of ADSCs was examined by co-culture with organotypic retinas. ADSCs were seeded on top of the RGCs allowing direct contact, or at the bottom of the well, sharing the same culture media and allowing a paracrine activity. The number of surviving RGCs was assessed using Brn3a staining and confocal microscopy. Cytokine secretion of ADSCs to medium was analyzed by cytokine array. When co-cultured with ADSCs, the number of surviving RGCs was similarly significantly higher in both treatment groups compared to controls. Analysis of ADSCs cytokines secretion profile, showed secretion of anti-apoptotic and pro-proliferative cytokines (threshold＞1.4). Transplantation of ADSCs in a co-culture system with organotypic ischemic retinas resulted in RGCs recovery. Since there was no advantage to direct contact of ADSCs with RGCs, the beneficial effect seen may be related to paracrine activity of ADSCs. Conclusions: These data correlated with secretion profile of ADSCs' anti-apoptotic and pro-proliferative cytokines.

CTGF/CCN2 has a possible detrimental role in the inflammation and the remyelination failure in the early stages of multiple sclerosis.

Connective tissue growth factor (CTGF/CCN2) is a proinflammatory and an oligodendrocyte-differentiating blocking agent. It is found in MS lesions, which raises the possibility of involvement in MS pathogenesis. We found that its CSF and serum levels were higher in RR-MS patients than in controls and for serum compared to PP and SP-MS. Immune cells of both RR-MS and controls secreted CTGF/CCN2, which was enhanced by CD3/CD28 stimulation or by LPS. Anti-CTGF treatment of mice with experimental autoimmune encephalitis ameliorated its clinical severity. CTGF/CCN2 may play a role in the immune pathogenesis of MS and in remyelination failure in early stages of MS.

Retinal Lineage Therapeutic Specific Effect of Human Orbital and Abdominal Adipose-Derived Mesenchymal Stem Cells.

Retinal degenerative diseases are one of the main causes of complete blindness in aged population. In this study, we compared the therapeutic potential for retinal degeneration of human mesenchymal stem cells derived from abdominal subcutaneous fat (ABASCs) or from orbital fat (OASCs) due to their accessibility and mutual embryonic origin with retinal tissue, respectively. OASCs were found to protect RPE cells from cell death and were demonstrated to increase early RPE precursor markers, while ABASCs showed a raise in retinal precursor marker expression. Subretinal transplantation of OASCs in a mouse model of retinal degeneration led to restoration of the RPE layer while transplantation of ABASCs resulted in a significant restoration of the photoreceptor layer. Taken together, we demonstrated a lineage-specific therapeutic effect for either OASCs or ABASCs in retinal regeneration.

Specific Blockade of Bone Morphogenetic Protein-2/4 Induces Oligodendrogenesis and Remyelination in Demyelinating Disorders.

Oligodendrocyte precursor cells (OPCs) are present in demyelinated lesions of multiple sclerosis (MS) patients. However, their differentiation into functional oligodendrocytes is insufficient, and most lesions evolve into nonfunctional astroglial scars. Blockade of bone morphogenetic protein (BMP) signaling induces differentiation of OPCs into myelin-producing oligodendrocytes. We studied the effect of specific blockade of BMP-2/4 signaling, by intravenous (IV) treatment with anti-BMP-2/4 neutralizing mAb in both the inflammatory model of relapsing experimental autoimmune encephalomyelitis (R-EAE) and the cuprizone-toxic model of demyelination in mice. Administration of anti-BMP-2/4 to R-EAE-induced mice, on day 9 post-immunization (p.i.), ameliorated R-EAE signs, diminished the expression of phospho-SMAD1/5/8, primarily within the astrocytic lineage, increased the numbers of de novo immature and mature oligodendrocytes, and reduced the numbers of newly generated astrocytes within the spinal cord as early as day 18 p.i. This effect was accompanied with elevated remyelination, manifested by increased density of remyelinating axons (0.8 < g-ratios < 1), and reduced fully demyelinated and demyelinating axons, in the anti-BMP-2/4-treated R-EAE mice, studied by electron microscopy. No significant immunosuppressive effect was observed in the CNS and in the periphery, during the peak of the first attack, or at the end of the experiment. Moreover, IV treatment with anti-BMP-2/4 mAb in the cuprizone-challenged mice augmented the numbers of mature oligodendrocytes and remyelination in the corpus callosum during the recovery phase of the disease. Based on our findings, the specific blockade of BMP-2/4 has a therapeutic potential in demyelinating disorders such as MS, by inducing early oligodendrogenesis-mediated remyelination in the affected tissue.

Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation.

The effect of the inflammatory response on regenerative processes in the brain is complex. This complexity is even greater when the cause of the tissue damage is an autoimmune response. Multiple sclerosis (MS) is an immune-mediated disease in which demyelination foci are formed in the central nervous system. The degree of repair through oligodendrocyte regeneration and remyelination is insufficient. Ephrins are membrane-bound ligands activating tyrosine kinase signaling proteins that are known to have an inhibitory effect on oligodendrocyte regeneration. In this study, we examined the expression of ephrins on immune cells of 43 patients with relapsing-remitting (RR) MS compared to 27 matched healthy controls (HC). We found an increased expression of ephrin-A2, -A3 and -B3, especially on T cell subpopulations. We also showed overexpression of ephrins on immune cells of patients with RR-MS that increases the forward signaling pathway and that expression of ephrins on immune cells has an inhibitory effect on the differentiation of oligodendrocyte precursor cells (OPCs) in vitro. Our study findings support the concept that the immune activity of T cells in patients with RR-MS has an inhibitory effect on the differentiation capacity of OPCs through the expression and forward signaling of ephrins.

Fingolimod Increases Brain-Derived Neurotrophic Factor Level Secretion from Circulating T Cells of Patients with Multiple Sclerosis.

BACKGROUND: The pathophysiology of multiple sclerosis involves an autoimmune and a neurodegenerative mechanism. Central nervous system-infiltrating immune cells in multiple sclerosis also possess a neuroprotective activity through secretion of neurotrophins, such as brain-derived neurotrophic factor. Fingolimod was shown to slow the progression of disability and loss of brain volume. OBJECTIVE: The objective of this study was to explore whether fingolimod induces secretion of neurotrophins by immune cells. METHODS: Blood was drawn from 21 patients before the initiation of treatment with fingolimod and at 6 and 12 months of follow-up. The levels of the neurotrophic factors brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, ß-nerve growth factor, neurotrophin-3, neurotrophin-4, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor were screened in the supernatants of separated T cells and monocyte cultures using a customized, multiplex enzyme-linked immunosorbent assay. Brain-derived neurotrophic factor levels were further validated by a specific enzyme-linked immunosorbent assay. RESULTS: Treatment with fingolimod significantly increased brain-derived neurotrophic factor secretion from T cells. A specific enzyme-linked immunosorbent assay confirmed these results in the supernatant of T cells after 6 and 12 months of therapy. CONCLUSIONS: T cells that reach the bloodstream of fingolimod-treated patients with multiple sclerosis may contribute to the neuroprotective effect of this therapy by increased secretion of brain-derived neurotrophic factor. This mechanism of action of fingolimod in patients with multiple sclerosis has not been previously reported.

Reduced levels of Coco in sera of multiple sclerosis patients: A potential role in neuro-regeneration failure.

Demyelination, axonal loss and failure of tissue repair characterize MS lesions. Bone morphogenetic proteins (BMPs) signaling is associated with remyelination failure. Coco is one of the BMP antagonists. We found reduced Coco serum levels in relapsing-remitting MS (RR-MS) and primary progressive MS (PP-MS) patients compared to matched healthy controls (HC) and patients with rheumatoid arthritis. Exposure of P19 cells, in the presence of retinoic acid, BMP-2, or BMP-4 to Coco, at average sera level of MS patients failed to induce neuronal phenotype, in contrast to the average sera level of HC. Coco may be a player in the BMP dysregulation and the tissue repair failure in MS.

Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress.

Oxidative stress leads to the degeneration of retinal pigment epithelial (RPE) and photoreceptor cells. We evaluated the potential of adipose-derived mesenchymal stem cells (ASCs) as a therapeutic tool by studying the migration capacity of ASCs in vitro and their protective effect against RPE cell death under oxidative stress in vitro and in vivo. ASCs exhibited enhanced migration when exposed to conditioned medium of oxidative stressed RPE cells obtained by hydrogen peroxide. Migration-related axis SDF-1/CXCR4 was studied, and upregulation of SDF-1 in stressed RPE and of CXCR4 in ASCs was detected. Moreover, ASCs' conditioned medium prevented H2O2-induced cell death of RPE cells. Early passage ASCs had high expression level of HGF, low VEGF levels, and unmodulated IL-1ß levels, compared to late passage ASCs. Thus, early passage ASCs show the potential to migrate towards damaged RPE cells and protect them in a paracrine manner from cell death induced by oxidative stress. In vivo, mice received systemic injection of NaIO3, and 72 h later, ASCs were transplanted in the subretinal space. Seven days after ASC transplantation, the eyes were enucleated fixed and frozen for immunohistochemical analysis. Under such conditions, ASC-treated mice showed preservation of nuclear layers in the outer nuclear layer and stronger staining of RPE and photoreceptor layer, compared to PBS-treated mice. Taken together, our results indicate that ASCs are able to home in on damaged RPE cells and protect against damage to the RPE and PR layers caused by oxidative stress. These data imply the potential that ASCs have in regenerating RPE under oxidative stress, providing the basis for a therapeutic approach to retinal degeneration diseases related to oxidative stress that could help save the eyesight of millions of people worldwide.