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    • 专利摘要:
    treatment method for mesenchymal stem cells and its application as a treatment for diseases related to oxidative stress the present invention refers to a method for the treatment of mesenchymal stem cells, preferably of adipose origin, comprising mainly two stages, first to collection and isolation of mesenchymal stem cells, and second, a period of growth and specific treatment of the cells in a conditioning medium or treatment with an oxidizing agent. the invention also comprises cells obtained directly by the method and their use in the treatment of diseases caused or associated with oxidative stress.
    公开号:BR112013032659B1
    申请号:R112013032659-0
    申请日:2011-07-06
    公开日:2021-04-06
    发明作者:María Begoña Castro；Javier Díez García
    申请人:Histocell S.L.；
    IPC主号:
    专利说明:

    [0001] [001] The present invention relates to a treatment method for mesenchymal stem cells, cells directly obtained by this method and its uses in the care of diseases caused or related to oxidative stress. BACKGROUND OF THE INVENTION
    [0002] [002] The potential of stem cells is based on their ability to differentiate into defined types of cells and integrate into the corresponding tissues and organs. Another advantageous feature of stem cells is their paracrine release of cytokines, interleukins, trophic factors and growth factors.
    [0003] [003] Current research and clinical experiments are being done projecting the therapeutic effect of stem cells in various pathologies, with an increasing demand for therapies based on stem cells.
    [0004] [004] Certain degenerative diseases of the respiratory system, cardiovascular system, immune system, endocrine system / function, central and peripheral nervous systems, spinal cord injury, ischemia / reperfusion injury and demyelinating diseases have an inflammatory component mediated by reactive species oxygen (ROS) called oxidative stress.
    [0005] [005] Reactive oxygen species, mainly the superoxide anion radical (O2 -) and its dismutation product H2O2, are by-products of natural elimination in the mitochondria of cells, where the respiratory chain occurs, a vital phenomenon for cell life, due to its function in the generation of the energy molecule (ATP).
    [0006] [006] Mitochondria are the most redox-active compartment of mammalian cells, accounting for more than 90% of electron transfer to O2 as the final electron acceptor. The predominant electronic transfer occurs through a central redox circuit that uses the potential energy available from the oxidation of various metabolic substrates (for example, pyruvate, fatty acids) to generate ATP. The regulation of this process is central to cellular function, because cells must produce ATP while, at the same time, maintaining adequate homeostasis in terms of supplying non-essential amino acids, eliminating excess amino acids, supplying glucose and interconverting energy precursors to allow long-term energy supply in the face of variable and intermittent food intake. Part of the regulation seems to occur through a low continuous rate of generation of ROS and molecular sensors. The associated redox circuit for this regulation, although poorly defined, requires a specialized redox environment.
    [0007] [007] Under excessive oxidative stress, the simultaneous collapse of the mitochondrial potential for ATP generation and a transient increase in the generation of ROS by the electron transfer chain, may result in the mitochondrial release of ROS in the cytosol. This can trigger “ROS-induced ROS release” in neighboring mitochondria. Thus, although a low rate of ROS generation is a normal process in the mitochondria, disruption of the electronic flow with excessive generation of ROS can result in senescence, apoptosis and cell death. Go and Jones, 2008. Redox compartmentalization in eukaryotic cells. Biochimica et Biophysica Acta 1780 (1273-1290); Zorov, Juhaszova and Sollott. 2006. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta 1757 (509-517).
    [0008] [008] In fact, these processes are directly relevant to mitochondrial diseases related to oxidative stress, such as Parkinson's disease, Friedrich's ataxia, Huntington's disease and diabetes. Go and Jones, 2008. Redox compartmentalization in eukaryotic cells. Biochimica et Biophysica Acta 1780 (1273–1290); Dringen, Gutterer and Hirrlinger, 2000. Glutathione metabolism in brain. Metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 267 (4912-4916); Chinta and Andersen, 2008. Redox imbalance in Parkinson's disease. Biochimica et Biophysica Acta 1780 (1362–1367); Cohen, 2000. Oxidative stress, mitochondrial respiration, and Parkinson's disease. Ann N Y Acad Sci 899 (112–120); Lodi, Tonon, Calabrese and Schapira, 2006. Friedreich's ataxia: from disease mechanisms to therapeutic interventions. Antioxid Redox Signal 8 (438–443); McGill and Beal, 2006. PGC-1alpha, a new therapeutic target in Huntington's disease Cell 127 (465–468); Donath, Ehses, Maedler, Schumann, Ellingsgaard, Eppler and Reinecke, 2005. Mechanisms of beta-cell death in type 2 diabetes. Diabetes 54 (Suppl 2) (S108 – S113).
    [0009] [009] Peroxides, including hydrogen peroxide (H2O2), are one of the main reactive oxygen species (ROS) that lead to oxidative stress. H2O2 is generated continuously by various enzymes (including superoxide dismutase, glucose oxidase, and monoamine oxidase) and must be degraded to avoid oxidative damage. It is believed that the cytotoxic effect of H2O2 must be caused by hydroxyl radicals generated from the catalyzed reactions of iron, causing subsequent damage to DNA, proteins and membrane lipids. H2O2 acts as a “suicide substrate” in high concentrations (> 100 μM), leading to irreversible inactivation of catalase. Hyslop, Zhang, Pearson and Phebus, 1995. Measurement of striatal H2O2 by microdyalysis following global forebrain ischemia and reperfusion in the rat: Correlation with the cytotoxic potential of H2O2 in vitro. Brain Res 671 (181-186). H2O2 causes intracellular depletion of glutathione, a molecule that removes H2O2 from the cell, suggesting that H2O2 enters cells and therefore can set in motion one or more toxic pathways in cells. Dringen, Pawlowski and Hirrlinger, 2005. Peroxide Detoxification by Brain Cells. J Neurosci Res 79 (157–165); Halliwell and Whiteman, 2004. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean British Journal of Pharmacology 142 (231–255); Baud, Greene, Li, Wang, Volpe and Rosenberg, 2004. Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes. J Neurosci 24 (1531-1540).
    [0010] [0010] The cells also synthesize the antioxidative molecules and have mechanisms to recycle them. Glutathione (GSH) is one of the main proteins involved in the antioxidant mechanism that eliminates H2O2, with its oxidized form GSSG and enzymes related to glutathione peroxidase (GPx), glutathione reductase (GR), glutaredoxin and NADPH / NADP +. A variety of studies using cell culture models support the crucial role played by GSH in mitochondria as a protective effect on apoptotic cell death. In apoptosis, programmed cell death, mitochondrial oxidation of GSH / GSSG stimulates GSH depletion that results in increased ROS, suggesting a role for GSH in controlling the generation of mitochondrial ROS. Dringen, Pawlowski and Hirrlinger, 2005. Peroxide Detoxification by Brain Cells. J Neurosci Res 79 (157–165); Dringen, Gutterer and Hirrlinger, 2000. Glutathione metabolism in brain. Metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 267 (4912-4916).
    [0011] [0011] Another enzyme in the antioxidant machinery that eliminates hydrogen peroxide is catalase. Catalase is a cytoplasmic enzyme that has special relevance when H2O2 clearance in high concentrations is required. Baud, Greene, Li, Wang, Volpe y Rosenberg, 2004. Glutathione Peroxidase – Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes. J Neurosci 24 (1531-1540).
    [0012] [0012] It was also proven that hMSCs have the main enzymatic and non-enzymatic mechanisms to detoxify the reactive species and correct the oxidative damage of the proteome and genome that guarantee the efficient administration of ROS. Valle-Prieto and Conget, 2010. Human Mesenchymal Stem Cells efficiently manage oxidative stress. Stem Cell Dev 19 (1885-1893). If this potential is maintained in vivo, hMSCs could also contribute to tissue regeneration, limiting tissue damage induced by ROS.
    [0013] [0013] Some successful attempts to modify the synthesis of enzymes involved in the elimination of ROS describe that Human Bone Marrow Stromal Cells cultured in the presence of ascorbate expressed higher levels of superoxide dismutase, catalase and glutathione (Stolzing and Scutt, 2006. Effect of reduced culture temperature on antioxidant defenses of mesenchymal stem cells.Free Radic Biol Med 41 (326-338) .In addition, in the article, Ebert, Ulmer, Zeck, Meissner-Weigl, Schneider, Stopper, Schupp, Kassem and Jacob, 2006 Selenium supplementation restores antioxidant capacity and prevents cell damage in bone marrow stromal cells In Stem cells 24 (1226-1235), the authors describe the upward regulation of the basal antioxidant capacity of BMMSCs by changing culture conditions cell with selenium supplementation or by reducing the temperature Stolzing and Scutt (2006) published that reducing the temperature in these BMMSC does not affect their viability, but the enhances your differentiation. On the other hand, stem cells directly obtained by the treatment method of the present invention, cells called HC016, do not show any evidence of differentiation, maintaining their undifferentiated phenotype, as well as their viability.
    [0014] [0014] In addition, Ebert et al., 2006 demonstrate that supplementing selenium from the BMMSC culture medium with 100 nM sodium selenite exclusively increases the activity of selenium-dependent intracellular enzymes, such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxRs). On the other hand, HC016 cells conserve viability, proliferative capacity and undifferentiated phenotype, and also activate the coding of genes for the main selenium-independent enzymes for ROS detoxification, such as superoxide dismutases (SODs) and catalase (Cat), fundamental for ROS detoxification. In addition, HC016 cells have higher levels of GSH.
    [0015] [0015] To conclude, HC016 cells directly obtained with the treatment method described in the present invention show a number of advantages over stem cells used in the prior art, which makes HC016 cells especially suitable for acting under stress conditions oxidative. These advantages are mainly 1) the generation of a superior intracellular set of the GSH detoxifying molecule, 2) a higher and increased expression of the genes that encode the enzymes involved in the elimination of reactive oxygen species, 3) a new cytoskeletal conformation and, therefore, a consequent greater capacity for migration to damaged areas, and 4) a greater expression of growth factors related to tissue regeneration processes.
    [0016] [0016] These effects obtained by HC016 cells increase their intra and extracellular defenses against ROS, without generating any modification regarding their viability and differentiation states.
    [0017] [0017] WO 2010/150094 describes a method for differentiating mesenchymal stem cells in vitro into adipocytes and their use as cell therapy. The described method consists of culturing these cells under hypoxic conditions.
    [0018] [0018] WO2007 / 030870 provides a method for the differentiation of stem cells, more precisely, cells from human embryos (hES cells), into cardiomyocytes and neural progenitors through the culture of hES cells in a medium without serum, which additionally contains prostaglandin or a p38MAP kinase inhibitory molecule.
    [0019] [0019] As a consequence, there is an important need in the technique to generate methods for obtaining mesenchymal stem cells with better or greater own enzymatic and non-enzymatic mechanisms focused on the elimination of reactive oxygen species, and as a consequence, generate cells that can be more effectively used in cell therapies for diseases associated with oxidative stress. OBJECTIVE OF THE INVENTION
    [0020] [0020] The present invention relates to a treatment method for mesenchymal stem cells, and also the use of these previously treated cells in the treatment of diseases caused or related to oxidative stress.
    [0021] [0021] The stem cells of the present invention can be obtained from different sources, among others, adipose tissue, bone marrow, umbilical cord and / or placenta, but preferably, the present invention is generated from mesenchymal stem cells derived from human adipose tissue, ASC.
    [0022] [0022] In one aspect of the present invention, diseases considered associated with or caused by oxidative stress, or conditions of degenerative stress by components mediated by reactive oxygen species are those selected from the group consisting of: periarthritis, diabetes mellitus, granulomatous diseases chronic, arteriosclerosis, pulmonary fibrosis (chronic obstructive pulmonary disease, COPD, idiopathic pulmonary fibrosis), ischemia, reperfusion syndrome, Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease: ulcerative colitis and Crohn's disease , adult respiratory distress syndrome, stroke, spinal cord injury, peripheral nerve injury, amyotrophic lateral syndrome, Huntington's disease, multiple sclerosis, Friedreich's ataxia, periodontitis, mucosal diseases, diseases and injuries that coexist with an inflammatory component, ulcers and acute and chronic injuries.
    [0023] [0023] The oxidative environment present in these pathologies induces the maintenance and even the intensification of the inflammatory process in the damaged area. This phenomenon is one of the factors that hinder tissue regeneration, which cannot recover due to the high amount of ROS that is produced. Mesenchymal stem cells treated with the method of the present invention obtain a greater ability to survive in the oxidative environment produced in the pathologies listed above. This fact produces an increase in the availability of soluble factors in living cells that promote the recovery of damaged tissue.
    [0024] [0024] Therefore, an aspect of the present invention is a treatment method for mesenchymal stem cells that includes obtaining and isolating mesenchymal stem cells from a human donor and culturing those cells in a defined medium of treatment.
    [0025] [0025] A preferred aspect of the present invention is the application of the previous treatment method for mesenchymal stem cells obtained from adipose tissue.
    [0026] [0026] It is also an aspect of the present invention a method for the functional modification of mesenchymal stem cells, for the promotion of their survival and to increase their capacity to produce molecules involved in the detoxification of reactive oxygen species.
    [0027] [0027] Another aspect of the present invention relates to mesenchymal stem cells obtained with the treatment method of the present invention that have a higher and increased expression of genes involved in the detoxification of reactive oxygen species, and / or higher intracellular levels of GSH and / or changes in cytoskeletal conformation and / or an increase in their cell migration capacity, compared to mesenchymal stem cells not treated with the preconditioning method of the invention.
    [0028] [0028] Another aspect of the present invention relates to the use of mesenchymal stem cells treated according to the present invention that have higher intracellular levels of GSH, more preferably, a higher and increased expression of genes involved in the elimination of reactive oxygen species , selected from a group consisting of: cytoplasmic superoxide dismutase SOD1, mitochondrial superoxide disututase SOD2 2, extracellular superoxide dismutase SOD3 3, cat calatase, glutathione peroxidase GPx and glutathione reductase GR, more preferably, a different cytoskeletal conformation and superior expression of the cytoskeletal structure and superior expression of the gene for beta-actin, and also the growth factor IGF-1, as therapeutic formulations / reagents in a cell therapy for the care of diseases caused or related to oxidative stress.
    [0029] [0029] The present invention also relates to the use of the mesenchymal cells of the present invention administered in an area adjacent to the damaged tissue, and / or at the epicenter of the lesion. DESCRIPTION OF THE DRAWINGS
    [0030] [0030] Figure 1. Proliferative capacity measured by the MTT method of ASC, HC016 (A), HOG and HOG treated with the method of the invention (B), and stressed with an oxidative environment (100 µM). It can be seen that HC016 cells have a significant increase in cell proliferation when they are stressed in an oxidative environment. This effect does not occur when the same treatment is applied to another mammalian cell type, such as HOG cells. The asterisk indicates p <0.05 in a t-Student test. (Example 3).
    [0031] [0031] Figure 2. Kinetic analysis of the intracellular levels of reactive oxygen species in ASC, HC016, HOG and HOG treated with the method of the invention. This analysis shows that only HC016 cells significantly reduce intracellular ROS levels, especially when they are exposed to high concentrations of oxidative stress. Asterisks indicate a statistically significant difference according to the two-way ANOVA test (*, P <0.05; **, P <0.01) (example 4).
    [0032] [0032] Figure 3. Intracellular levels of total glutathione (GSHtotal) in ASC and HC016 cells (Example 5). Under control conditions, not stressed, treatment induces a 10% increase in baseline GSH HC016 levels compared to ASC.
    [0033] [0033] Figure 4A. Expression levels of the genes involved in the detoxification of reactive oxygen species in ASC and HC016 cells (Example 6).
    [0034] [0034] Figure 4B. Quantification of the expression of genes involved in the detoxification of reactive oxygen species (SOD1, SOD2, SOD3, Cat, GR, GPx) in ASC and HC016 cells. The values are expressed as an HC016 / ASC ratio (Example 6).
    [0035] [0035] Figure 5A. Expression levels of the genes involved in the cytoskeletal composition (β-Actin) in ASC and HC016 cells (Example 7).
    [0036] [0036] Figure 5B. Quantification of the expression of the genes involved in the cytoskeletal composition (β-Actin) in ASC and HC016 cells (Example 7). The value is expressed as an HC016 / ASC ratio (Example 7).
    [0037] [0037] Figure 5C. Expression levels of the IGF-I growth factor gene coding in ASC and HC016 cells (Example 7).
    [0038] [0038] Figure 5D. Quantification of the expression of the IGF-I growth factor gene coding in ASC and HC016 cells (Example 7). The value is expressed as an HC016 / ASC ratio (Example 7).
    [0039] [0039] Figure 6. Fluorescence microscopy images of the F-actin immunostaining. An increase in the presence of F-actin in HC016 cells can be observed, in relation to ASC, and its distribution in stress fibers, which means changes in the cytoskeletal conformation in HC016 in relation to its superior migration and chemotactic capacity (Example 8) .
    [0040] [0040] Figure 7. Growth rate of HOG cells of neural lineage after the insult of oxidative stress and effect of the application ASC and HC016 on this growth rate. Co-culture of HOG cells stressed with an oxidative environment together with ASC and HC016, increases the survival of HOG cells. However, only HC016 co-culture sustains this protective effect 48 hours after exposure of HOG cells to the oxidative environment. The asterisk indicates a statistically significant difference according to a Student's t test (p <0.05) (Example 9).
    [0041] [0041] Figure 8. Representative images and quantitative bar graph showing the significant superior migration capacity of HC016 cells, relative to ASC cells, targeted at cells suffering from an oxidative stress insult (Example 10).
    [0042] [0042] Figure 9. Evolution table of the BBB classification (Basso-Beattie-Bresnahan) of three experimental groups of rats with spinal cord injury consisting of untreated, ASC-treated and HC016-treated rats (Example 12). DETAILED DESCRIPTION OF THE INVENTION
    [0043] [0043] The present invention relates in one aspect to a treatment method for mesenchymal stem cells, preferably from adipose tissue, meaning obtained and / or isolated from adult adipose tissue, and more precisely from animal origin, preferably from humans. This procedure mainly requires two defined steps, first, obtaining and isolating the mesenchymal stem cells, and second, a period of growth and specific treatment of cells in a defined treatment medium that includes an oxidative agent.
    [0044] [0044] Acquisition of ASC cells:
    [0045] [0045] With respect to this stage, the procedure first includes the acquisition and isolation of mesenchymal stem cells.
    [0046] [0046] The origin of mesenchymal stem cells can be selected from a group consisting of adipose tissue, bone marrow, umbilical cord and / or placenta, preferably, the present invention is generated from mesenchymal stem cells derived from adipose tissue human, ASC.
    [0047] Therefore, preferably, in the present invention, the fraction of mesenchymal stem cells derived from adipose tissue is extracted from the liposuction of healthy human patients under anesthesia. The liposuction is donated by patients after the corresponding informed consent. The lipoaspirates are then washed with 1x PBS and digested with collagenase type I for 30 minutes at 37 ° C and then centrifuged to obtain a cell granule. This granule is resuspended in lysis of erythrocyte buffer and the purified cell suspension is filtered through a 100 µm nylon sieve centrifuged again. After resuspension of the cells, they are planted in culture flasks, to continue the expansion of the cell colony.
    [0048] [0048] Expansion of the cell colony or subculture processes that are used in the present invention include removing cells from culture vessels by incubating with a trypsin / EDTA solution, centrifuging the harvested cell suspension, determining cell density and viability and seed these cells in new cell culture vessels.
    [0049] [0049] The cell harvest of the present invention follows the methodology described in the prior art, as indicated in the publications: Yoshimura, Shigeura, Matsumoto, Sato, Takaki, Aiba-Kojima, Sato, Inoue, Nagase, Koshima y Gonda, 2006. Characterization of Freshly Isolated and Cultured Cells Derived From the Fatty and Fluid Portions of Liposuction Aspirates. J Cell Physiol 208 (64–76); Almeida, Campa, Alonso-Vale, Lima, Daud and Stocchero, 2008. Stromal vascular fraction of adipose fabric. Cir.plást. iberolatinoam. 34 (71-79); Wagner, Wein, Seckinger, Frankhauser, Wirkner, Krause, Blake, Schwager, Eckstein, Ansorge y Ho, 2005. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Experimental Hematology 33 (1402-1416).
    [0050] [0050] Method of treatment of cells. Acquisition of HC016 cells:
    [0051] [0051] Once the appropriate number of cells has been obtained, between 300,000 - 2,000,000, they are processed with a treatment that requires cells in contact with a defined concentration of an oxidizing agent following specific periods of treatment.
    [0052] [0052] Oxidizing agents are considered, for example, oxides and / or peroxides, among others, hydrogen peroxide (H2O2), calcium peroxide (CaO2), magnesium peroxide (MgO2), zinc peroxide (ZnO2), peroxide manganese (MnO2), lead peroxide (PbO2), nitric oxide (NO), nitrous oxide (N2O), ozone (O3), sodium perborate (NaBO3), selenium dioxide (SeO2), silver oxide (Ag2O), ferric salts like ferric chloride (FeCl3), copper salts like copper hydroxide (CuOH, Cu (OH) 2), percarbonates like sodium percarbonate (2Na2CO3), permanganates like potassium permanganate (K2Mn2O8), dichromates like potassium dichromate (K2Cr2O7), lithium, sodium and calcium salts of hypochlorous acid (HClO-), sodium chlorite (NaClO2), chloric acid (HClO3), potassium chlorate (KClO3), aluminum hydroxide (Al2O3), aluminum hydroxide co-precipitated with magnesium carbonate (MgCO3), arsenic trioxide (As (OH) 3), benzoylperoxide ((C6H5CO) 2O2), calcium hydroxide (Ca (OH) 2), chloride chlorodiazepoxide rat, cupric acid (CuO), iron oxides, magnesium oxide (MgO), magnesium dioxide, magnesium hydroxide (Mg (OH) 2), potassium hydroxide (KOH), sodium hydroxide (NaOH), titanium oxide (TiO2), zinc oxide (ZnO) and other oxidizing agents, preferably hydrogen peroxide (H2O2) which belongs to the group of reactive oxygen species (ROS) which is a product of disposal of the mitochondrial respiratory chain and a molecule signaling in inflammatory processes. An increase in H2O2 above certain tolerance values induces cell death. Nevertheless, the present method of treatment includes culturing cells with H2O2 in a controlled manner; this means with controlled periods and methodology and with defined concentrations that trigger new functionalities and characteristics in the mesenchymal stem cells directly obtained with this treatment.
    [0053] [0053] More precisely, the treatment method developed in the present invention includes culturing cells in a moderate oxidative environment, following defined periods of treatment.
    [0054] [0054] In general terms, the treatment method includes two consecutive treatment cycles over an interval of 48-72 hours, following a third treatment cycle of 24-48 hours in an experimental container.
    [0055] a) Primeiro ciclo: Semear células em um recipiente de cultura e esperar o período de adaptação celular entre 4 a 8 horas para permitir a aderência das células e a obtenção da morfologia típica. b) Adicionar um meio de tratamento, composto por DMEM mais 10% FBS e uma solução de H2O2, até a obtenção de uma concentração final na faixa de 0,01 e 0,05 mM. c) Manter 48-72 horas no incubador a 37°C e uma atmosfera de 5% CO2. d) Segundo ciclo: Renovar o meio de tratamento substituindo-o por DMEM mais 10% FBS e uma solução de H2O2, até a obtenção de uma concentração final na faixa de 0,01 e 0,05 mM. e) Incubar essas células por 48-72 horas a 37°C e uma atmosfera de 5% CO2. f) Terceiro ciclo: Renovar o meio de tratamento substituindo-o por DMEM mais 10% FBS e uma solução de H2O2, até a obtenção de uma concentração final na faixa de 0,01 e 0,05 mM. g) Incubar essas células por 48-72 horas a 37°C e uma atmosfera de 5% CO2. [0055] In detail, the treatment period includes the following steps: a) First cycle: Sow cells in a culture vessel and wait for the cell adaptation period between 4 to 8 hours to allow the cells to adhere and obtain the typical morphology. b) Add a treatment medium, composed of DMEM plus 10% FBS and a solution of H2O2, until a final concentration in the range of 0.01 and 0.05 mM is obtained. c) Keep 48-72 hours in the incubator at 37 ° C and an atmosphere of 5% CO2. d) Second cycle: Renew the treatment medium by replacing it with DMEM plus 10% FBS and a solution of H2O2, until a final concentration in the range of 0.01 and 0.05 mM is obtained. e) Incubate these cells for 48-72 hours at 37 ° C and an atmosphere of 5% CO2. f) Third cycle: Renew the treatment medium by replacing it with DMEM plus 10% FBS and a solution of H2O2, until a final concentration in the range of 0.01 and 0.05 mM is obtained. g) Incubate these cells for 48-72 hours at 37 ° C and an atmosphere of 5% CO2.
    [0056] [0056] After this treatment period, the cells changed their morphological and functional characteristics. From that point on, the acronym HC016 is now implemented for these cells.
    [0057] [0057] Culture or growth media include components normally known in the prior art, which are therefore media with a high concentration of glucose (DMEM, Invitrogen) at 85-95% of the total volume, with fetal bovine serum in concentrations 5-15% of the total volume (Biochrom) and an antibiotic PSA solution in a concentration of 1% of the total volume (Invitrogen).
    [0058] [0058] Also, the treatment medium used in the method of the invention described above includes a high concentration of glucose (DMEM, Invitrogen) at 85-95% of the total volume, with fetal bovine serum in concentrations of 5-15% of the total volume (Biochrom), an antibiotic PSA solution in a concentration of 1% of the total volume (Invitrogen) and hydrogen peroxide (H2O2) in concentrations between 0.01 to 0.05 mM of the total volume (Panreac).
    [0059] [0059] Characterization of HC016 cells compared to ASCs not treated with the method of the invention ...
    [0060] [0060] As a consequence of the treatment method of the invention, the HC016 cells of the present invention were obtained and show higher and expanded levels of expression of defined genes involved in the elimination of reactive oxygen species, such as the coding of genes for the following proteins: SOD1 Cytoplasmic superoxide dismutase 1, mitochondrial SOD2 superoxide dimutase 2, extracellular SOD3 superoxide dismutase 3, GPx glutathione peroxidase, GR glutathione reductase and Cat catalase, compared with ASCs not treated with the method of the invention.
    [0061] • Cu-Zn(n+1)+-SOD + O2− → Cu-Zn n+-SOD + O2 • Cu-Znn+-SOD + O2− + 2H+ → Cu-Zn (n+1)+-SOD + H2O2 [0061] SOD1: The enzyme superoxide dismutase 1 is a dimeric protein that contains copper (Cu) and zinc (Zn) as cofactors. SOD1 is in the cell cytoplasm and catalyzes the dismutation of superoxide, a product of the respiratory chain and the enzyme xanthine oxidase, into oxygen and hydrogen peroxide through the following reactions. • Cu-Zn (n + 1) + - SOD + O2− → Cu-Zn n + -SOD + O2 • Cu-Znn + -SOD + O2− + 2H + → Cu-Zn (n + 1) + - SOD + H2O2
    [0062] • Mn(n+1)+-SOD + O2− → Mn n+-SOD + O2 • Mnn+-SOD + O2− + 2H+ → Mn (n+1)+-SOD + H2O2 [0062] SOD2: The enzyme superoxide dismutase 2 is a tetrameric protein that contains manganese (Mn) as a cofactor. SOD2 is located in the cellular mitochondria and catalyzes the dismutation of superoxide, a product of the respiratory chain and the enzyme xanthine oxidase, into oxygen and hydrogen peroxide through the following reactions. • Mn (n + 1) + - SOD + O2− → Mn n + -SOD + O2 • Mnn + -SOD + O2− + 2H + → Mn (n + 1) + - SOD + H2O2
    [0063] • Cu-Zn(n+1)+-SOD + O2− → Cu-Zn n+-SOD + O2 • Cu-Znn+-SOD + O2− + 2H+ → Cu-Zn (n+1)+-SOD + H2O2 [0063] SOD3: The enzyme superoxide dismutase 3 is a homotetrameric protein that contains copper (Cu) and zinc (Zn) as cofactors. SOD3 is released into the extracellular medium where it is joined to the extracellular matrix by heparan proteoglycan sulfate and type I collagen to catalyze the dismutation of the superoxide present in the medium, generating oxygen and hydrogen peroxide through the following reaction. • Cu-Zn (n + 1) + - SOD + O2− → Cu-Zn n + -SOD + O2 • Cu-Znn + -SOD + O2− + 2H + → Cu-Zn (n + 1) + - SOD + H2O2
    [0064] • H2O2 + Fe(III)-E → H2O + O=Fe(IV)-E(.+) • H2O2 + O=Fe(IV)-E(.+) → H2O + Fe(III)-E + O2 [0064] Cat: The catalase enzyme is a tetrameric protein with four peptide chains and four porphyrin hemegroups (Ferro, Fe) that are present in the peroxisomes of most aerobic cells as a key enzyme in the defense against oxidative stress. Catalase reacts with hydrogen peroxide and converts it to water. Although its mechanisms are not fully understood, its activity has been described according to the following reactions. • H2O2 + Fe (III) -E → H2O + O = Fe (IV) -E (. +) • H2O2 + O = Fe (IV) -E (. +) → H2O + Fe (III) -E + O2
    [0065] [0065] Chelikani, Fita y Loewen, 2004. Diversity of structures and properties among catalases. Cell Mol Life Sci 61 (192–208).
    [0066] [0066] GPx: The enzyme glutathione peroxidase is one of the small proteins known in upper vertebrates that contains selenocysteine. GPx is mainly found in the cytoplasm and takes part in the detoxification of hydrogen peroxide generated by superoxide dismutase and monoamine oxidase by the H2O2 catalysis that binds to reduced glutathione (GSH) molecules.
    [0067] [0067] GR: The enzyme glutathione reductase is a homodimeric flavoprotein. GR belongs to the pyridine nucleotide-disulfide oxidoreductase class I family. This enzyme takes part in a fundamental cycle of antioxidant defense. Its activity consists of reducing oxidized glutathione (GSSG) to its sulfhydryl form (GSH), which is the key molecule in antioxidant defense.
    [0068] [0068] The increase in the expression of genes involved in the detoxification of ROS has been quantified in relation to untreated ASC cells, confirming that HC016 cells show an increase in the expression of the SOD1 gene of at least 30%, preferably 53%, an an increase in SOD2 gene expression of at least 25%, preferably 37%, an increase in SOD3 gene expression of at least 50%, preferably 77% and / or an increase in Cat gene expression of at least 50% , preferably 78%.
    [0069] [0069] The methodology used to quantify the superior expression of HC016 genes in relation to ASC, has been the following: Cell lysis, mRNA extraction and purification of each experimental group following the protocol included and described in the commercial kit of the SuperScript membrane filters -III® First Strand. One volume of mRNA serves as a template for cDNA generation with the Retro Transcription Polymerase Chain Reaction (RT-PCR) following the reagents and protocols included in the Pure Link ™ RNA Micro Kit. Corresponding volumes of cDNA from each experimental group are processed with Polymerase Chain Reaction (PCR) including specific primers that locate the DNA fragments present in the genes encoding Superoxide Dismutase 1, 2 and 3 (SOD1-3), Catalase (Cat ), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR). The PCR products of each experimental group are migrated by electrophoresis, the size of the amplified fragment is determined and the intensity of each band is quantified. The resulting value is normalized to the intensity value of a defined constitutive gene, Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH.
    [0070] [0070] Furthermore, it has been shown that HC016 cells have a higher level of intracellular GSH of at least 8%, preferably 10%, compared to ASC cells. The method used to quantify the upper level of GSH in HC016 cells with respect to ASCs was the Tietze enzymatic method, as described below: The experimental groups are two independent batches of cells, ASC and HC016. After treatment, cells are harvested, their proteins extracted by incubating the cells in lysis buffer and the total protein in the supernatant is quantified following the protocol and reagents provided with the BioRad DC protein assay kit. In a different aliquot of the supernatant, the proteins are precipitated and the supernatant is transferred for total quantification GSH and GSSG. The samples for the GSH and total GSSG assays are processed in triplicates on a 96-well plate. For the GSH measurement, the samples are incubated with glutathione reductase and afterwards the absorbance is measured at 405 nm every 15 seconds for 2.5 minutes. For the GSSG measurement, the purified sample proteins are pretreated with 2-vinylpyridine, then with glutathione reductase and finally the absorbance is measured at 405 nm every 15 seconds for 30 minutes.
    [0071] [0071] The absorbance values are extrapolated into a standard curve generated by repeating these steps previously described, but instead of using the cell protein samples, use the known concentrations of GSH. Values are expressed as nmol / mg of protein. Total glutathione is calculated as: GSHtotal = GSHreduced + 2GSSGoxidated
    [0072] [0072] In addition, HC016 cells are characterized by presenting lower levels of intracellular ROS levels by at least 10%, preferably 11%, more preferably 15% when compared to ASC.
    [0073] [0073] The method selected to quantify the superiority in the intracellular levels of ROS of HC016 over ASC was that of a fluorimetric quantification based on a DCFA probe: The experimental groups consist of four independent populations, one of ASCs, a second of HC016 , a third of intact HOG cells, and a fourth of HOG cultured with the same treatment that generates HC016 from ASC. The cells are washed with 1x PBS and then 10 μM of 2 ', 7'-dichlorodihydrofluorescein (DCFA) diacetate (DCFA) is added for 30 minutes. Then, the DCFA is washed and a new medium is added. Then, the cells are cultured in an oxidizing medium by adding H2O2 to the medium in a concentration gradient of 0, 0.1, 0.25, 0.5 and 1 mM and shortly thereafter, the evolution of intracellular ROS levels is measured at every 5 minutes and for a total of 60 minutes in a fluorometric plate reader. The graphs are represented as arbitrary units of intensity fluorescence over time (minutes).
    [0074] [0074] In addition, HC016 cells treated according to the method of the present invention show superior migration capacity. This property determines that HC016 can more efficiently access the damaged tissue area and initiate its topical action to protect the damaged cells and influence the control of an adverse environment. This significant greater capacity for migration in relation to ASC is determined by conformational changes that occur in the cytoskeleton of HC016, and also an increase in the type of microfilaments (beta-actin) organized at the limit of cell expansion. These cell membrane projections are the physical substrate for the movement of cells during migration. At that point, analysis of the betaactin gene expression shows a 59% increase in HC016 compared to ASC (Figure 5A-B). In addition, the polymerized actin or the immunostaining of F-actin indicates relevant morphological changes related to a greater capacity for movement, such as the formation of stress fibers, a fundamental element in migration events. Figure 6 shows the immunostaining of F-actin disposed in the stress fibers, which is one of the critical aspects of the cell migration process (Mitchison et al., 1996). Also, the experiment carried out exclusively to analyze the cell migration capacity through Boyden's chambers (Figure 8, example 10), shows that HC016 cells have a 30 times greater migration capacity compared to ASC.
    [0075] [0075] In addition, HC016 cells show a 64% increase in gene expression of insulin-like growth factor type 1 (IGF-1) compared to ASC (Figure 5 C-D). Previous studies have shown that IGF-1 significantly intervenes the regenerative processes that occur after damage. IGF-1 is a potent neurotrophic factor produced by non-neuronal cells after damage to nervous tissue, stimulating tissue regeneration. IGF-1 promotes neuronal survival, neurite growth, nerve cell proliferation, myelination and improves the interaction between axon and Schwann cell (Apel et al., 2010). In addition, in other experimental models, it has been proven that the local application of IGF-1 allows the repair of damaged skeletal muscle without the formation of scar tissue and greater recruitment of stem cells to the damaged area (Spangenburg et al., 2010 ). For these reasons, the induction of IGF-1 expression in HC016 cells can promote the ASC regenerative capacity, in such a way as to increase the capacity of cell proliferation, the functional recovery of cells from damaged tissue and the recruitment of stem cells to the area. damaged in order to contribute to the recovery process.
    [0076] [0076] The method selected for the analysis of gene expression with respect to ASC has been the following: This comparative analysis requires two experimental groups consisting of ASC and HC016 cells. Once the treatment method has been completed, each cell group is harvested, lysed, the RNA is extracted and purified, and processed to obtain the cDNA. The PCR reaction is carried out including the specific primers that locate the DNA fragments present in the genes that encode β-actin and the insulin-like growth factor type 1 (IGF-1). The PCR products of each experimental group are migrated by electrophoresis and the size of the amplified fragment is determined, and the intensity of each band is quantified. The resulting value is normalized to the intensity value of a defined constitutive gene, GAPDH.
    [0077] [0077] This data set indicates that the treatment method that generates HC016 induces the synthesis of a "pool" of intra- and extracellular molecular machinery required for the elimination of H2O2 and the control of an adverse oxidative environment. HC016 cells have a greater capacity for migration to reach the damaged tissue area and also produce a greater amount of trophic factors that exert their effects on the regenerative processes of the damaged tissue cells and sustain their survival.
    [0078] [0078] Use of HCO16 cells in the treatment of diseases associated or resulting from oxidative stress.
    [0079] [0079] As mentioned above, the characteristics of the HC016 cells indicated in the previous section make them especially suitable for the treatment of diseases caused by oxidative stress or by degenerative conditions of evolution due to components mediated by reactive oxygen species.
    [0080] [0080] One of the pathologies that affect the central nervous system and that involves oxidative stress, resulting in degenerative changes due to components mediated by reactive oxygen species, is spinal cord injury.
    [0081] [0081] Spinal cord injury constitutes damage to nervous tissue in which pathogens are not involved, and is not induced by external or genetic factors. At the point of injury, the immediate consequences of trauma are tissue compression, hemorrhage, edema, oxygen and nutrient depletion in the impact zone (Lu, Liang, Chen, Chen, Hsu, Liliang, Lin y Cho, 2004. Injury severity and cell death mechanisms: Effects of concomitant hypovolemic hypotension on spinal cord ischemia-reperfusion in rats (Exp Neurol 185: 120–132; Fehlings y Tator, 1995). The relationship between the severity of spinal cord injury, residual neurological function, axon count and neuron count retrograde labeled after experimental spinal cord injury. (Exp Neurol 132: 220–228). These biological phenomena induce two cellular defense mechanisms: programmed cell death or apoptosis (Yukawa, Lou, Fukui and Lenke, 2002. Optimal treatment timing to attenuate neuronal apoptosis via Bcl-2 gene transfer in vitro and in vivo. J Neurotrauma 19: 1091-1103) and the innate immune reaction (Carpentier y Palmer, 2009. Immune influence on adult neural stem cell regulation and function. Neuron 64: 79-92) that extends into the surrounding undamaged areas and persist for weeks or even months after the injury, in terms of tissue ischemia and inflammation, in other words, producing secondary damage (Lu, Liang, Chen, Chen, Hsu, Liliang, Lin y Cho, 2004. Injury severity and cell death mechanisms: Effects of concomitant hypovolemic hypotension on spinal cord ischemia-reperfusion in rats (Exp Neurol 185: 120–132).
    [0082] [0082] These two types of tissue response are expanded along the spine and initially affect healthy tissue, as the mechanisms available for nervous tissue to respond against its physiological consequences are insufficient.
    [0083] [0083] The expansion is carried out mainly by means of reactive oxygen metabolites (MROs) characteristic of the innate immune reaction that causes oxidative stress and, depending on its intensity, can induce apoptosis (Liu, Liu, y Wen, 1999. Elevation of hydrogen peroxide after spinal cord injury detected by using the Fenton reaction.Free Rad Biol & Medicine 27: 478-482; Yukawa et al., 2002). Among these, the most abundant MRO is hydrogen peroxide (H2O2), which diffuses into the extracellular environment that surrounds nerve cells and spreads in the form of secondary damage. H2O2 reacts and degrades cell membranes, proteins and DNA (Braughler and Hall, 1989. Central Nervous System trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Radic Biol Med. 6: 289–301) and at the extreme it induces them to programmed cell death or apoptosis (Yukawa et al., 2002). H2O2 is also a waste product from normal cells, so it has some defenses against this molecule (Phillis, 1994. The “radical” view of cerebral ischemic injury. Prog Neurobiol 42: 441–448). However, the levels of H2O2 that are generated after the injury are higher than the physiologically tolerable ones (Hyslop, Zhang, Pearson and Phebus, 1995. Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: Correlation with the cytotoxic potential of H2O2 in vitro, Brain Res 671: 181- 186). Among nerve cells, oligodendrocytes, cells that generate the myelin sheath that covers the axon of neurons are more vulnerable to MROs because they have less defenses against them, containing molecules that make them targets of secondary damage (Dringen R, Pawlowski P y Hirrlinger J. 2005. Peroxide detoxification by brain cells (J Neurosci Res 79: 157–165). Therefore, in addition to the initial cell death after the injury, oxidative damage due to subsequent H2O2 causes persistent demyelization of nerve fibers, which reduces the conduction of the nerve signal by them (Nashmi R y Fehlings MG. 2001. Changes in axonal physiology and morphology after chronic compressive injury of the rat thoracic spinal cord (Neuroscience 104: 235-251).
    [0084] [0084] Other diseases are those selected from the group consisting of periarteritis nodosa, diabetes mellitus, chronic granulomatous disease, arteriosclerosis, stroke, pulmonary fibrosis (chronic obstructive pulmonary disease, COPD, idiopathic pulmonary fibrosis), ischemia and reperfusion syndrome, Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease: ulceratitis colitis and Chron's disease, adult respiratory distress syndrome, atherosclerosis, spinal cord injury, peripheral nerve injury, amyotrophic lateral sclerosis, Huntington's disease, Friedeich's ataxia, periodontitis, mucosal diseases and diseases and injuries that occur with a component inflammation, ulcers and acute and chronic injuries.
    [0085] [0085] The administration of HC016 cells in the affected area reduces the level of oxidative stress caused as a result of the activation of immune cells during inflammatory processes.
    [0086] [0086] Additionally, the administration of HC016 cells in the affected area reduces the levels of oxidative stress that arise as a result of the production of MROs after internal bleeding, improves the secretion of paracrine cytokines, interleukins, chemokines, trophic factors and growth factors , increased survival and proliferation of other mammalian cells, reduces levels of extracellular MROs in the vicinity of mammalian cells near administered HC016 cells and reduces extracellular levels of signaling molecules such as pro-inflammatory TNFalpha, IL-1 beta , etc.
    [0087] [0087] In addition, HC016 cells have a significantly greater chemotactic capacity for cells damaged by extracellular H2O2, as demonstrated in Example 10 of the present invention.
    [0088] [0088] The use of HC016 cells includes the administration of previously treated mesenchymal cells in an area adjacent to the damage site, rather than at the epicenter of the lesion, in order to avoid irradiation of tissue damage, the extent of the lesion and functional loss.
    [0089] [0089] The tissue in the lesion undergoes severe compression force that breaks the cell membranes and nerve cells of the vascular system. Also, as a result of the injury, it could be bleeding due to the rupture of arteries and veins, a disruption of the organelles, cytoplasm, vesicles and cell membrane, facts that lead to tissue necrosis. These events are inherent to the injury. Then, if they are not targeted, the molecules released by the necrotic cells, along with other molecules also released by the cells of the immune system, expand the damage to the areas adjacent to the lesion by means of cell signaling molecules, such as H2O2. The application of this therapy based on mesenchymal cells is designed to reduce or minimize the spread of tissue damage. Therefore, the application of cell therapy will preferably be in an area adjacent to the epicenter of the lesion.
    [0090] [0090] In another aspect of the invention, the treated cells will be applied using an administration route, which allows them to directly reach the epicenter of the lesion, in order to metabolize the reactive oxygen species in the area, reduce oxidative stress and control the condition inflammatory, to avoid the situation of mass cell death in that area.
    [0091] [0091] Routes of administration can be any parenteral route (such as intra-arterial, intravenous, intralymphatic, intraracid, epidural, intramedullary), subcutaneous, intramuscular, intraperitoneal, transdermal (percutaneous), intra-articular, intratracheal, intra-alveolar, intrathecal, intraocular, conjunctival, intracardiac, intranasal, vaginal, urethral, cutaneous, rectal, sublingual, oral, oral transmucosal. To carry out these applications, the cells obtained by the method of the present invention are formulated in suitable pharmaceutical acceptable vehicles that are already known to those skilled in the art, depending on the route.
    [0092] [0092] Among others, the formulation provides a solution that in addition to HC016 cells contains, among others, RingerLactate, human albumin (CSL-Behring), etc., contained in glass vials for administration, being sterile and non-pyrogenic (Sword Scientific) .
    [0093] [0093] Similarly, HC016 cells can be incorporated into biomaterials of natural and / or synthetic origin, for the generation of cell and tissue engineering therapies such as hydrogels, foams and polymeric materials, compounds, calcium phosphate derivatives and metallic materials, which allow for better administration of cells at the injury site and increase cell survival and functionality, as appropriate.
    [0094] [0094] After the administration of HC016 cells to mammals, the mesenchymal cells migrate towards the lesion site, where they activate the proliferation of cells adjacent to the injection site. Preferably, these cells have the same phenotype adjacent to the parenchyma in which the injection was applied, being a precursor of cells. Even more preferably, the cell phenotype combines with that of the cells of the adjacent parenchyma and that of the cell precursor.
    [0095] [0095] In another aspect of the invention, the administered HC016 mesenchymal cells remain in the tissue. In addition, the presence of the administered mesenchymal cells in the patient's tissue does not induce an immune response against those of the administered mesenchymal cells. EXAMPLES Example 1. Obtaining adipose tissue (ASC) mesenchymal cells.
    [0096] [0096] Mesenchymal stem cells from adipose tissue are isolated from human tissue following the methodology described by Yoshimura et al., 2006, Almeida et al., 2008, Wagneret al., 2005.
    [0097] [0097] The fraction of adipose tissue mesenchymal stem cells is obtained from liposuction of healthy patients under anesthesia. The liposuction is washed with 1x PBS and digested with collagenase type I for 30 minutes at 37 ° C and then centrifuged to obtain a cell granule. This granule is resuspended in erythrocyte lysis buffer and purified and the cell suspension is passed through a 100 µm filter and centrifuged again. After resuspending the cells, they are planted in culture medium for cell expansion.
    [0098] [0098] The cells are grown as primary cultures for a period of 5 days in a growth medium composed of DMEM (Invitrogen) with 10% fetal calf serum (Biochrom) and 1% antibiotic-antimycotic PSA (Invitrogen), in the incubator at 37 ° C and 5% CO2.
    [0099] [0099] Consecutively, the cells are expanded to obtain semiconfluence. For this process, the cells are detached from the culture surface using a 0.05% trypsin / EDTA solution, centrifuged and resuspended in fresh medium. Cell density and cell viability are determined in the cell suspension obtained and planted on a new cell culture surface. Example 2. Application of the treatment of the invention to ASC cells: Obtaining HC016 cells.
    [0100] [00100] 350,000 subculture ASC cells are planted in a T25 culture flask with 5 ml of growth medium with the following composition: DMEM (Invitrogen) with 10% fetal calf serum (Biochrom) and 1% antibiotic antimycotic PSA (Invitrogen ) and incubated at 37 ° C and 5% CO2, until adhered.
    [0101] [00101] First cycle: add the treatment medium that contains the following composition: DMEM (Invitrogel) with 10% fetal calf serum (Biochrom), 1% antibiotic antimicotic PSA (Invitrogen) and 0.01% H2O2 (Panreac). These cells are incubated in this medium for 48 hours.
    [0102] [00102] Second cycle: after 48 hours, cells are obtained and again 350,000 cells are seeded in a second flask of T25 culture and incubated for 4 hours at 37 ° C and 5% CO2 until adherence. Consecutively, the treatment medium is added and the cells are incubated for 48 hours.
    [0103] [00103] Third cycle: after these 48 hours, the cells are obtained and again 350,000 cells are seeded in a second flask of T25 culture and incubated for 4 hours at 37 ° C and 5% CO2 until adherence. Consecutively, the treatment medium is added and the cells are incubated for 48 hours.
    [0104] [00104] After these 48 hours, the treated ASC cells are renamed HC016. Example 3. Comparative analysis of cell proliferation of HC016 cells with respect to ASC.
    [0105] [00105] The analysis of cell proliferation is made in the following four experimental groups: ASC, HC016, intact HOG cells and HOG cells cultured with the treatment of the invention that generates HC016.
    [0106] [00106] HOG cells are human cells of an oligodendroglioma considered as an experimental oligodendroglial model of neural lineage. HOG cells are cultured undifferentiated with proliferative capacity and an oligodendroglial genetic background.
    [0107] [00107] ASCs are grown with the methodology and growth medium defined and described in Example 1 to generate the cell population of this experiment. A batch with the same number of HC016 cells was generated according to Example 2. The HOG cell population was generated by the methods for ASC and HC016 according to Example 1 and Example 2, respectively.
    [0108] [00108] Once the four cell populations have been prepared, the cells are seeded in 96-well plates with growth medium, until they attach to the well. Then, the cells are grown in an oxidative environment by exposure to 0.1 mM H2O2. Proliferation was measured following the protocols and reagents of the MTT Cell Proliferation method on different occasions: 0, 24, 48 and 72 hours. According to the kit manufacturer, this test produces a colorimetric estimate of cell proliferation and, therefore, this parameter is analyzed and compared in four cell types.
    [0109] [00109] The figures are represented in arbitrary units of fluorescence intensity over time (hours). Results of
    [0110] [00110] The proliferative capacity of HC016 is 1.23 times greater than that of the ASC control population in 72 hours after the oxidative stimulus (23% increase) (Student's t test p <0.05; n = 3 samples) (Figure 1A). This effect is not shared by the other human cells, the HOG oligodendrocytes (Figure 1B). The treatment of the present invention is effective in ASC, but it might not be effective in other types of cells such as HOG oligodendrocytes.
    [0111] [00111] Example 4. Levels of intracellular reactive oxygen species in HC016 cells.
    [0112] [00112] The analysis of the intracellular levels of reactive oxygen species is made in four experimental cell groups: ASC, HC016, intact HOG cells and HOG cells cultured with the treatment of the invention that generates HC016.
    [0113] [00113] Once the four cell populations are prepared, the cells are planted in well plates with growth medium, until they are fixed to the well. Then, the cells are quickly washed with 1x PBS, and then the PBS is replaced with 100 µl of 1x PBS with the probe 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFA) 10 µM in the cells incubated for 30 minutes. DCFA was subsequently removed and fresh vehicle added. Then, the cells were cultured in a medium with H2O2 in different concentrations: 0, 0.1, 0.25, 0.5 and 1 mM. A time period of MROs is measured every 5 minutes for a total of 60 minutes in a fluorimeter plate reader. The excitation / emission wavelength ranges are 485/538 nm.
    [0114] [00114] The figures are represented in arbitrary units of fluorescence intensity over time (minutes). Results of
    [0115] [00115] 55 minutes after exposure to an H2O2 gradient, HC016 cells contain significant low levels of intracellular MROs with 1 mM and 0.5 mM H2O2, compared to ASC (11% lower), with two-way ANOVA test P <0.031 and (15% smaller), with two-way ANOVA test (P <0.039, respectively) (Figure 2). The treatment that generates HC016 from ASC does not induce the same response in other mammalian cells, such as HOG oligodendroglial cells (Figure 2). In addition, the presence of lower intracellular levels of ROS in HC016 cells indirectly indicates that the extracellular levels of this molecule are also reduced, so that HC016 cells have a greater capacity to remove hydrogen peroxide from the environment, endowing the cells with a greater H2O2 detoxification capacity at the application site as cell therapy. Example 5. Levels of total intracellular glutathione (GSH).
    [0116] [00116] The experimental groups consist of two separate lots of ASCs and HC016. The ASC was cultivated using the methodology and means described above. ASCs were extracted and isolated from human adipose tissue using the methods described and subcultured in growth medium until the necessary cell population has been obtained for this experiment. Also a batch of HC016 cells was generated with the same number of cells using the methodology of the invention.
    [0117] [00117] After the cell conditioning step, the cells are collected by digestion with 0.05 trypsin / EDTA, the proteins being extracted by incubating the cells with a lysis buffer (protease inhibitor, EDTA and Triton X-100 in sodium phosphate buffer with pH 7.5) and the total protein in the supernatant is quantified according to the protocol and reagents provided by the BioRad DC protein assay kit.
    [0118] [00118] In another aliquot of the supernatant, the proteins are precipitated with 5% sulfosalicylic acid and centrifuged, and small mass peptides dissolved in the supernatant transferred to the total GSH and GSSG assays.
    [0119] [00119] Samples for the total GSH and GSSG assays are processed in triplicates in a 96-well plate. For the measurement of GSH, the samples contain 5% purified protein sample, 45% distilled water and 50% reaction buffer (0.2 M EDTA, DTNB 1.2 mg / 100 μl, 3.6 mg NADPH and 4.5 Units of glutathione reductase in 0.1 M sodium phosphate buffer with pH 7.5). Then, the absorbance at 405 nm is measured every 15 seconds for 2.5 minutes. For the GSSG measurement, the purified protein samples are pre-treated with 2-vinylpyridine in a 96.3% / 3.7% (protein / reagent) ratio and are incubated for 1 hour at 4ºC. Then, samples are prepared for analysis containing 10% of the pre-treated sample, 40% distilled water and 50% reaction buffer (0.2 M EDTA, DTNB 1.2 mg / 100 μl, 3.6 mg NADPH and 4.5 Units of glutathione reductase in 0.1 M sodium phosphate buffer with pH 7.5). Then, the absorbance at 405 nm is measured every 15 seconds for 30 minutes.
    [0120] [00120] The absorbance values are extrapolated to a standard linear adjustment generated by repeating the same steps with known concentrations of GSH. Values are expressed as nmol / mg of protein. Total GSH is calculated as GSH + 2GSSG. Results of
    [0121] [00121] HC016 cells show a higher baseline content in total GSH compared to ASC (Figure 3) (10% higher). The presence of these higher levels of total intracellular GSH in HC016 cells also confirms the greater capacity for detoxification of toxic agent cells, providing greater resistance to environmental stress or to increased cellular metabolic activity. Example 6. Levels of expression of the genes involved in the detoxification of species reactive to metabolites.
    [0122] [00122] This comparative analysis required two experimental groups consisting of ASC and HC016 cells. Conventional ASC cells are cultured using the methodology and medium described above to obtain the cell population needed for this experiment. A batch of similar number of HC016 cells is generated using the method described in example 2.
    [0123] [00123] Once the conditioning procedure is finished, each cell group is harvested from the cell culture vessel by trypsin / EDTA digestion. The cells are lysed and the total mRNA of each experimental group is extracted separately and purified using a commercially available membrane filter system that retains the cell membrane fragments and proteins. The eluted mRNA is converted to cDNA by a Retro Transcription Polymerase Chain Reaction (RT-PCR) following an established protocol and reagents included in a commercial kit (Pure LinkTM RNA Micro Kit; Invitrogen; Ref. 911811). The corresponding volumes of the cDNA obtained from each experimental group are mixed with the appropriate volumes and concentrations to perform the Polymerase Chain Reaction (PCR) including tailor-made primer sequences that bind to specific cDNA fragments located in the exon regions of the Super gene. Oxide Dismutase 1, 2 and 3, Catalase, Glutathione Peroxidase and Glutathione Reductase. The PCR products from each experimental group are migrated on an agarose gel by the electrophoresis technique (4% agarose in 1x TAE buffer) plus a volume of defined concentration of commercial DNA labeling reagent (SYBR Safe DN gel stain; Invitrogen; S33102). The gels are transilluminated with UV light and optical density by a digital imaging system that are obtained to measure the optical density of each migrated band. The optical density value of each band of each experimental group and each selected gene has background subtraction, being normalized to the value of the optical density of a defined constitutive expression of the gene. Results of
    [0124] [00124] The levels of expression of the genes involved in the oxidative metabolism of HC016 cells are different from the ASC (Figure 4A). Quantification of the expression of the different genes shows that HC016 cells show a 53% increase in the SOD1 gene when compared to ASC, 37% in the SOD2 gene, 77% in SOD3, 78% in the Cat gene, 18% in the GR gene and 6 % of the GPx gene (Figure 4B). These results lead us to conclude that HC016 cells show an improvement in intra and extracellular antioxidant defenses in relation to ASC (Figure 4A). Example 7. Levels of expression of the genes and proteins involved in the cytoskeleton, and the secretion of growth factors.
    [0125] [00125] This comparative analysis required two experimental groups consisting of ASC cells that are cultured with the methodology and medium described above to obtain the cell population required for this experiment and a batch of similar number of HC016 cells that is generated by the methodology described in example 2. As in example 6, when the conditioning procedure ends, each cell group is harvested by trypsin / EDTA digestion, the cells are lysed, the total mRNA is extracted separately and purified and the mRNA is converted to cDNA. PCR is performed with the cDNA including custom-designed primer sequences that bind to specific cDNA fragments located in the exon regions of the β-actin and IGF-1 gene. As in Example 6, the PCR products from each experimental group are migrated on an agarose gel by the electrophoresis technique. The value of the optical density of each migrated band is measured and its value is subtracted from the background and normalized to the value of the optical density of a defined expression constituting the gene, GADPH. Results of
    [0126] [00126] The levels of expression of genes associated with cytoskeletal components and growth factors in HC016 cells when compared to ASC (Figures 5A and C). Quantifying the expression of the different genes shows that HC016 cells show a 59% increase in the βactin gene (Figure 5B) and a 64% increase in the IGF-I gene compared to ASC (Figure 5D). Example 8. Composition and arrangement of cytoskeletal molecules.
    [0127] [00127] This comparative analysis required two experimental groups that again consist of conventional ASC cells that are cultured with the methodology and medium described above to obtain the cell population needed for this experiment and a batch of similar number of HC016 cells that is generated by the methodology described in example 2.
    [0128] [00128] The cells of both populations are seeded in 24-well plates with the medium and methodology described above. Once adhered to the cells, the medium is removed and the cells are quickly washed with 1x PBS. Then, the cells are fixed with 4% formaldehyde in PBS 1x for 12 minutes, permeabilized with 0.1% Triton X-100 in PBS 1x for 10 minutes at 4 ° C, washed again in 1x PBS and incubated with 100 μg / ml Phalloidin-FITC (Sigma-Aldrich; Ref. P5282) in PBS 1x for 1 hour at room temperature (≈23 ° C). Then, the cells were washed extensively with 1x PBS, the nuclei marked with Hoescht 33258 (Invitrogen; Ref. H1398) and finally covered with a thin layer of Fluoromont-G (Southern Biotech; Ref. 0100-01), and visualized in a fluorescence microscope. Results of
    [0129] [00129] The cytoskeleton of HC016 cells has more filaments of F-actin and are thicker than ASC cells, and are organized in stress fibers. Observation of ASC and HC016 cells under the microscope shows qualitatively that HC016 cells have more F-actin filaments and are thicker than ASC (Figure 6), which provides a more robust and better prepared cytoskeleton for possible structural remodeling. Example 7 shows the quantification of the expression of the genes that encode the monomers F-actin and β-actin, (Fig 5A-B). Both results indicate the increased synthesis of F-actin, and the latter result indicates the effect on the cell cytoskeleton. Example 9. Ability to protect cells of neural lineage by HC016 cells.
    [0130] [00130] Experimental groups consist of control oligodendroglial HOG cells without any modification in the normal culture procedure, HOG cells cultured in an oxidizing environment, co-cultured ASC cells with HOG in an oxidizing environment and cocultivated HC016 cells with HOG cells in an oxidizing environment.
    [0131] [00131] HOG cells are human oligodendroglioma cells considered as an experimental oligodendroglial model of neural lineage. HOG cells are cultured without differentiation with proliferative capacity and an oligodendroglial genetic background.
    [0132] [00132] Populations with similar cell numbers of ASC and HC016 cells are generated using the methods described in Example 1 and 2. On the day of the experiment, the cells are harvested by 0.05 trypsin / EDTA digestion before being included in the system. in vitro coculture based on Boyden chambers inserted in 24-well plates (transpo chamber inserts) that prevent physical contact between two cell populations.
    [0133] [00133] On the day of the experiment, the oligodendrocytes are plated and adhered to the bottom of the 24-well plates and cultured in an oxidizing environment for 1 hour by adding 0.5 ml of 0.5 mM H2O2 in culture medium. After the toxic insult, the medium is replaced with fresh culture medium and grown in a medium composed of DMEM containing 10% fetal bovine serum and antibiotics at 37 ° C.
    [0134] [00134] In the next stage, in co-culture situations, ASC or HC016 cells were included sown in a Boyden chamber (transposable chamber inserts) that corresponds to the experimental groups explained above.
    [0135] [00135] Up to a period of 24 and 48 hours, the oligodendroglial viability is quantified by the blue trypan exclusion method.
    [0136] [00136] The growth rate (GR) of living cells is calculated as:% living cells -% dead cells. The growth rate of oxidized HOG in relation to normal HOG is calculated as: HOG GRoxidated / HOG GRcontrol.
    [0137] [00137] The resulting values are normalized with respect to oxidized HOG and are represented as percentages in bar graphs. Results of
    [0138] [00138] After 48 hours of oxidative stimulation in HOG cells, cells in coculture with HC016 cells, have a normalized growth rate in relation to oxidized HOGs 21% higher (Figure 7) in relation to HOG cells in coculture with ASC. While in 24 h both cell types promote the viability of oxidized HOGs (Figure 7) in a similar way, in the middle term HC016 cells have a longer lasting effect on oxidized HOGs. For this reason, the application of these cells as cell therapy in vitro has more benefits compared to conventional ASC therapy, since HC016 cells have an improvement and an increase in the effect over time, related to ASCs, compared to other cell types under stress conditions, such as tissue damage in the diseases mentioned in this document. Example 10. Ability to migrate HC016 cells to inflammatory / oxidative stress signaling cells.
    [0139] [00139] Experimental groups consist of control oligodendroglial cells without any modification in the normal culture procedure, oligodendroglial cells cultured in an oxidizing environment, ASC co-culture with oligodendroglial cells cultured in an oxidizing environment and HC016 cell culture with oligodendroglial cells grown in an oxidizing environment.
    [0140] [00140] To analyze the migration capacity of ASC and HC016 cells, an oxidative stress model is generated with cells from test mammals (oligodendroglial cells) whose proliferation and viability is sensitive to oxidative stress conditions. The oxidative stress model consists of oligodendroglial HOG cells plated and adhered to the bottom of a 24-well plate grown in an oxidizing environment for 1 hour by adding 0.5 ml of 0.5 mM H2O2 to the culture medium. After the toxic insult, the medium is replaced with fresh culture medium and cultured with a medium composed of DMEM containing 10% fetal bovine serum and antibiotics at 37 ° C.
    [0141] [00141] In a next step, in co-culture situations, the ASC or HC016 cells were incubated and seeded in a Boyden chamber (transposable chamber inserts) as corresponds to the experimental groups explained above.
    [0142] [00142] Up to a period of 24, 48 and 72 hours, the transposed inserts are washed in 1x PBS and fixed with 4% formaldehyde in 1x PBS for 12 minutes. The cells on the upper surface of the transposable membrane are removed with a Q-tip cotton swab and the remaining cells are marked with a 0.1% violet cresyl solution for 1 hour at room temperature. Then, they are completely washed in 1x PBS and finally cut from the inserts, mounted flat on glass plates covered with a thin layer of DPX half-riser (Sigma-Aldrich; Ref. 44581), and viewed under a microscope. The membranes are observed under the microscope, obtaining representative images of each situation and the number of cells per area is counted directly for each experimental group.
    [0143] [00143] The quantification is expressed in migrated number of cells per mm2. Results of
    [0144] [00144] We analyzed a total of 10 fields of 0.57 mm2 each (Figure 8) for a total of 5.7 mm2. In this area, in 48 and 72 hours, HC016 cells have a migration capacity 32.75 times (3.275%) and 20.61 times (2061%) greater (Figure 8) with respect to ASC towards the area of cells damaged by stress oxidative. In conclusion, the treatment of HC016 cells generates a significantly more robust and superior chemotactic capacity in the direction of HOG cells damaged by extracellular H2O2, in relation to ASC. The effect of chemotaxis it can have on HOG cells after H2O2 application may be related to a phenomenon such as oxidative stress and the induction of inflammation components that occur after a release of the reactive oxygen metabolites. Example 11. Manufacturing of the pharmaceutical composition of HC016 cells.
    [0145] [00145] HC016 cells were prepared according to the pharmaceutical formulation that takes the medicinal product for cell therapy to be used in the animal model and to determine its effectiveness:
    [0146] [00146] Thus, once the treatment has been carried out to obtain the HC016 cells from ASC, as specified in Example 2, the cells are placed in non-pyrogenic glass vials.
    [0147] [00147] For this purpose, the HC016 cells are detached from the culture flask by the application of a 0.05% solution of trypsin-EDTA, the enzymatic activity is neutralized by the addition of FBS (Biochrom), and the centrifugation at 400g is performed to obtain cell suspension. The supernatant is removed, the cell granule resuspended in saline (Grifols) and a new centrifugation is performed to remove all traces of the previous solutions. The supernatant is discarded and the cells are resuspended in an injectable solution (Ringer-Lactate 95% (Grifols) and 5% human albumin (CSL-Behring). We did the cell count and viability analysis using a hemocytometer, and the solution cell concentration was adjusted to a concentration of 200,000 cells / µl.
    [0148] [00148] The pharmaceutical formulation for stereotactic injection in the animal model is composed of: a solution of 50 µl of viable HC016 cells in a concentration of 200,000 cells / µl in 95% of Ringer-Lactate (Grifols) and 5% of human albumin (CSL-Behring), placed in glass bottles, sterile and non-pyrogenic (Sword Scientific). Example 12. Ability to protect and / or functional motor rehabilitation of cell therapy based on the application of HC016 cells in an animal model of spinal cord injury.
    [0149] [00149] The experimental groups consist of three groups of 10 adult Sprague-Dawley rats weighing 250-300 grams. A laminectomy was performed on these animals, under general anesthesia with 3-4% isoflurane at the thoracic level, so that the spine was exposed. A moderate spinal cord injury was applied to the dorsal spine by means of a calibrated contusion and defined using a set of parameters such as the distance and weight loaded on a metallic piston of known diameter. The first group of 10 animals received an injury and no therapy. The second group of 10 animals received an injury and was treated with ASC-based cell therapy. The third group of 10 animals received the lesion and was treated with cell therapy based on HC016. Cellular therapies, ASC and HC016, are applied 48 hours after the injury by stereotaxic injection at 6 points in the spinal levels above and below the injury. Each injection consists of 1 ml of saline containing 200,000 cells, making a total dose of 1,200,000 cells per animal. The rats are always supervised in the animal facilities and receive food and drink at will. The capacity for protection and / or motor functional rehabilitation in each group is determined by a functional test after 1, 2, 3, 4, 6 and 8 weeks of exploring locomotion in the open field. This test is known as Basso-BeattieBresnahan (BBB rating), being a reliable and sensitive method that obtains a score of 21 and provides a semi-quantitative measure of recovery in the short, medium and long term (Basso, Beattie and Bresnahan, 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12, 1-21).
    [0150] [00150] Quantification is expressed as a statistical average of the BBB scale value of each experimental group at each moment of exploration. Results of
    [0151] [00151] The application of therapy based on HC016 cells after spinal cord injury promotes the recovery of motor skills verified in the BBB test. According to the scale of this test, the values indicate that this recovery is at least one point above conventional ASC-based therapy on all examined occasions, 1, 2, 3, 4, 6 and 8 weeks after the injury (Figure 9 ). The evolution of recovery shows that therapy with HC016 cells shortens the time to obtain the best classification obtained in the eighth week with HC016 cells.
    权利要求:
    Claims (7)
    [0001]
    METHOD FOR TREATING MESENQUIMAL STEM CELLS, WHICH UNDERSTANDS THE OBTAINING AND ISOLATION OF MESENQUIMAL STEM CELLS AND CULTURE OF CELLS IN A TREATMENT MEDIUM, characterized by the culture of cells in a treatment medium comprising two consecutive treatment cycles of treatment 48-72 hours, followed by a third 24-48 hour preconditioning cycle on an experimental support.
    [0002]
    METHOD, according to claim 1, characterized by the mesenchymal cells being isolated from adipose tissue, bone marrow, umbilical cord and / or placenta.
    [0003]
    METHOD according to either of claims 1 or 2, characterized in that the mesenchymal cells are isolated from human adipose tissue.
    [0004]
    METHOD according to any one of claims 1 to 3, characterized in that the oxidizing agent is selected from the group consisting of: hydrogen peroxide (H2O2), calcium peroxide (CaO2), magnesium peroxide (MgO2), zinc peroxide ( ZnO2), manganese peroxide (MnO2), lead peroxide (PbO2) and nitric oxide (NO), nitrous oxide (N2O), ozone (O3), sodium perborate (NaBO3), selenium dioxide (SeO2), oxide silver (Ag2O), ferric salts like ferric chloride (FeCl3), copper salts like sodium percarbonate (2Na2CO3), permanganates like potassium permanganate (K2Mn2O8), dichromates like potassium dichromate (K2Cr2O7), lithium salts, sodium and lithium salts, sodium hypochloric acid (HClO), sodium chlorite (NaClO2), chloric acid (HClO3), potassium chlorate (KClO3), aluminum hydroxide (Al2O3), aluminum hydroxide co-precipitated with magnesium carbonate (MgCO3), arsenic trioxide ( As (OH) 3), benzoylperoxide ((C6H5CO) 2O2), calcium hydroxide (Ca (OH) 2), chlorodiazepoxide hydrochloride acid, cupric oxide (CuO), iron oxides, magnesium oxide (MgO), magnesium dioxide, magnesium hydroxide (Mg (OH) 2), potassium hydroxide (KOH), sodium hydroxide (NaOH), oxide titanium (TiO2) and / or zinc oxide (ZnO).
    [0005]
    METHOD, according to claim 4, characterized in that said cell culture in a treatment medium comprises the following steps: a) first cycle: cultivate the cells on a culture surface and allow a conditioning time between 4 and 8 hours to allow the cells to adhere and obtain their typical morphologies; b) add a treatment medium until a final H2O2 concentration between 0.01 and 0.05 mM is obtained; c) keep for 48-72 hours in the incubator at 37 ° C and in a 5% CO2 atmosphere; d) second cycle: renew the treatment medium until the final H2O2 concentration between 0.01 and 0.05 mM is reached again; e) incubate these cells for 48-72 hours at 37 ° C with 5% CO2; f) third cycle: renew the treatment medium by applying again to said medium until obtaining a final concentration of H2O2 between 0.01 and 0.05 mM once again; g) incubate these cells for 24-48 hours at 37 ° C with 5% CO2.
    [0006]
    METHOD according to claim 5, characterized by the treatment medium used in steps b), d) and f) comprising a conventional 85-95% cell culture medium, 5-15% fetal calf serum, antibiotics at 0 , 5-5% and 0.01-0.05 mM hydrogen peroxide.
    [0007]
    USE OF CELLS, obtained according to the method of claim 1, characterized for being for the manufacture of a medicine for the treatment of diseases related or caused by oxidative stress and / or by the conditions of degenerative evolution due to compounds mediated by oxygen-reactive species.
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    同族专利:
    公开号 | 公开日
    MX342200B|2016-08-25|
    AU2011372711B2|2016-07-14|
    KR101811481B1|2017-12-21|
    WO2013004859A1|2013-01-10|
    EP2730650A1|2014-05-14|
    JP2014520521A|2014-08-25|
    KR20140148284A|2014-12-31|
    CA2839106C|2018-01-02|
    CN103687940A|2014-03-26|
    AU2011372711A1|2014-01-16|
    CA2839106A1|2013-01-10|
    CN103687940B|2016-10-12|
    MX2013014307A|2014-05-27|
    US20140154221A1|2014-06-05|
    EP2730650B1|2017-01-04|
    JP5908582B2|2016-04-26|
    PT2730650T|2017-03-23|
    US9080153B2|2015-07-14|
    ES2620258T3|2017-06-28|
    DK2730650T3|2017-04-10|
    BR112013032659A2|2017-01-24|
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    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
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    2020-12-22| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-03-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-04-06| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/07/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    PCT/ES2011/070489|WO2013004859A1|2011-07-06|2011-07-06|Method for processing mesenchymal stem cells and the use thereof in the treatment of diseases associated with oxidative stress|
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