WO2022254075A1 - Mesenchymal stem cells with increased therapeutic potential for the treatment of cancer - Google Patents

Mesenchymal stem cells with increased therapeutic potential for the treatment of cancer Download PDF

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WO2022254075A1
WO2022254075A1 PCT/ES2022/070346 ES2022070346W WO2022254075A1 WO 2022254075 A1 WO2022254075 A1 WO 2022254075A1 ES 2022070346 W ES2022070346 W ES 2022070346W WO 2022254075 A1 WO2022254075 A1 WO 2022254075A1
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cells
cell
msc
mscs
melatonin
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PCT/ES2022/070346
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Spanish (es)
French (fr)
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Vivian CAPILLA GONZALEZ
Yolanda AGUILERA GARCIA
Laura OLMEDO MORENO
Nuria MELLADO-DAMAS SANZ
Alejandro MARTIN-MONTALVO SANCHEZ
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Fundación Pública Andaluza Progreso Y Salud
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is within the field of regenerative medicine, and relates to the use of melatonin to enhance the therapeutic effects of mesenchymal stem cells (MSCs) in cancer, particularly glioma.
  • Melatonin pretreatment is generally associated with anti-inflammatory actions, antioxidant effects, and protection of mitochondria, thus promoting MSC survival and preventing MSC senescence.
  • This method aims to provide a cellular (ie MSC) product with superior anticancer properties.
  • MSCs mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • antioxidants such as melatonin could solve this limitation.
  • melatonin exhibits anticancer effects against a variety of tumors. Therefore, the combination of MSC and melatonin could offer a more effective cell therapy for its application in cancer.
  • FIG. 1 Characterization of MT-pretreated human MSCs.
  • A Viability of MSCs after 1 hour exposure to various concentrations of H2O2, as measured by the Alamar Blue assay.
  • B Viability of MSCs after a 1 hour exposure to 1000 pm of H2O2 and various concentrations of MT, as measured by the Alamar Blue assay.
  • C Representative microscope images of MSCs pretreated or not with MT that differentiated into adipocytes (demonstrated by the presence of lipid droplets stained with Oil Red O), osteocytes (demonstrated by the presence of calcium deposits stained with Alizarin Red ) and chondrocytes (demonstrated by the presence of cartilage-specific extracellular matrix components in alcian blue-stained sections of paraffin-embedded chondrocyte spheroids).
  • D Quantification of stainings shown in C.
  • E Representative flow cytometric analysis of cultured MSCs pretreated or not with MT, showing positive expression of MSC-specific markers CD13, CD29, CD73, CD105, and CD90, while there was a negative expression for CD31, CD45, CD34 and HLA II.
  • F Detection of apoptosis using annexin V and the non-vital dye propidium iodide by flow cytometry.
  • G Bar graph representing the number of colonies generated in a colony-forming unit assay.
  • H Bar graph representing the size of colonies generated in a colony-forming unit assay.
  • I Representative diagram of Seahorse analysis of OCR on MSCs pretreated or not with MT. Data are represented as mean ⁇ SEM.
  • FIG. 1 Graph depicting the number of migrated glioma cells via the Transwell assay shown in A.
  • C Metabolic activity determined by the Alamar Blue assay on glioma cells.
  • D Immunocytochemistry against Ki67 (green) and OCT 2/3 (red) in cultured glioma cells. Cell nuclei were stained with Hoechst stain (blue).
  • E Quantification of immunocytochemistry against OCT 2/3 shown in D.
  • F Quantification of immunocytochemistry against Ki67 shown in D.
  • G Analysis of apoptotic glioma cells by flow cytometry after annexin V staining.
  • H Representative Western blot for caspase 3 and Bcl-XL protein levels in glioma cells. GAPDH was used as an internal control. Data are represented as mean ⁇ SEM.
  • FIG. 3 Effects of MSCs pretreated or not with MT on the growth of subcutaneous gliomas in athymic nude mice.
  • Figure 4 Effects of MSCs pretreated or not with MT on the histological phenotype of subcutaneous gliomas in athymic nude mice.
  • A Representative images of tumor sections stained with hematoxylin and eosin, CD34, TUNEL, Ki67, and Sirius Red.
  • B Quantification of the percentage of TUNEL+ cells shown in A.
  • C Correlation between the percentage of TUNEL+ cells and the volume of the tumor.
  • D Quantification of the area with Ki67+ cells shown in A.
  • FIG. 5 Analysis of inflammatory cytokines secreted in the tumor environment in vitro. Cytokine profile in the supernatant of cocultured cells. Bar graphs show the concentration levels of the cytokines.
  • Basic FGF fibroblast growth factor
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IFN-g interferon gamma
  • I L-1a interleukin 1 alpha
  • I L-1b interleukin 1 beta
  • IL-5 interleukin 5
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • IL-9 interleukin 9
  • MCP-1 monocyte chemoattractant protein 1
  • MIP-1a macrophage inflammatory protein 1 alpha
  • MIP-1 b macrophage inflammatory protein 1 beta
  • TNF-a tumor necrosis factor a
  • VEGF vascular endothelial growth factor
  • FIG. 6 Effects of MSCs pretreated or not with MT on glioma cells (direct system).
  • A Representative images of U87, U87+MSC, and U87+MSCMT spheroids seeded on fibronectin scaffolds and allowed to migrate for 24 h, 48 h, and 72 h.
  • B Quantification of area of migration shown in A.
  • C Representative images of U87, U87+MSC, and U87+MSCMT spheroids seeded on Matrigel scaffolds and allowed to migrate for 24 h, 48 h, 72 h and 96 h.
  • D Quantification of the area of invasion shown in C. Data are represented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to U87; One-way ANOVA.
  • Figure 8. Histological analysis of subcutaneous tumor xenografts. Subcutaneous tumors were formed from U87 glioma cells that were implanted in combination or not with MSC or MSCMT.
  • a first aspect of the invention refers to a stem cell obtained or obtainable by a process that comprises placing the cell in contact with melatonin.
  • the stem cell is a mesenchymal stem cell.
  • the stem cell of the invention is an adult stem cell, and more preferably, it is a mesenchymal stem cell.
  • adult stem cell refers to that stem cell that is isolated from a tissue or an organ of an animal in a state of growth subsequent to the embryonic state.
  • the stem cells of the invention are isolated in a postnatal state.
  • they are isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents, and adults.
  • ⁇ stem cells can be isolated from a wide variety of tissues and organs, such as bone marrow (mesenchymal stem cells, multipotent adult progenitor cells, and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, cardiac muscle, intestine , liver, neural.
  • MSCs may be obtained from, but not limited to, bone marrow, adipose tissue (such as subcutaneous adipose tissue), liver, spleen, testes, menstrual blood, amniotic fluid, pancreas, periosteum, synovium, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, dental pulp, and peripheral blood.
  • adipose tissue such as subcutaneous adipose tissue
  • liver spleen
  • testes menstrual blood
  • amniotic fluid pancreas
  • periosteum synovium
  • skeletal muscle skeletal muscle
  • dermis pericytes
  • trabecular bone human umbilical cord
  • the activated stem cell of the invention can be genetically modified by any conventional method including, by way of illustration, without limitation, transgenesis processes, deletions or insertions in its genome that modify the expression of genes that are important for its properties. basic (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences that encode proteins of interest, such as proteins with therapeutic properties. Therefore, in another preferred embodiment, the activated stem cell of the invention has been genetically modified.
  • this aspect of the invention refers to an activated mesenchymal stem cell, hereinafter activated mesenchymal stem cell of the invention, obtained or obtainable by a method comprising contacting the cell with melatonin.
  • activated mesenchymal stem cell is understood to be a cellular product based on MSCs that presents enhanced one or some of its cellular functions (survival, antioxidant activity, mitochondrial function, immunomodulatory capacity, etc.).
  • Another aspect of the invention relates to a population of activated stem cells, hereinafter the activated stem cell population of the invention, comprising at least one activated stem cell of the invention.
  • the stem cell is an MSC.
  • the cell population of the invention comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% adult stem cells. of the invention.
  • the term "isolated” indicates that the cell or cell population of the invention to which it refers is not found in its natural environment. That is, the cell or cell population has been separated from its surrounding tissue.
  • the activated stem cells of the invention, as well as the cells present in the cell population of the invention, can be autologous, allogeneic or xenogeneic cells. In a particular embodiment, said cells are of autologous origin, thus reducing potential complications associated with/or antigenic and immunogenic responses of said cells when administered to the individual.
  • Mesenchymal stem cells activated according to this aspect of the invention will preferably be obtained by pre-treating this cell product with a solution of, preferably 25 mM, melatonin.
  • the pre-treatment will preferably take place during the 24 hours prior to use, but it can also be carried out for a longer period of time (e.g. 48 hours).
  • another preferred embodiment of this aspect of the invention relates to activated mesenchymal stem cells obtainable by a method or methodology for obtaining activated stem cells, which comprises contacting the stem cell with a melatonin solution.
  • the stem cell is a mesenchymal stem cell.
  • the melatonin is in a concentration between 10 pM and 50 pM, more preferably between 15 pM and 40 pM, even more preferably between 20 pM and 30 pM, and most preferably still 25 pM.
  • the pretreatment time is at least 12 hours, preferably at least 16 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.
  • a third aspect of the invention refers to a composition, hereinafter composition of the invention, comprising at least one activated stem cell of the invention.
  • the composition of the invention further comprises a pharmaceutically acceptable carrier.
  • the composition of the invention also comprises another active principle.
  • the stem cells are mesenchymal stem cells.
  • the cell composition of the invention is at least 50%, at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, and still more preferably , 95% of the isolated stem cells activated of the invention.
  • composition of adult cells of the invention may contain a medium in which the cells of the invention are found; said medium must be compatible with said cells.
  • a medium in which the cells of the invention are found; said medium must be compatible with said cells.
  • isotonic solutions optionally supplemented with serum; cell culture media or, alternatively, a solid, semi-solid, gelatinous or viscous support medium.
  • the composition of the invention is preferably a pharmaceutical composition for administration to a subject.
  • pharmaceutically acceptable vehicle means a vehicle that must be approved by a federal or state government regulatory agency or listed in the US Pharmacopeia or European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more specifically in humans.
  • vehicle refers to a diluent, adjuvant, excipient or carrier with which the cells or cell population of the invention or said composition comprising stem cells of the invention obtainable according to the process of the invention must be administered; obviously, said vehicle must be compatible with said cells.
  • Illustrative, non-limiting examples of such a vehicle include any physiologically compatible vehicle, for example, isotonic solutions (for example, sterile 0.9% NaCI saline, phosphate buffered saline (PBS), Ringerlactate solution, etc.), optionally supplemented with serum, preferably with autologous serum; cell culture media (eg, DMEM, etc.); or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, such as collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polyornithine, hydrogels, agarose, silicone dextran sulfate.
  • the support medium may, in specific embodiments, contain growth factors or other agents.
  • the cells can be introduced into a liquid phase of the vehicle which is subsequently treated such that it becomes a more solid phase.
  • the pharmaceutical composition of the invention may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the cells, which said pharmaceutical composition comprises, and/or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators.
  • the stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by adding additional substances, such as, for example, aspartic acid, glutamic acid, etc.
  • Said pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions.
  • active substance means any component that potentially provides a pharmacological activity or other different effect in diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • active ingredient means any component that potentially provides a pharmacological activity or other different effect in diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • the term includes those components that promote a chemical change in the preparation of the drug and are present in the drug in an intended modified form that provides the specific activity or effect.
  • Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, for use as a medicament. More preferably the stem cells are mesenchymal stem cells.
  • drug refers to any substance used for the prevention, diagnosis, alleviation, treatment or cure of diseases in humans and animals.
  • the disease is cancer.
  • the pharmaceutical composition of the invention will contain a prophylactically or therapeutically effective amount of the cells of the invention or of the cell population of the invention, preferably a substantially homogeneous cell population, to provide! desired therapeutic effect.
  • the term "therapeutically or prophylactically effective amount” refers to the amount of cells of the invention contained in the pharmaceutical composition that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, due to the characteristics of the cells and the desired therapeutic effect pursued.
  • the therapeutically effective amount of cells of the invention that must be administered will depend, among other factors, on the characteristics of the subject, the severity of the disease, the form of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the person skilled in the art, who must adjust this dose depending on the factors described above.
  • the pharmaceutical composition of the invention can be administered as a single dose, containing approximately between 1x10 5 and 10x10 6 cells of the invention per kilogram of body weight of the recipient, and more preferably between 5x10 5 and 5x10 6 cells of the invention per kilo of the recipient's body weight, in an even more preferred embodiment said composition pharmaceutical will contain approximately between 1x10 6 and 2x10 6 cells of the invention per kilogram of the recipient's body weight, depending on the factors described above.
  • the dose of cells of the invention can be repeated, depending on the state and evolution of the patient, at time intervals of days, weeks or months that must be established by the specialist in each case.
  • Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, to increase, restore or partially or totally replace the functional activity of a diseased tissue or organ. or damaged.
  • Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, for the treatment of cancer.
  • Activated mesenchymal stem cells will be obtained by pretreating this cell product with a solution of, preferably 25 mM, melatonin.
  • the pre-treatment will preferably take place during the 24 hours prior to use, but it can also be carried out for a longer period of time (e.g. 48 hours).
  • another aspect of the invention refers to a procedure or methodology for obtaining activated stem cells, from now on the procedure of the invention, which comprises contacting the stem cell with a melatonin solution.
  • the stem cell is a mesenchymal stem cell.
  • the melatonin is in a concentration between 10 mM and 50 pM, more preferably between 15 pM and 40 pM, even more preferably between 20 pM and 30 pM, and most preferably still 25 pM.
  • the pretreatment time is at least 12 hours, preferably at least 16 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.
  • MSC mesenchymal stem cells
  • MT melatonin
  • EXAMPLE 1 Development of an in vitro coculture model to mimic the in vivo biological interaction between MSCs and glioma cells.
  • MSC culture Adipose tissue-derived MSCs were purchased from ATCC (PCS-500-011). Briefly, MSCs are grown in a growth medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% C02 until 70-80% confluence. The media were changed every 2-3 days. Passages were performed using a 0.25% trypsin solution. Cells from passages 3 to 6 were used for all experiments.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • MT Pretreatment Subconfluent MSC cultures were pretreated with 25 mM MT for 24 hours. The cells were then used for the different experiments proposed here. To determine whether MT pretreatment modifies MSC identity, we evaluated the potential for multiple MSC lineages (adipocytes, osteocytes, and chondrocytes) and the expression of established MSC markers (MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II) (1-3). We will also determine if MT pretreatment affects cell proliferation (e.g. Alamar Blue), apoptosis (e.g. Annexin V), antioxidant activity (e.g. catalase activity) and mitochondrial function (e.g. Sehorse).
  • cell proliferation e.g. Alamar Blue
  • apoptosis e.g. Annexin V
  • antioxidant activity e.g. catalase activity
  • mitochondrial function e.g. Se
  • Glioma cell culture Glioma cells were obtained from ATCC (U87MG; HTB-14). Briefly, U87 cells are grown in a growth medium composed of Eagle's Minimal Essential Medium (EMEM; ATCC), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells are incubated at 37 °C in a humidified atmosphere with 20% 02 and 5% C02 to 70-80% confluence. The media were changed every 2-3 days. Passaging is done using a 0.25% trypsin solution. Cells from passages 3 to 6 were used for all experiments.
  • EMEM Eagle's Minimal Essential Medium
  • D. Co-cultivation system To study the interaction between glioma cells and MSCs (pretreated or not with MT), the transwell co-cultivation system (0.4 pm pore size) is used. MSCs are seeded on top, while glioma cells are seeded on the bottom. This system allows to easily separate the two cell populations for perform specific studies with glioma cells.
  • the experimental groups are: U87 (U87 cells without MSC), U87 + MSC (U87 cells co-cultured with MSC) and U87 + MSC + MT (U87 cells co-cultured with MSC that were pre-treated with MT).
  • EXAMPLE 2 To study the interactions between MSCs and glioma cells using the in vitro coculture model.
  • glioma cells After 24 hours in the transwell coculture system, glioma cells are used to study proliferation using the Alamar Blue assay and immunocytochemistry.
  • Apoptosis After 24 hours in the transwell coculture system, glioma cells are used to study apoptosis by flow cytometry (Annexin V kit).
  • glioma cells are harvested to investigate the molecular pathways that are modulated by MSCs, using RNA-Seq, RT-PCR, and western blotting.
  • Cytokine profile The cell supernatant generated after cocultivation will be analyzed by multiplex ELISA (Bio-Plex Pro TM Human Cytokine) to identify survival, angiogenic and inflammatory cytokines, among others.
  • EXAMPLE 3 To study the interactions between MSCs and glioma cells using an in vivo model.
  • mice and experimental groups To avoid rejection of the transplanted cells, athymic nude mice (10 weeks old) are used. Animals are randomly assigned to three experimental groups: mice injected with glioma cells (U87 group), mice injected with glioma cells and MSC (U87 + MT group) and control mice (U87 + MSC + MT group).
  • Tumor growth Mice are monitored daily to study tumor evolution throughout the project. Once sacrificed, the tumors are excised for subsequent histological and immunohistochemical analysis.
  • Example 4 Pretreatment of mesenchymal stem cells (MSC) with melatonin (MT) enhances the anticancer properties of the cells.
  • Adipose tissue-derived MSCs were purchased from ATCC (PCS-500-011). Briefly, MSCs were grown in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 to 70-80% confluence. The media were changed every 2-3 days. Passages were carried out using a 0.25% trypsin solution. For all experiments cells from passages 3-6 were used.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II.
  • MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II.
  • Glioma cell culture Glioma cells were obtained from ATCC (U87MG; HTB-14). Briefly, U87 cells were grown in growth medium consisting of Eagle's minimal essential medium (EMEM; ATCC), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 to 70-80% confluence. The media were changed every 2-3 days. Passages were carried out using a 0.25% trypsin solution. For all experiments cells from passages 3-6 were used.
  • EMEM Eagle's minimal essential medium
  • Co-culture system To study the interaction between glioma cells and MSCs (pre-treated or not with MT) two approaches were used:
  • the Transwell co-culture system was used (with a pore size of 0.4 pm). MSCs were seeded on top, while glioma cells were seeded on the bottom. This system made it possible to easily separate the two cell populations for specific studies with glioma cells.
  • the experimental groups were: U87 (U87 cells without MSC), U87+MSC (U87 cells cocultured with MSC) and U87+MSC+MT (U87 cells cocultured with MSC that were pretreated with MT).
  • a. Proliferation After 24 hours in the co-culture system, glioma cells were used to study proliferation using the Alamar Blue assay and immunocytochemistry.
  • b. Apoptosis After 24 hours in the co-culture system, glioma cells were used to study apoptosis by flow cytometry (Annexin V kit).
  • c. Migration assay To study how MSCs interfere with the migration ability of U87, the Transwell system (with 0.8 pm pore size) was used. U87 cells were seeded on top with serum-free medium, while MSCs were seeded on the bottom with FBS medium to encourage cell migration through the membrane pores.
  • Tumor beads cells were placed in a 20 ⁇ l hanging drop with growth medium. For each spheroid, 21,000 U87 cells were used, which were cocultivated or not with MSC or MSCMT in a 3:1 ratio.
  • spheroids were transferred to fibronectin precoated wells for migration assays or fibronectin precoated wells. Matrigel for invasion assays. Spheroids were imaged every 24 hours and the area of migration or invasion was analyzed using ImageJ software (version 1.4r; National Institute of Health, Bethesda, MD). and. Molecular study: After 24 hours in the coculture system, glioma cells were harvested to investigate the molecular pathways modulated by MSCs, using RNA-Seq, RT-PCR, and Western blotting. F.
  • Cytokine profiling Cell supernatants generated after cocultivation were analyzed by multiplex ELISA (Bio-Plex ProTM human cytokine) to identify survival, angiogenic, and inflammatory cytokines, among others. Direct and indirect cocultures (Transwell system) were used.
  • mice To avoid rejection of the transplanted cells, athymic nude mice (10 weeks old) were used. Animals were randomly assigned to three experimental groups: mice injected with glioma cells (U87 group), mice injected with glioma cells and MSC (U87+MT group), and control mice (U87+MSC+MT group).
  • U87 group mice injected with glioma cells
  • U87+MT group mice injected with glioma cells and MSC
  • U87+MSC+MT group mice
  • mice were injected subcutaneously into the flank of the right hind paw with 150 ⁇ l of DMEM:Matrigel (1:2) mixture containing T 106 glioma cells, with or without T 106 MSC (pretreated or not with MT). Mice were sacrificed when the tumors reached an average volume of 4000 mm 3 (greater volumes may affect animal welfare).
  • Tumor Growth Mice were monitored daily to study tumor evolution over time, until tumors reached an average volume of 4000 mm 3 . Tumors were measured with a digital caliper every 5 days to estimate their volume using the formula (minor diameter 2 c major diameter)/2. d.
  • mice were subjected to intracardiac perfusion with 0.9% saline solution and 4% paraformaldehyde.
  • Half of each tumor was paraffin-embedded and processed for histological staining (eg, hematoxylin and eosin and Syrian red) as well as immunohistological techniques.
  • Specific markers of proliferation eg, hematoxylin and eosin and Syrian red
  • specific markers of proliferation eg, hematoxylin and eosin and Syrian red

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Abstract

The present invention relates to activated stem cells that are obtained by a method that comprises putting the cell in contact with melatonin, a method for obtaining same, and uses thereof.

Description

Células madre mesenquimales con potencial terapéutico aumentado para el tratamiento del cáncer Mesenchymal stem cells with increased therapeutic potential for cancer treatment
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se encuentra dentro del campo de la medicina regenerativa, y se refiere al uso de melatonina para mejorar los efectos terapéuticos de las células madre mesenquimales (MSC) en el cáncer, particularmente el glioma. El pre-tratamiento con melatonina generalmente se asocia con acciones antiinflamatorias, efectos antioxidantes y protección de las mitocondrias, lo que promueve la supervivencia de las MSC y previene la senescencia de las MSC. Este método tiene como objetivo proporcionar un producto celular (es decir, MSC) con propiedades anticancerígenas superiores. The present invention is within the field of regenerative medicine, and relates to the use of melatonin to enhance the therapeutic effects of mesenchymal stem cells (MSCs) in cancer, particularly glioma. Melatonin pretreatment is generally associated with anti-inflammatory actions, antioxidant effects, and protection of mitochondria, thus promoting MSC survival and preventing MSC senescence. This method aims to provide a cellular (ie MSC) product with superior anticancer properties.
ESTADO DEL ARTE STATE OF THE ART
El uso de medicamentos de terapia avanzada, incluidas las células madre, se ha convertido en una alternativa prometedora en la medicina regenerativa. Aunque las células madre mesenquimales (de aquí en adelante referidas como “MSCs”) han mostrado un enorme potencial terapéutico en diversas enfermedades, su aplicación para el tratamiento del cáncer sigue siendo controvertida. Si bien algunos estudios indican que las MSCs pueden contribuir a la patogénesis del cáncer, los datos emergentes respaldan los efectos beneficiosos de las MSCs para el tratamiento oncológico. Además del efecto antitumoral que poseen las MSC per se, muchos investigadores han utilizado estas células como vehículo para administrar agentes anticancerígenos de una manera dirigida al tumor. Sin embargo, el potencial terapéutico de las MSC en el tratamiento del cáncer puede verse afectado por el entorno tumoral. Por ejemplo, el entorno oxidativo en el que crecen los tumores puede acelerar la senescencia de las MSC, afectando las propiedades terapéuticas de estas células. En este contexto, el uso de antioxidantes como la melatonina podría solucionar esta limitación. Además, la melatonina exhibe efectos anticancerígenos contra una variedad de tumores. Por tanto, la combinación de MSC y melatonina podría ofrecer una terapia celular más eficaz para su aplicación en el cáncer. The use of advanced therapy drugs, including stem cells, has become a promising alternative in regenerative medicine. Although mesenchymal stem cells (hereinafter referred to as "MSCs") have shown enormous therapeutic potential in various diseases, their application for cancer treatment remains controversial. While some studies indicate that MSCs may contribute to cancer pathogenesis, emerging data supports the beneficial effects of MSCs for cancer treatment. In addition to the antitumor effect that MSCs possess per se, many investigators have used these cells as a vehicle to deliver anticancer agents in a tumor-directed manner. However, the therapeutic potential of MSCs in cancer treatment may be affected by the tumor environment. For example, the oxidative environment in which tumors grow can accelerate the senescence of MSCs, affecting the therapeutic properties of these cells. In this context, the use of antioxidants such as melatonin could solve this limitation. Furthermore, melatonin exhibits anticancer effects against a variety of tumors. Therefore, the combination of MSC and melatonin could offer a more effective cell therapy for its application in cancer.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Figura 1. Caracterización de las MSC humanas pretratadas con MT. (A) Viabilidad de MSC después de una exposición de 1 hora a diversas concentraciones de H2O2, medida mediante el ensayo Alamar Blue. (B) Viabilidad de MSC después de una exposición de 1 hora a 1000 pm de H2O2 y diversas concentraciones de MT, medida mediante el ensayo Alamar Blue. (C) Imágenes de microscopio representativas de MSC pretratadas o no con MT que se diferenciaron en adipocitos (demostrado por la presencia de gotitas de lípidos teñidas con rojo aceite O), osteocitos (demostrado por la presencia de depósitos de calcio teñidos con rojo de alizarina) y condrocitos (demostrado por la presencia de componentes de la matriz extracelular específicos de cartílago en secciones de esferoides de condrocitos incrustadas en parafina teñidas con azul alcián). (D) Cuantificación de las tinciones mostradas en C. (E) Análisis de citometría de flujo representativo de MSC cultivadas pretratadas o no con MT, que muestra la expresión positiva de los marcadores específicos de MSC CD13, CD29, CD73, CD105 y CD90, mientras que había una expresión negativa para CD31, CD45, CD34 y HLA II. (F) Detección de apoptosis usando anexina V y el colorante no vital yoduro de propidio mediante citometría de flujo. (G) Barra gráfica que representa el número de colonias generadas en un ensayo de unidades formadoras de colonias. (H) Barra gráfica que representa el tamaño de las colonias generadas en un ensayo de unidades formadoras de colonias. (I) Diagrama representativo de análisis Seahorse de OCR en las MSC pretratadas o no con MT. Los datos se representan como media ± EEM. Figure 1. Characterization of MT-pretreated human MSCs. (A) Viability of MSCs after 1 hour exposure to various concentrations of H2O2, as measured by the Alamar Blue assay. (B) Viability of MSCs after a 1 hour exposure to 1000 pm of H2O2 and various concentrations of MT, as measured by the Alamar Blue assay. (C) Representative microscope images of MSCs pretreated or not with MT that differentiated into adipocytes (demonstrated by the presence of lipid droplets stained with Oil Red O), osteocytes (demonstrated by the presence of calcium deposits stained with Alizarin Red ) and chondrocytes (demonstrated by the presence of cartilage-specific extracellular matrix components in alcian blue-stained sections of paraffin-embedded chondrocyte spheroids). (D) Quantification of stainings shown in C. (E) Representative flow cytometric analysis of cultured MSCs pretreated or not with MT, showing positive expression of MSC-specific markers CD13, CD29, CD73, CD105, and CD90, while there was a negative expression for CD31, CD45, CD34 and HLA II. (F) Detection of apoptosis using annexin V and the non-vital dye propidium iodide by flow cytometry. (G) Bar graph representing the number of colonies generated in a colony-forming unit assay. (H) Bar graph representing the size of colonies generated in a colony-forming unit assay. (I) Representative diagram of Seahorse analysis of OCR on MSCs pretreated or not with MT. Data are represented as mean ± SEM.
Figura 2. Efectos de las MSC pretratadas o no con MT en células de glioma (sistema indirecto).Figure 2. Effects of MSCs pretreated or not with MT on glioma cells (indirect system).
(A) Tinción con violeta de cresilo de células de glioma que transmigraron a través de una membrana Transwell después de 48 h en presencia o no de MSC o MSC pretratadas con MT.(A) Cresyl violet staining of glioma cells that transmigrated across a Transwell membrane after 48 h in the presence or absence of MSCs or MT-pretreated MSCs.
(B) Gráfico que representa el número de células de glioma migradas a través del ensayo Transwell mostrado en A. (C) Actividad metabólica determinada mediante el ensayo Alamar Blue en células de glioma. (D) Inmunocitoquímica contra Ki67 (verde) y OCT 2/3 (rojo) en células de glioma cultivadas. Los núcleos celulares se tiñeron con colorante Hoechst (azul). (E) Cuantificación de la inmunocitoquímica contra OCT 2/3 mostrada en D. (F) Cuantificación de la inmunocitoquímica contra Ki67 mostrada en D. (G) Análisis de células de glioma apoptóticas mediante citometría de flujo después de la tinción con anexina V. (H) Inmunotransferencia de tipo Western representativa para caspasas 3 y niveles de proteína Bcl-XL en células de glioma. Se usó GAPDH como control interno. Los datos se representan como media ± EEM. (B) Graph depicting the number of migrated glioma cells via the Transwell assay shown in A. (C) Metabolic activity determined by the Alamar Blue assay on glioma cells. (D) Immunocytochemistry against Ki67 (green) and OCT 2/3 (red) in cultured glioma cells. Cell nuclei were stained with Hoechst stain (blue). (E) Quantification of immunocytochemistry against OCT 2/3 shown in D. (F) Quantification of immunocytochemistry against Ki67 shown in D. (G) Analysis of apoptotic glioma cells by flow cytometry after annexin V staining. (H) Representative Western blot for caspase 3 and Bcl-XL protein levels in glioma cells. GAPDH was used as an internal control. Data are represented as mean ± SEM.
Figura 3. Efectos de las MSC pretratadas o no con MT en el crecimiento de gliomas subcutáneos en ratones desnudos atímicos. (A) Peso corporal de los animales durante el transcurso del experimento n = 5 por grupo. * p<0,05, ** p<0,01 para la comparación U87 frente a U87+MSC; ANOVA bidireccional. (B) Aumento de peso corporal el día 40 tras la implantación del tumor. Los valores del aumento de peso corporal se corrigieron restando el peso del tumor el día 40 tras la implantación del tumor n = 5 por grupo. * p<0,05, ** p<0,01, **** p<0,0001; ANOVA unidireccional. (C) Curva de crecimiento tumoral a lo largo del tiempo n = 5 por grupo. * p<0,05, *** p<0,001, **** p<0,0001 en comparación con U87; ANOVA bidireccional. (D) Diagramas de Kaplan-Meier de la proporción de ratones con un volumen tumoral inferior a 1000 mm2. n = 5 por grupo. (E) Imágenes de los ratones 40 días tras la implantación del tumor. (F) Volumen de los tumores disecados n = 5 por grupo. * p<0,05 en comparación con U87; ANOVA unidireccional. (G) Peso de los tumores disecados n = 5 por grupo. * p<0,05 en comparación con U87; ANOVA unidireccional. (H) Densidad de los tumores disecados n = 5 por grupo. ANOVA unidireccional. (I) Imágenes de los tumores disecados. El código de color de las estadísticas corresponde al color de cada grupo experimental. Figure 3. Effects of MSCs pretreated or not with MT on the growth of subcutaneous gliomas in athymic nude mice. (A) Body weight of the animals during the course of the experiment n = 5 per group. * p<0.05, ** p<0.01 for comparison U87 vs. U87+MSC; Two-way ANOVA. (B) Body weight gain on day 40 after tumor implantation. Body weight gain values were corrected by subtracting tumor weight at day 40 post tumor implantation n=5 per group. * p<0.05, ** p<0.01, **** p<0.0001; One-way ANOVA. (C) Tumor growth curve over time n = 5 per group. *p<0.05, ***p<0.001, ****p<0.0001 compared to U87; Two-way ANOVA. (D) Kaplan-Meier plots of the proportion of mice with a tumor volume less than 1000 mm 2 . n = 5 per group. (E) Images of mice 40 days after tumor implantation. (F) Volume of dissected tumors n = 5 per group. * p<0.05 compared to U87; One-way ANOVA. (G) Weight of dissected tumors n = 5 per group. * p<0.05 compared to U87; One-way ANOVA. (H) Density of dissected tumors n = 5 per group. One-way ANOVA. (I) Images of the dissected tumors. The color code of the statistics corresponds to the color of each experimental group.
Figura 4. Efectos de las MSC pretratadas o no con MT en el fenotipo histológico de los gliomas subcutáneos en ratones desnudos atímicos. (A) Imágenes representativas de secciones tumorales teñidas con hematoxilina y eosina, CD34, TUNEL, Ki67 y Sirius Red. (B) Cuantificación del porcentaje de células TUNEL + que se muestra en A. (C) Correlación entre el porcentaje de células TUNEL + y el volumen del tumor. (D) Cuantificación del área con células Ki67 + que se muestra en A. (E) Cuantificación del porcentaje de área teñida con Sirius Red que se muestra en A. n = 5 por grupo. ANOVA unidireccional. Figure 4. Effects of MSCs pretreated or not with MT on the histological phenotype of subcutaneous gliomas in athymic nude mice. (A) Representative images of tumor sections stained with hematoxylin and eosin, CD34, TUNEL, Ki67, and Sirius Red. (B) Quantification of the percentage of TUNEL+ cells shown in A. (C) Correlation between the percentage of TUNEL+ cells and the volume of the tumor. (D) Quantification of the area with Ki67+ cells shown in A. (E) Quantification of the percentage of area stained with Sirius Red shown in A. n = 5 per group. One-way ANOVA.
Figura 5. Análisis de citocinas inflamatorias secretadas en el entorno tumoral in vitro. Perfil de citocinas en el sobrenadante de células cocultivadas. Los gráficos de barras muestran los niveles de concentración de las citocinas. FGF básico, factor de crecimiento de fibroblastos; G-CSF, factor estimulante de colonias de granulocitos; GM-CSF, factor estimulante de colonias de granulocitos y macrófagos; IFN-g, interferón gamma; I L- 1 a , interleucina 1 alfa; I L- 1 b , interleucina 1 beta; IL-5, interleucina 5; IL-6, interleucina 6; IL-8, interleucina 8; IL-9, interleucina 9; MCP-1 , proteína quimioatrayente de monocitos 1 ; MIP-1a, proteínas inflamatorias de macrófagos 1 alfa; MIP-1 b, proteínas inflamatorias de macrófagos 1 beta; TNF-a, factor de necrosis tumoral a; VEGF, factor de crecimiento endotelial vascular. Figure 5. Analysis of inflammatory cytokines secreted in the tumor environment in vitro. Cytokine profile in the supernatant of cocultured cells. Bar graphs show the concentration levels of the cytokines. Basic FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-g, interferon gamma; I L-1a, interleukin 1 alpha; I L-1b, interleukin 1 beta; IL-5, interleukin 5; IL-6, interleukin 6; IL-8, interleukin 8; IL-9, interleukin 9; MCP-1, monocyte chemoattractant protein 1; MIP-1a, macrophage inflammatory protein 1 alpha; MIP-1 b, macrophage inflammatory protein 1 beta; TNF-a, tumor necrosis factor a; VEGF, vascular endothelial growth factor.
Figura 6. Efectos de las MSC pretratadas o no con MT en células de glioma (sistema directo). (A) Imágenes representativas de los esferoides de U87, U87+MSC y U87+MSCMT sembrados en andamiajes de fibronectina y a los que se les permitió migrar durante 24 h, 48 h y 72 h. (B) Cuantificación del área de migración mostrada en A. (C) Imágenes representativas de los esferoides de U87, U87+MSC y U87+MSCMT sembrados en andamiajes de Matrigel y a los que se les permitió migrar durante 24 h, 48 h, 72 h y 96 h. (D) Cuantificación del área de invasión mostrada en C. Los datos se representan como media ± EEM. *p<0,05, **p<0,01, ***p<0,001 en comparación con U87; ANOVA unidireccional. Figure 6. Effects of MSCs pretreated or not with MT on glioma cells (direct system). (A) Representative images of U87, U87+MSC, and U87+MSCMT spheroids seeded on fibronectin scaffolds and allowed to migrate for 24 h, 48 h, and 72 h. (B) Quantification of area of migration shown in A. (C) Representative images of U87, U87+MSC, and U87+MSCMT spheroids seeded on Matrigel scaffolds and allowed to migrate for 24 h, 48 h, 72 h and 96 h. (D) Quantification of the area of invasion shown in C. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to U87; One-way ANOVA.
Figura 7. Análisis de las citocinas inflamatorias secretadas en el entorno tumoral in vitro. Perfil de citocinas en el sobrenadante de las células cocultivadas de manera directa (A) e indirecta (B). Los gráficos de barras muestran los niveles de concentración de las citocinas evaluadas. Los datos se representan como media ± EEM. n = 4-5 por grupo. *p<0,05, **p<0,01, ***p<0,001, ****p<0 ,0001 en comparación con el grupo U87, a menos que se indique lo contrario, ANOVA unidireccional. Figura 8. Análisis histológico de los xenoinjertos tumorales subcutáneos. Los tumores subcutáneos se formaron a partir de células de glioma U87 que se implantaron en combinación o no con MSC o MSCMT. Los tumores se extirparon después de sacrificar a los ratones el día 30 tras el trasplante (n = 6-7 por grupo). (A) Imágenes histológicas representativas de la tinción con hematoxilina y eosina de los tumores que muestran el área necrótica, los vasos sanguíneos y las áreas proliferativas en los diferentes grupos experimentales (punta de flecha roja para células mitóticas). Obsérvese que los ratones U87 muestran una gran área necrótica con infiltración de células inflamatorias (punta de flecha negra) y vasos sanguíneos (asterisco) que estaban frecuentemente atrofiados (flecha negra), en contraste con los ratones U87+MSC y U87+MSCMT. (B) Fibras de colágeno teñidas con rojo sirio y visualizadas con microscopía de campo claro y de luz polarizada. Las fibras de colágeno también se detectaron mediante microscopía electrónica (rosa, flechas). Obsérvese que el colágeno era más abundante en el grupo U87+MSCMT. (C) Cuantificación del área positiva de las fibras de colágeno teñidas con rojo sirio en tumores subcutáneos n = 6-7 por grupo. Los datos se representan como media ± EEM. * p<0,05 en comparación con el grupo U87; ANOVA unidireccional. (D) Representación esquemática de los tumores que se hicieron crecer en cada grupo. Barra de escala: 1,10, 50,90 y 200 pm. BF: campo claro; L: tumor vivo; MSC: células madre mesenquimales; MSCMT: células madre mesenquimales pretratadas con melatonina; N: área necrótica. Figure 7. Analysis of inflammatory cytokines secreted in the tumor environment in vitro. Cytokine profile in the supernatant of cells cocultivated directly (A) and indirectly (B). The bar graphs show the concentration levels of the cytokines evaluated. Data are represented as mean ± SEM. n = 4-5 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the U87 group, unless otherwise stated, one-way ANOVA. Figure 8. Histological analysis of subcutaneous tumor xenografts. Subcutaneous tumors were formed from U87 glioma cells that were implanted in combination or not with MSC or MSCMT. Tumors were excised after sacrificing mice on day 30 post-transplantation (n=6-7 per group). (A) Representative histological images of hematoxylin and eosin staining of tumors showing the necrotic area, blood vessels, and proliferative areas in the different experimental groups (red arrowhead for mitotic cells). Note that U87 mice show a large necrotic area with inflammatory cell infiltration (black arrowhead) and blood vessels (asterisk) that were frequently atrophied (black arrow), in contrast to U87+MSC and U87+MSCMT mice. (B) Collagen fibers stained with Syrian red and visualized with brightfield and polarized light microscopy. Collagen fibers were also detected by electron microscopy (pink, arrows). Note that collagen was more abundant in the U87+MSCMT group. (C) Quantification of the positive area of Syrian red-stained collagen fibers in subcutaneous tumors n=6-7 per group. Data are represented as mean ± SEM. * p<0.05 compared to the U87 group; One-way ANOVA. (D) Schematic representation of the tumors grown in each group. Scale bar: 1.10, 50.90 and 200 pm. BF: bright field; L: live tumor; MSC: mesenchymal stem cells; MSCMT: melatonin-pretreated mesenchymal stem cells; N: necrotic area.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
CÉLULAS Y POBLACIÓN CELULAR DE LA INVENCIÓN CELLS AND CELL POPULATION OF THE INVENTION
Un primer aspecto de la invención se refiere a una célula madre obtenida u obtenible por un procedimiento que comprende poner la célula en contacto con melatonina. A first aspect of the invention refers to a stem cell obtained or obtainable by a process that comprises placing the cell in contact with melatonin.
En una realización más preferida, este aspecto de la invención la célula madre es una célula madre mesenquimal. In a more preferred embodiment, this aspect of the invention the stem cell is a mesenchymal stem cell.
La célula madre de la invención es una célula madre adulta, y más preferiblemente, es una célula madre mesenquimal. El término "célula madre adulta" se refiere a aquella célula madre que es aislada de un tejido o un órgano de un animal en un estado de crecimiento posterior al estado embrionario. Preferiblemente, las células madre de la invención son aisladas en un estado postnatal. Preferiblemente son aisladas de un mamífero, y más preferiblemente de un humano, incluyendo neonatos, juveniles, adolescentes y adultos. Se pueden aislar células madre adultas de una gran variedad de tejidos y órganos, como médula ósea (células madre mesenquimales, células progenitoras adultas multipotentes y células madre hematopoyéticas), tejido adiposo, cartílago, epidermis, folículo piloso, músculo esquelético, músculo cardíaco, intestino, hígado, neuronal. El término "célula madre mesenquimal" o "MSC", tal como se usa en el presente documento, se refiere a una célula de estroma multipotente, originada a partir de la capa germinal mesodermal, que puede diferenciarse en una variedad de tipos de células, incluyendo osteocitos (células de hueso), condrocitos (células de cartílago) y adipocitos (células de grasa). Las MSCs pueden ser obtenidas a partir de, sin quedar limitado a, médula ósea, tejido adiposo (tal como el tejido adiposo subcutáneo), hígado, bazo, testículos, sangre menstrual, fluido amniótico, páncreas, periostio, membrana sinovial, músculo esquelético, dermis, pericitos, hueso trabecular, cordón umbilical humano, pulmón, pulpa dental y sangre periférica. Las MSCs de acuerdo con la invención pueden obtenerse a partir de cualquiera de los tejidos anteriores, tal como a partir de médula ósea, de tejido adiposo subcutáneo o de cordón umbilical. Las células madre de la invención también pueden diferenciarse a partir de células madre de pluripotencia inducida.The stem cell of the invention is an adult stem cell, and more preferably, it is a mesenchymal stem cell. The term "adult stem cell" refers to that stem cell that is isolated from a tissue or an organ of an animal in a state of growth subsequent to the embryonic state. Preferably, the stem cells of the invention are isolated in a postnatal state. Preferably they are isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents, and adults. Adult stem cells can be isolated from a wide variety of tissues and organs, such as bone marrow (mesenchymal stem cells, multipotent adult progenitor cells, and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, cardiac muscle, intestine , liver, neural. The term "mesenchymal stem cell" or "MSC", as used herein, refers to a multipotent stromal cell, originating from the germinal layer mesodermal, which can differentiate into a variety of cell types, including osteocytes (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells). MSCs may be obtained from, but not limited to, bone marrow, adipose tissue (such as subcutaneous adipose tissue), liver, spleen, testes, menstrual blood, amniotic fluid, pancreas, periosteum, synovium, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, dental pulp, and peripheral blood. MSCs according to the invention can be obtained from any of the above tissues, such as from bone marrow, subcutaneous adipose tissue or umbilical cord. The stem cells of the invention can also differentiate from stem cells of induced pluripotency.
Si se desea, la célula madre activada de la invención puede ser modificada genéticamente por cualquier método convencional incluyendo, a modo ilustrativo, no limitativo, procesos de transgénesis, deleciones o inserciones en su genoma que modifiquen la expresión de genes que sean importantes para sus propiedades básicas (proliferación, migración, diferenciación, etc.), o mediante la inserción de secuencias de nucleótidos que codifiquen proteínas de interés como, por ejemplo, proteínas con propiedades terapéuticas. Por tanto, en otra realización preferida, la célula madre activada de la invención ha sido modificada genéticamente. If desired, the activated stem cell of the invention can be genetically modified by any conventional method including, by way of illustration, without limitation, transgenesis processes, deletions or insertions in its genome that modify the expression of genes that are important for its properties. basic (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences that encode proteins of interest, such as proteins with therapeutic properties. Therefore, in another preferred embodiment, the activated stem cell of the invention has been genetically modified.
Por tanto, preferiblemente este aspecto de la invención se refiere a una célula madre mesenquimal activada, de ahora en adelante célula madre mesenquimal activada de la invención, obtenida u obtenible por un procedimiento que comprende poner la célula en contacto con melatonina. Therefore, preferably this aspect of the invention refers to an activated mesenchymal stem cell, hereinafter activated mesenchymal stem cell of the invention, obtained or obtainable by a method comprising contacting the cell with melatonin.
En esta memoria se entiende por “célula madre mesenquimal activada" un producto celular basado en MSCs que presenta potenciada una o algunas de sus funciones celulares (supervivencia, actividad antioxidante, función mitocondrial, capacidad inmunomoduladora, etc).In this specification, "activated mesenchymal stem cell" is understood to be a cellular product based on MSCs that presents enhanced one or some of its cellular functions (survival, antioxidant activity, mitochondrial function, immunomodulatory capacity, etc.).
Otro aspecto de la invención se refiere a una población de células madre activadas, de ahora en adelante población de células madre activadas de la invención, que comprende al menos una célula madre activada de la invención. Preferiblemente la célula madre es una MSC. Another aspect of the invention relates to a population of activated stem cells, hereinafter the activated stem cell population of the invention, comprising at least one activated stem cell of the invention. Preferably the stem cell is an MSC.
En una realización preferida la población celular de la invención comprende al menos un 20%, preferiblemente un 40%, y aún más preferiblemente un 50%, 60%, 80%, 90%, 95%, o un 99% de células madre adultas de la invención. In a preferred embodiment, the cell population of the invention comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% adult stem cells. of the invention.
El término "aislada" indica que la célula o la población celular de la invención a la que se refiere, no se encuentran en su ambiente natural. Esto es, la célula o la población celular ha sido separada de su tejido circundante. Las células madre activadas de la invención, así como las células presentes en la población celular de la invención, pueden ser células de autólogo, alogénico o xenogénico. En una realización particular, dichas células son de origen autólogo, reduciendo así las complicaciones potenciales asociados con / o respuestas antigénicas e inmunogénicas de dichas células cuando se administran al individuo. The term "isolated" indicates that the cell or cell population of the invention to which it refers is not found in its natural environment. That is, the cell or cell population has been separated from its surrounding tissue. The activated stem cells of the invention, as well as the cells present in the cell population of the invention, can be autologous, allogeneic or xenogeneic cells. In a particular embodiment, said cells are of autologous origin, thus reducing potential complications associated with/or antigenic and immunogenic responses of said cells when administered to the individual.
Las células madre mesenquimales activadas de acuerdo a este aspecto de la invención se obtendrán, preferiblemente, pretratando este producto celular con una solución de, preferiblemente 25 mM, de melatonina. El pretratamiento tendrá lugar preferentemente durante las 24 horas previas a su uso, pero también podrá realizarse por un periodo de tiempo superior (e.g. 48 horas). Mesenchymal stem cells activated according to this aspect of the invention will preferably be obtained by pre-treating this cell product with a solution of, preferably 25 mM, melatonin. The pre-treatment will preferably take place during the 24 hours prior to use, but it can also be carried out for a longer period of time (e.g. 48 hours).
Por tanto, otra realización preferida de este aspecto de la invención se refiere a células madre mesenquimales activadas obtenibles por un procedimiento o metodología de obtención de células madre activadas, que comprende poner en contacto la célula madre con una solución de melatonina. Preferiblemente la célula madre es una célula madre mesenquimal. Aún más preferiblemente, la melatonina se encuentra en una concentración de entre 10 pM y 50 pM, más preferiblemente entre 15 pM y 40 pM, aún más preferiblemente entre 20 pM y 30 pM, y aún mucho más preferiblemente es de 25 pM. Therefore, another preferred embodiment of this aspect of the invention relates to activated mesenchymal stem cells obtainable by a method or methodology for obtaining activated stem cells, which comprises contacting the stem cell with a melatonin solution. Preferably the stem cell is a mesenchymal stem cell. Even more preferably, the melatonin is in a concentration between 10 pM and 50 pM, more preferably between 15 pM and 40 pM, even more preferably between 20 pM and 30 pM, and most preferably still 25 pM.
En otra realización de este aspecto de la invención, el tiempo de pretratamiento es de al menos 12 horas, preferiblemente de al menos 16 horas, aún más preferiblemente de al menos 20 horas, y aún mucho más preferiblemente de al menos 24 horas. In another embodiment of this aspect of the invention, the pretreatment time is at least 12 hours, preferably at least 16 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.
COMPOSICIÓN DE LA INVENCIÓN COMPOSITION OF THE INVENTION
La célula madre aislada activada de la invención, o la población celular de la invención, pueden formar parte de una composición. Por tanto, un tercer aspecto de la invención se refiere a una composición, de ahora en adelante composición de la invención, que comprende al menos una célula madre activada de la invención. En una realización preferida de este aspecto la composición de la invención además comprende un vehículo farmacéuticamente aceptable. En una realización preferida de este aspecto la composición de la invención además comprende otro principio activo. The isolated activated stem cell of the invention, or the cell population of the invention, may form part of a composition. Therefore, a third aspect of the invention refers to a composition, hereinafter composition of the invention, comprising at least one activated stem cell of the invention. In a preferred embodiment of this aspect the composition of the invention further comprises a pharmaceutically acceptable carrier. In a preferred embodiment of this aspect, the composition of the invention also comprises another active principle.
En otra realización aún más preferida de este aspecto de la invención, las células madre son células madre mesenquimales. In another even more preferred embodiment of this aspect of the invention, the stem cells are mesenchymal stem cells.
Preferiblemente, la composición de células de la invención tiene, al menos, el 50%, al menos, el 60%, preferiblemente el 70%, más preferiblemente el 80%, aún más preferiblemente, el 90%, y, todavía aún más preferiblemente, el 95% de las células madre aisladas activadas de la invención.Preferably, the cell composition of the invention is at least 50%, at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, and still more preferably , 95% of the isolated stem cells activated of the invention.
Dicha composición de células adultas de la invención puede contener un medio en el que se encuentran las células de la invención; dicho medio debe ser compatible con dichas células. Por ejemplo, pero sin limitarse, soluciones isotónicas, opcionalmente suplementadas con suero; medios de cultivo celular o, alternativamente, un medio soporte sólido, semisólido, gelatinoso o viscoso. La composición de la invención, preferiblemente, es una composición farmacéutica para su administración a un sujeto. Said composition of adult cells of the invention may contain a medium in which the cells of the invention are found; said medium must be compatible with said cells. By example, but not limited to, isotonic solutions, optionally supplemented with serum; cell culture media or, alternatively, a solid, semi-solid, gelatinous or viscous support medium. The composition of the invention is preferably a pharmaceutical composition for administration to a subject.
El término "vehículo farmacéuticamente aceptable" se refiere a un vehículo que debe estar aprobado por una agencia reguladora del gobierno federal o un gobierno estatal o enumerado en la Farmacopea Estadounidense o la Farmacopea Europea, u otra farmacopea reconocida generalmente para su uso en animales, y más concretamente en humanos. The term "pharmaceutically acceptable vehicle" means a vehicle that must be approved by a federal or state government regulatory agency or listed in the US Pharmacopeia or European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more specifically in humans.
El término "vehículo" se refiere a un diluyente, coadyuvante, excipiente o portador con el que se deben administrar las células o la población celular de la invención o de dicha composición que comprende células madre de la invención obtenible según el procedimiento de la invención; obviamente, dicho vehículo debe ser compatible con dichas células. Ejemplos ilustrativos, no limitativos, de dicho vehículo incluyen cualquier vehículo fisiológicamente compatible, por ejemplo, soluciones isotónicas (por ejemplo, solución salina estéril al 0,9% NaCI, solución salina tamponada con fosfatos (PBS), solución Ringerlactato, etc.), opcionalmente suplementadas con suero, preferiblemente con suero autólogo; medios de cultivo celular (por ejemplo, DMEM, etc.); o, alternativamente, un medio soporte sólido, semisólido, gelatinoso o viscoso, tal como colágeno, colagen-glicosamino-glicano, fibrina, cloruro de polivinilo, poliaminoácidos, tales como polilisina, o poliornitina, hidrogeles, agarosa, sulfato de dextrano silicona. Asimismo, si se desea, el medio de soporte puede, en realizaciones específicas, contener factores de crecimiento u otros agentes. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which the cells or cell population of the invention or said composition comprising stem cells of the invention obtainable according to the process of the invention must be administered; obviously, said vehicle must be compatible with said cells. Illustrative, non-limiting examples of such a vehicle include any physiologically compatible vehicle, for example, isotonic solutions (for example, sterile 0.9% NaCI saline, phosphate buffered saline (PBS), Ringerlactate solution, etc.), optionally supplemented with serum, preferably with autologous serum; cell culture media (eg, DMEM, etc.); or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, such as collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polyornithine, hydrogels, agarose, silicone dextran sulfate. Also, if desired, the support medium may, in specific embodiments, contain growth factors or other agents.
Si el soporte es sólido, semisólido, o gelatinoso, las células pueden ser introducidas en una fase líquida del vehículo que es tratada posteriormente de forma tal que se convierte en una fase más sólida. If the support is solid, semi-solid, or gelatinous, the cells can be introduced into a liquid phase of the vehicle which is subsequently treated such that it becomes a more solid phase.
La composición farmacéutica de la invención, si se desea, puede contener también, cuando sea necesario, aditivos para aumentar, controlar o dirigir de otro modo el efecto terapéutico deseado de las células, los cuales comprenden dicha composición farmacéutica, y/o sustancias auxiliares o sustancias farmacéuticamente aceptables, tales como agentes tamponantes, tensioactivos, codisolventes, conservantes, etc. También, para estabilizar la suspensión celular, es posible añadir quelantes de metales. La estabilidad de las células en el medio líquido de la composición farmacéutica de la invención puede mejorarse mediante la adición de sustancias adicionales, tales como, por ejemplo, ácido aspártico, ácido glutámico, etcétera. Dichas sustancias farmacéuticamente aceptables que pueden usarse en la composición farmacéutica de la invención son conocidas, en general, los técnicos en la materia y se usan normalmente en la elaboración de composiciones celulares. Ejemplos de vehículos farmacéuticos adecuados se describen, por ejemplo, en "Remington's Pharmaceutical Sciences", de E.W. Martin. Puede encontrarse información adicional sobre dichos vehículos en manuales de tecnología farmacéutica (Farmacia Galénica). Como se emplea aquí, el término "principio activo", "sustancia activa", "sustancia farmacéuticamente activa", "ingrediente activo" o "ingrediente farmacéuticamente activo" significa cualquier componente que potencialmente proporcione una actividad farmacológica u otro efecto diferente en el diagnóstico, cura, mitigación, tratamiento, o prevención de una enfermedad, o que afecta a la estructura o función del cuerpo del hombre u otros animales. El término incluye aquellos componentes que promueven un cambio químico en la elaboración del fármaco y están presentes en el mismo de una forma modificada prevista que proporciona la actividad específica o el efecto. The pharmaceutical composition of the invention, if desired, may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the cells, which said pharmaceutical composition comprises, and/or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators. The stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by adding additional substances, such as, for example, aspartic acid, glutamic acid, etc. Said pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions. Examples of suitable pharmaceutical carriers are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin. Additional information on such vehicles can be found in pharmaceutical technology manuals (Farmacia Galénica). As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect in diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present in the drug in an intended modified form that provides the specific activity or effect.
USOS MÉDICOS DE LA INVENCIÓN MEDICAL USES OF THE INVENTION
Otro aspecto de la invención se refiere a una célula madre activada de la invención, la población celular de la invención, o de una composición de la invención, para su uso como medicamento. Más preferiblemente las células madre son células madre mesenquimales. Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, for use as a medicament. More preferably the stem cells are mesenchymal stem cells.
El término "medicamento", tal y como se usa en esta memoria, hace referencia a cualquier sustancia usada para prevención, diagnóstico, alivio, tratamiento o curación de enfermedades en el hombre y los animales. En el contexto de la presente invención, la enfermedad es un cáncer.The term "drug", as used in this specification, refers to any substance used for the prevention, diagnosis, alleviation, treatment or cure of diseases in humans and animals. In the context of the present invention, the disease is cancer.
La composición farmacéutica de la invención contendrá una cantidad profiláctica o terapéuticamente efectiva de las células de la invención o de la población celular de la invención, preferentemente, una población celular sustancialmente homogénea, para proporcionare! efecto terapéutico deseado. The pharmaceutical composition of the invention will contain a prophylactically or therapeutically effective amount of the cells of the invention or of the cell population of the invention, preferably a substantially homogeneous cell population, to provide! desired therapeutic effect.
Tal como se usa en la presente descripción, el término "cantidad terapéutica o profilácticamente efectiva" se refiere a la cantidad de células de la invención contenida en la composición farmacéutica que es capaz de producir el efecto terapéutico deseado y, en general, se determinará, entre otros factores, por las propias características de las células y el efecto terapéutico deseado que se persigue. En general, la cantidad terapéuticamente efectiva de células de la invención que debe administrarse dependerá, entre otros factores, de las propias características del sujeto, la gravedad de la enfermedad, la forma de administración, etc. Por este motivo, las dosis mencionadas en esta invención deben tenerse en cuenta sólo como guía para la persona conocedora de la técnica, que debe ajustar esta dosis dependiendo de los factores anteriormente descritos. Como ejemplo ilustrativo y no limitativo, la composición farmacéutica de la invención puede administrarse como una dosis única, que contenga aproximadamente entre 1x105 y 10x106 células de la invención por kilo de peso corporal del receptor, y más preferentemente entre 5x105 y 5x106 células de la 25 invención por kilo del peso corporal del receptor, en una forma de realización más preferente aún dicha composición farmacéutica contendrá aproximadamente entre 1x106 y 2x106 células de la invención por kilo del peso corporal del receptor, dependiendo de los factores descritos anteriormente. La dosis de células de la invención puede repetirse, dependiendo del estado y evolución del paciente, en intervalos temporales de días, semanas o meses que debe establecer el especialista en cada caso. As used in the present description, the term "therapeutically or prophylactically effective amount" refers to the amount of cells of the invention contained in the pharmaceutical composition that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, due to the characteristics of the cells and the desired therapeutic effect pursued. In general, the therapeutically effective amount of cells of the invention that must be administered will depend, among other factors, on the characteristics of the subject, the severity of the disease, the form of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the person skilled in the art, who must adjust this dose depending on the factors described above. As an illustrative and non-limiting example, the pharmaceutical composition of the invention can be administered as a single dose, containing approximately between 1x10 5 and 10x10 6 cells of the invention per kilogram of body weight of the recipient, and more preferably between 5x10 5 and 5x10 6 cells of the invention per kilo of the recipient's body weight, in an even more preferred embodiment said composition pharmaceutical will contain approximately between 1x10 6 and 2x10 6 cells of the invention per kilogram of the recipient's body weight, depending on the factors described above. The dose of cells of the invention can be repeated, depending on the state and evolution of the patient, at time intervals of days, weeks or months that must be established by the specialist in each case.
Otro aspecto de la invención se refiere a una célula madre activada de la invención, la población celular de la invención, o de una composición de la invención, para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de un tejido o un órgano enfermo o dañado.Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, to increase, restore or partially or totally replace the functional activity of a diseased tissue or organ. or damaged.
Otro aspecto de la invención se refiere a una célula madre activada de la invención, la población celular de la invención, o de una composición de la invención, para el tratamiento del cáncer. Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, for the treatment of cancer.
PROCEDIMIENTO DE OBTENCIÓN DE CÉLULAS MADRE MESENQUIMALES ACTIVADAS PROCEDURE FOR OBTAINING ACTIVATED MESENCYMAL STEM CELLS
Las células madre mesenquimales activadas se obtendrán pretratando este producto celular con una solución de, preferiblemente 25 mM, de melatonina. El pretratamiento tendrá lugar preferentemente durante las 24 horas previas a su uso, pero también podrá realizarse por un periodo de tiempo superior (e.g. 48 horas). Activated mesenchymal stem cells will be obtained by pretreating this cell product with a solution of, preferably 25 mM, melatonin. The pre-treatment will preferably take place during the 24 hours prior to use, but it can also be carried out for a longer period of time (e.g. 48 hours).
Por tanto, otro aspecto de la invención se refiere a un procedimiento o metodología de obtención de células madre activadas, de ahora en adelante procedimiento de la invención, que comprende poner en contacto la célula madre con una solución de melatonina. Preferiblemente la célula madre es una célula madre mesenquimal. Aún más preferiblemente, la melatonina se encuentra en una concentración de entre 10 mM y 50 pM, más preferiblemente entre 15 pM y 40 pM, aún más preferiblemente entre 20 pM y 30 pM, y aún mucho más preferiblemente es de 25 pM.Therefore, another aspect of the invention refers to a procedure or methodology for obtaining activated stem cells, from now on the procedure of the invention, which comprises contacting the stem cell with a melatonin solution. Preferably the stem cell is a mesenchymal stem cell. Even more preferably, the melatonin is in a concentration between 10 mM and 50 pM, more preferably between 15 pM and 40 pM, even more preferably between 20 pM and 30 pM, and most preferably still 25 pM.
En otra realización de este aspecto de la invención, el tiempo de pretratamiento es de al menos 12 horas, preferiblemente de al menos 16 horas, aún más preferiblemente de al menos 20 horas, y aún mucho más preferiblemente de al menos 24 horas. In another embodiment of this aspect of the invention, the pretreatment time is at least 12 hours, preferably at least 16 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. EJEMPLOS DE LA INVENCIÓN Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages, and features of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES OF THE INVENTION
Los autores de la presente invención han desarrolado una serie de ensayos para mostrar que el tratamiento previo de las células madre mesenquimales (MSC) con melatonina (MT) mejora sus propiedades anticancerígenas. The authors of the present invention have developed a series of tests to show that the pretreatment of mesenchymal stem cells (MSC) with melatonin (MT) improves their anticancer properties.
EJEMPLO 1. Desarrollo de un modelo de cocultivo in vitro para imitar la interacción biológica in vivo entre las CMM y las células de glioma. EXAMPLE 1. Development of an in vitro coculture model to mimic the in vivo biological interaction between MSCs and glioma cells.
A. Cultivo de MSC: las MSC derivadas de tejido adiposo se compraron en ATCC (PCS-500- 011). Brevemente, las MSC se cultivan en un medio de crecimiento compuesto por medio de Eagle modificado de Dulbecco (DMEM) complementado con suero bovino fetal (FBS) al 10% y penicilina-estreptomicina al 1%. Las células se incubaron a 37°C en una atmósfera humidificada con 20% de 02 y 5% de C02 hasta una confluencia del 70-80%. Los medios se cambiaron cada 2-3 días. Los pases se realizaron utilizando una solución de tripsina al 0,25%. Se utilizaron células de los pasajes 3 a 6 para todos los experimentos. A. MSC culture: Adipose tissue-derived MSCs were purchased from ATCC (PCS-500-011). Briefly, MSCs are grown in a growth medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% C02 until 70-80% confluence. The media were changed every 2-3 days. Passages were performed using a 0.25% trypsin solution. Cells from passages 3 to 6 were used for all experiments.
B. Pretratamiento de MT: los cultivos de MSC subconfluentes se trataron previamente con 25 mM de MT durante 24 horas. Luego, las células se utilizaron para los diferentes experimentos propuestos aquí. Para determinar si el tratamiento previo con MT modifica la identidad de las MSC, se evaluó el potencial de múltiples linajes de las MSC (adipocitos, osteocitos y condrocitos) y la expresión de marcadores de MSC establecidos (las MSC expresan los marcadores CD29, CD13, CD73, CD105 y CD90, mientras que son negativos para la expresión de CD31, CD45, CD34 y HLA II) (1-3). También determinaremos si el pretratamiento con MT afecta la proliferación celular (e.j. Alamar Blue), la apoptosis (e.j. Anexina V), la actividad antioxidante (e.j. actividad catalasa) y la función mitocondrial (e.g. Sehorse). B. MT Pretreatment: Subconfluent MSC cultures were pretreated with 25 mM MT for 24 hours. The cells were then used for the different experiments proposed here. To determine whether MT pretreatment modifies MSC identity, we evaluated the potential for multiple MSC lineages (adipocytes, osteocytes, and chondrocytes) and the expression of established MSC markers (MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II) (1-3). We will also determine if MT pretreatment affects cell proliferation (e.g. Alamar Blue), apoptosis (e.g. Annexin V), antioxidant activity (e.g. catalase activity) and mitochondrial function (e.g. Sehorse).
C. Cultivo de células de glioma: las células de glioma se obtuvieron de ATCC (U87MG; HTB- 14). Brevemente, las células U87 se cultivan en un medio de crecimiento compuesto de Medio Esencial Mínimo de Eagle (EMEM; ATCC), suplementado con FBS al 10% y penicilina- estreptomicina al 1%. Las células se incuban a 37 ° C en una atmósfera humidificada con 20% de 02 y 5% de C02 hasta una confluencia del 70-80%. Los medios se cambiaron cada 2-3 días. Los pases se realizan utilizando una solución de tripsina al 0,25%. Se utilizaron células de los pasajes 3 a 6 para todos los experimentos. C. Glioma cell culture: Glioma cells were obtained from ATCC (U87MG; HTB-14). Briefly, U87 cells are grown in a growth medium composed of Eagle's Minimal Essential Medium (EMEM; ATCC), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells are incubated at 37 °C in a humidified atmosphere with 20% 02 and 5% C02 to 70-80% confluence. The media were changed every 2-3 days. Passaging is done using a 0.25% trypsin solution. Cells from passages 3 to 6 were used for all experiments.
D. Sistema de co-cultivo: Para estudiar la interacción entre las células de glioma y las MSC (pretratadas o no con MT), se utiliza el sistema de co-cultivo transwell (tamaño de poro de 0,4 pm). Las MSCs se siembran en la parte superior, mientras que las células de glioma se siembran en la parte inferior. Este sistema permite separar fácilmente las dos poblaciones celulares para realizar estudios específicos con células de glioma. Los grupos experimentales son: U87 (células U87 sin MSC), U87 + MSC (células U87 cocultivadas con MSC) y U87 + MSC + MT (células U87 cocultivadas con MSC que fueron pretratadas con MT). D. Co-cultivation system: To study the interaction between glioma cells and MSCs (pretreated or not with MT), the transwell co-cultivation system (0.4 pm pore size) is used. MSCs are seeded on top, while glioma cells are seeded on the bottom. This system allows to easily separate the two cell populations for perform specific studies with glioma cells. The experimental groups are: U87 (U87 cells without MSC), U87 + MSC (U87 cells co-cultured with MSC) and U87 + MSC + MT (U87 cells co-cultured with MSC that were pre-treated with MT).
EJEMPLO 2. Estudiar las interacciones entre las CMM y las células de glioma mediante el modelo de cocultivo in vitro. EXAMPLE 2. To study the interactions between MSCs and glioma cells using the in vitro coculture model.
A. Proliferación y stemnes: después de 24 horas en el sistema de cocultivo transwell, las células de glioma se utilizan para estudiar la proliferación mediante el ensayo Alamar Blue y mediante inmunocitoquímica. A. Proliferation and stemnes: After 24 hours in the transwell coculture system, glioma cells are used to study proliferation using the Alamar Blue assay and immunocytochemistry.
B. Apoptosis: después de 24 horas en el sistema de cocultivo transwell, las células de glioma se utilizan para estudiar la apoptosis mediante citometría de flujo (kit de Annexin V). B. Apoptosis: After 24 hours in the transwell coculture system, glioma cells are used to study apoptosis by flow cytometry (Annexin V kit).
C. Ensayo de migración: para estudiar cómo las MSC interfieren en la capacidad de migración de U87, se utiliza el sistema transwell (tamaño de poro de 0,8 pm). Las células U87 se siembran en la parte superior utilizando medio sin suero, mientras que las MSC se siembran en la parte inferior con medio FBS para promover la migración celular a través de los poros de la membrana. Las células se incuban a 37°C en una atmósfera humidificada de 20% de 02 y 5% de C02 y se dejarán migrar durante 48 h. Después de la incubación, las células no migradas en la parte superior de los insertos se eliminan con un hisopo de algodón. Las células migradas en la parte inferior de los insertos se fijan con paraformaldehído al 4% y se tiñen con violeta de cresilo al 0,5%. Luego, se toman imágenes de 9 campos aleatorios por membrana y se cuentan para su comparación utilizando un microscopio Olympus 1X71. C. Migration Assay: To study how MSCs interfere with the migration ability of U87, the transwell system (0.8 pm pore size) is used. U87 cells are seeded on top using serum-free medium, while MSCs are seeded on bottom with FBS medium to promote cell migration through the membrane pores. Cells are incubated at 37°C in a humidified atmosphere of 20% O2 and 5% C02 and allowed to migrate for 48 h. After incubation, non-migrated cells on top of the inserts are removed with a cotton swab. The migrated cells at the bottom of the inserts are fixed with 4% paraformaldehyde and stained with 0.5% cresyl violet. Then, images of 9 random fields per membrane are taken and counted for comparison using an Olympus 1X71 microscope.
D. Estudio molecular: después de 24 horas en el sistema de cocultivo transwell, se recolectan células de glioma para investigar las vías moleculares que son moduladas por las MSC, utilizando RNA-Seq, RT-PCR y western blot. D. Molecular study: After 24 hours in the transwell coculture system, glioma cells are harvested to investigate the molecular pathways that are modulated by MSCs, using RNA-Seq, RT-PCR, and western blotting.
A. Perfil de citocinas: el sobrenadante celular generado después del cocultivo se analizará mediante ELISA multiplex (Bio-Plex Pro ™ Human Cytokine) para identificar citocinas de supervivencia, angiogénicas e inflamatorias, entre otras. A. Cytokine profile: The cell supernatant generated after cocultivation will be analyzed by multiplex ELISA (Bio-Plex Pro ™ Human Cytokine) to identify survival, angiogenic and inflammatory cytokines, among others.
EJEMPLO 3. Estudiar las interacciones entre las CMM y las células de glioma mediante un modelo in vivo. EXAMPLE 3. To study the interactions between MSCs and glioma cells using an in vivo model.
A. Animales y grupos experimentales: Para evitar el rechazo de las células trasplantadas, se utilizan ratones desnudos atímicos (10 semanas de edad). Los animales se asignan aleatoriamente a tres grupos experimentales: ratones inyectados con células de glioma (grupo U87), ratones inyectados con células de glioma y MSC (grupo U87 + MT) y ratones de control (grupo U87 + MSC + MT). B. Trasplante de células (modelo de xenoinjerto subcutáneo): se inyectan ratones anestesiados por vía subcutánea en el costado de la pata trasera derecha con 150 pl_ de mezcla de DMEM: matrigel (1 :2) que contiene T 106 células de glioma, con o sin T 106 MSC (pretratados o no con MT). Los ratones son sacrificados cuando los tumores alcancen un volumen promedio de 4000 mm3 (volúmenes más altos pueden afectar el bienestar animal). A. Animals and experimental groups: To avoid rejection of the transplanted cells, athymic nude mice (10 weeks old) are used. Animals are randomly assigned to three experimental groups: mice injected with glioma cells (U87 group), mice injected with glioma cells and MSC (U87 + MT group) and control mice (U87 + MSC + MT group). B. Cell transplantation (subcutaneous xenograft model): Anesthetized mice are injected subcutaneously into the flank of the right hind paw with 150 pl_ of a mixture of DMEM: matrigel (1:2) containing T 10 6 glioma cells, with or without T 10 6 MSC (pretreated or not with MT). Mice are sacrificed when tumors reach an average volume of 4000 mm3 (higher volumes may affect animal welfare).
C. Crecimiento del tumor: los ratones son monitoreados diariamente para estudiar la evolución del tumor durante todo el proyecto. Una vez sacrificados, los tumores son extirpados para su posterior análisis histológico e inmunohistoquímico. C. Tumor growth: Mice are monitored daily to study tumor evolution throughout the project. Once sacrificed, the tumors are excised for subsequent histological and immunohistochemical analysis.
Ejemplo 4. El pretratamiento de las células madre mesenquimales (MSC) con melatonina (MT) mejora las propiedades anticancerígenas de las células. Example 4. Pretreatment of mesenchymal stem cells (MSC) with melatonin (MT) enhances the anticancer properties of the cells.
4.1. Desarrollo de un modelo de co-cultivo in vitro para imitar la interacción biológica in vivo entre las MSC y las células de glioma. 4.1. Development of an in vitro co-culture model to mimic the in vivo biological interaction between MSCs and glioma cells.
Cultivo de MSC: las MSC derivadas de tejido adiposo se adquirieron de ATCC (PCS-500-011). Brevemente, las MSC se cultivaron en medio de crecimiento que se componía de medio de Eagle modificado por Dulbecco (DMEM) complementado con suero bovino fetal (FBS) al 10% y penicilina-estreptomicina al 1%. Las células se incubaron a 37°C en una atmósfera humidificada con el 20% de O2 y el 5% de CO2 hasta un 70-80% de confluencia. Los medios se cambiaron cada 2-3 días. Los pases se llevaron a cabo usando una disolución de tripsina al 0,25%. Para todos los experimentos se utilizaron células de los pases 3-6. MSC culture: Adipose tissue-derived MSCs were purchased from ATCC (PCS-500-011). Briefly, MSCs were grown in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 to 70-80% confluence. The media were changed every 2-3 days. Passages were carried out using a 0.25% trypsin solution. For all experiments cells from passages 3-6 were used.
Pretratamiento con MT: los cultivos de MSC subconfluentes se pretrataron con 25 mM de MT durante 24 horas. Luego, las células pretratadas obtenidas se usaron para los siguientes experimentos. MT pretreatment: subconfluent MSC cultures were pretreated with 25 mM MT for 24 hours. Then, the obtained pretreated cells were used for the following experiments.
Para determinar si el pretratamiento con MT modifica la identidad de MSC, se evaluó el potencial de múltiples linajes de las MSC (adipocitos, osteocitos y condrocitos) y la expresión de marcadores de MSC establecidos (las MSC expresan los marcadores CD29, CD13, CD73, CD105 y CD90, mientras que son negativas para la expresión de CD31 , CD45, CD34 y HLA II) (1-3). También se determinó si el pretratamiento con MT afectaba a la proliferación y apoptosis de las MSC cultivadas. To determine whether MT pretreatment modifies MSC identity, we evaluated the potential for multiple MSC lineages (adipocytes, osteocytes, and chondrocytes) and the expression of established MSC markers (MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II) (1-3). We also determined whether MT pretreatment affected the proliferation and apoptosis of cultured MSCs.
Cultivo de células de glioma: las células de glioma se obtuvieron de ATCC (U87MG; HTB-14). Brevemente, las células U87 se cultivaron en medio de crecimiento que se componía de medio esencial mínimo de Eagle (EMEM; ATCC), complementado con FBS al 10% y penicilina- estreptomicina al 1%. Las células se incubaron a 37°C en una atmósfera humidificada con el 20% de O2 y el 5% de CO2 hasta un 70-80% de confluencia. Los medios se cambiaron cada 2-3 días. Los pases se llevaron a cabo usando una disolución de tripsina al 0,25%. Para todos los experimentos se utilizaron células de los pases 3-6. Glioma cell culture: Glioma cells were obtained from ATCC (U87MG; HTB-14). Briefly, U87 cells were grown in growth medium consisting of Eagle's minimal essential medium (EMEM; ATCC), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 to 70-80% confluence. The media were changed every 2-3 days. Passages were carried out using a 0.25% trypsin solution. For all experiments cells from passages 3-6 were used.
Sistema de co-cultivo: para estudiar la interacción entre las células de glioma y las MSC (pretratadas o no con MT) se usaron dos enfoques: Co-culture system: To study the interaction between glioma cells and MSCs (pre-treated or not with MT) two approaches were used:
- Co-cultivos directos: se sembraron U87 y MSC (pretratadas o no con MT) en la misma placa con MSC y se les permitió que se adhirieran durante la noche. Luego, después de 24 horas, se recogió el sobrenadante y se almacenó a -80°C hasta su uso. - Direct co-cultures: U87 and MSC (pretreated or not with MT) were seeded on the same plate with MSC and allowed to adhere overnight. Then, after 24 hours, the supernatant was collected and stored at -80°C until use.
- Co-cultivos indirectos: se usó el sistema de cocultivo Transwell (con un tamaño de poro de 0,4 pm). Las MSC se sembraron en la parte superior, mientras que las células de glioma se sembraron en la parte inferior. Este sistema permitió separar fácilmente las dos poblaciones celulares para realizar estudios específicos con células de glioma. Los grupos experimentales fueron: U87 (células U87 sin MSC), U87+MSC (células U87 cocultivadas con MSC) y U87+MSC+MT (células U87 cocultivadas con MSC que se pretrataron con MT). - Indirect co-cultures: the Transwell co-culture system was used (with a pore size of 0.4 pm). MSCs were seeded on top, while glioma cells were seeded on the bottom. This system made it possible to easily separate the two cell populations for specific studies with glioma cells. The experimental groups were: U87 (U87 cells without MSC), U87+MSC (U87 cells cocultured with MSC) and U87+MSC+MT (U87 cells cocultured with MSC that were pretreated with MT).
4.2. Estudio de las interacciones entre las MSC y las células de glioma usando el modelo de cocultivo in vitro. a. Proliferación: después de 24 horas en el sistema de co-cultivo, las células de glioma se usaron para estudiar la proliferación mediante el ensayo Alamar Blue e inmunocitoquímica. b. Apoptosis: después de 24 horas en el sistema de co-cultivo, las células de glioma se usaron para estudiar la apoptosis mediante citometría de flujo (kit de anexina V). c. Ensayo de migración: para estudiar cómo las MSC interfieren en la capacidad de migración de U87, se usó el sistema Transwell (con un tamaño de poro de 0,8 pm). Las células U87 se sembraron en la parte superior con medio libre de suero, mientras que las MSC se sembraron en la parte inferior con medio FBS para fomentar la migración celular a través de los poros de la membrana. Las células se incubaron a 37°C en una atmósfera humidificada con el 20% de O2 y el 5% de CO2 y se les permitió que migraran durante 48 h. Después de la incubación, las células no migradas de la parte superior de los insertos se retiraron usando un hisopo de algodón. Las células migradas en la parte inferior de los insertos se fijaron con paraformaldehído al 4% y se tiñeron con violeta de cresilo al 0,5%. Luego, se tomaron imágenes de 9 campos aleatorios por membrana y se contaron para su comparación usando un microscopio Olympus 1X71. d. Esferas tumorales: las células se colocaron en una gota colgante de 20 pl con medio de crecimiento. Para cada esferoide se usaron 21000 células U87 que se cocultivaron o no con MSC o MSCMT en una proporción 3:1. Después de 72 h, los esferoides se transfirieron a pocilios prerrecubiertos de fibronectina para los ensayos de migración o a pocilios prerrecubiertos de Matrigel para los ensayos de invasión. Se tomaron imágenes de los esferoides cada 24 horas y se analizó el área de migración o invasión usando el software ImageJ (versión 1.4r; National Institute of Health, Bethesda, MD). e. Estudio molecular: después de 24 horas en el sistema de cocultivo, se recogieron las células de glioma para investigar las rutas moleculares que modulan las MSC, usando RNA-Seq, RT- PCR e inmunotransferencia de tipo Western. f. Perfil de citocinas: los sobrenadantes celulares generados después del cocultivo se analizaron mediante ELISA múltiple (citocina humana Bio-Plex Pro™) para identificar citocinas de supervivencia, angiogénicas e inflamatorias, entre otras. Se usaron cocultivos directos e indirectos (sistema Transwell). 4.2. Study of interactions between MSCs and glioma cells using the in vitro coculture model. a. Proliferation: After 24 hours in the co-culture system, glioma cells were used to study proliferation using the Alamar Blue assay and immunocytochemistry. b. Apoptosis: After 24 hours in the co-culture system, glioma cells were used to study apoptosis by flow cytometry (Annexin V kit). c. Migration assay: To study how MSCs interfere with the migration ability of U87, the Transwell system (with 0.8 pm pore size) was used. U87 cells were seeded on top with serum-free medium, while MSCs were seeded on the bottom with FBS medium to encourage cell migration through the membrane pores. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 and allowed to migrate for 48 h. After incubation, non-migrated cells from the top of the inserts were removed using a cotton swab. The migrated cells at the bottom of the inserts were fixed with 4% paraformaldehyde and stained with 0.5% cresyl violet. Then, 9 random fields per membrane were imaged and counted for comparison using an Olympus 1X71 microscope. d. Tumor beads: cells were placed in a 20 µl hanging drop with growth medium. For each spheroid, 21,000 U87 cells were used, which were cocultivated or not with MSC or MSCMT in a 3:1 ratio. After 72 h, the spheroids were transferred to fibronectin precoated wells for migration assays or fibronectin precoated wells. Matrigel for invasion assays. Spheroids were imaged every 24 hours and the area of migration or invasion was analyzed using ImageJ software (version 1.4r; National Institute of Health, Bethesda, MD). and. Molecular study: After 24 hours in the coculture system, glioma cells were harvested to investigate the molecular pathways modulated by MSCs, using RNA-Seq, RT-PCR, and Western blotting. F. Cytokine profiling: Cell supernatants generated after cocultivation were analyzed by multiplex ELISA (Bio-Plex Pro™ human cytokine) to identify survival, angiogenic, and inflammatory cytokines, among others. Direct and indirect cocultures (Transwell system) were used.
4.3. Estudio de las interacciones entre las MSC y las células de glioma usando un modelo in vivo. a. Animales y grupos experimentales: para evitar el rechazo de las células trasplantadas, se usaron ratones desnudos atímicos (de 10 semanas de edad). Los animales se asignaron aleatoriamente a tres grupos experimentales: ratones inyectados con células de glioma (grupo U87), ratones inyectados con células de glioma y MSC (grupo U87+MT) y ratones de control (grupo U87+MSC+MT). b. Trasplante de células (modelo de xenoinjerto subcutáneo): a los ratones anestesiados se les inyectó por vía subcutánea en el flanco de la pata trasera derecha con 150 pl de mezcla DMEM:Matrigel (1:2) que contenía T 106 células de glioma, con o sin T 106 MSC (pretratadas o no con MT). Los ratones se sacrificaron cuando los tumores alcanzaron un volumen promedio de 4000 mm3 (volúmenes mayores pueden afectar al bienestar de los animales). c. Crecimiento del tumor: los ratones se monitorizaron diariamente para estudiar la evolución del tumor a lo largo del tiempo, hasta que los tumores alcanzaron un volumen promedio de 4000 mm3. Los tumores se midieron con un compás calibrador digital cada 5 días para estimar su volumen usando la fórmula (diámetro menor 2 c diámetro mayor)/2. d. Estudio celular de los tumores: los ratones se sometieron a perfusión intracardíaca con solución salina al 0,9% y paraformaldehído al 4%. La mitad de cada tumor se incrustó en parafina y se procesó para la tinción histológica (por ejemplo, hematoxilina y eosina y rojo sirio), así como para técnicas inmunohistológicas. Se estudiaron marcadores específicos de proliferación (anticuerpo anti-K¡67), apoptosis (anticuerpo anti-caspasa 3), pluripotencialidad (anticuerpo anti- nestina) y angiogénesis (anticuerpo anti-CD31). La otra mitad de cada tumor se procesó para microscopía electrónica. Bibliografía 4.3. Study of the interactions between MSCs and glioma cells using an in vivo model. a. Experimental animals and groups: To avoid rejection of the transplanted cells, athymic nude mice (10 weeks old) were used. Animals were randomly assigned to three experimental groups: mice injected with glioma cells (U87 group), mice injected with glioma cells and MSC (U87+MT group), and control mice (U87+MSC+MT group). b. Cell transplantation (subcutaneous xenograft model): Anesthetized mice were injected subcutaneously into the flank of the right hind paw with 150 µl of DMEM:Matrigel (1:2) mixture containing T 106 glioma cells, with or without T 106 MSC (pretreated or not with MT). Mice were sacrificed when the tumors reached an average volume of 4000 mm 3 (greater volumes may affect animal welfare). c. Tumor Growth: Mice were monitored daily to study tumor evolution over time, until tumors reached an average volume of 4000 mm 3 . Tumors were measured with a digital caliper every 5 days to estimate their volume using the formula (minor diameter 2 c major diameter)/2. d. Cellular study of tumors: mice were subjected to intracardiac perfusion with 0.9% saline solution and 4% paraformaldehyde. Half of each tumor was paraffin-embedded and processed for histological staining (eg, hematoxylin and eosin and Syrian red) as well as immunohistological techniques. Specific markers of proliferation (anti-K¡67 antibody), apoptosis (anti-caspase 3 antibody), pluripotency (anti-nestin antibody) and angiogenesis (anti-CD31 antibody) were studied. The other half of each tumor was processed for electron microscopy. Bibliography
1 Capilla-Gonzalez, V., Lopez-Beas, J., Escacena, N., Aguilera, Y., de la Cuesta, A. et al. Molecular therapy : the journal of the American Society of Gene Therapy (2018). 1 Capilla-Gonzalez, V., Lopez-Beas, J., Escacena, N., Aguilera, Y., de la Cuesta, A. et al. Molecular therapy : the journal of the American Society of Gene Therapy (2018).
2 Aguilera, Y., Mellado-Damas, N., Olmedo-Moreno, L, López, V., Panadero-Morón, C. et al. Cancers (2021). 2 Aguilera, Y., Mellado-Damas, N., Olmedo-Moreno, L, López, V., Panadero-Morón, C. et al. Cancers (2021).
3 Soria, B., Martin-Montalvo, A., Aguilera, Y., Mellado-Damas, N., Lopez-Beas, J. et al. Frontiers in cellular neuroscience (2019). 3 Soria, B., Martin-Montalvo, A., Aguilera, Y., Mellado-Damas, N., Lopez-Beas, J. et al. Frontiers in cellular neuroscience (2019).

Claims

REIVINDICACIONES
1.- Una célula madre mesenquimal (MSC) activada obtenida u obtenible por un procedimiento que comprende poner la célula en contacto con melatonina. 1.- An activated mesenchymal stem cell (MSC) obtained or obtainable by a process that comprises placing the cell in contact with melatonin.
2.- La célula mesenquimal activada según la reivindicación anterior, donde la célula ha estado en contacto con melatonina al menos 24 horas a una concentración de entre 10 mM y 50 mM.2. The activated mesenchymal cell according to the preceding claim, wherein the cell has been in contact with melatonin for at least 24 hours at a concentration between 10 mM and 50 mM.
3.- La célula mesenquimal activada según la reivindicación anterior, donde la célula ha estado en contacto con melatonina al menos 24 horas a una concentración de entre 15 pM y 40 pM.3. The activated mesenchymal cell according to the preceding claim, wherein the cell has been in contact with melatonin for at least 24 hours at a concentration between 15 pM and 40 pM.
4 - La célula mesenquimal activada según cualquiera de las reivindicaciones 1-3, donde la concentración de la melatonina es de entre 20 pM y 30 pM. 4 - The activated mesenchymal cell according to any of claims 1-3, wherein the concentration of melatonin is between 20 pM and 30 pM.
5.- La célula mesenquimal activada según cualquiera de las reivindicaciones 1-4, donde la concentración de la melatonina es de 25 pM. 5. The activated mesenchymal cell according to any of claims 1-4, wherein the concentration of melatonin is 25 pM.
6.- Una población celular que comprende al menos una célula madre aislada activada obtenible según cualquiera de las reivindicaciones 1-5. 6. A cell population comprising at least one isolated activated stem cell obtainable according to any of claims 1-5.
7.- Una composición que comprende una célula mesenquimal activada según cualquiera de las reivindicaciones 1-5, o una población celular según la reivindicación 6. 7. A composition comprising an activated mesenchymal cell according to any of claims 1-5, or a cell population according to claim 6.
8.- La composición según la reivindicación anterior, que además comprende otro principio activo. 8. The composition according to the preceding claim, which also comprises another active ingredient.
9.- La composición según cualquiera de las reivindicaciones 7-8, que además comprende un vehículo farmacéuticamente aceptable. 9. The composition according to any of claims 7-8, further comprising a pharmaceutically acceptable carrier.
10.- La célula mesenquimal activada según cualquiera de las reivindicaciones 1-5, la población celular según la reivindicación 6, o la composición según cualquiera de las reivindicaciones 7-9, para su uso como medicamento. The activated mesenchymal cell according to any of claims 1-5, the cell population according to claim 6, or the composition according to any of claims 7-9, for use as a medicament.
11.- La célula mesenquimal activada según cualquiera de las reivindicaciones 1-5, la población celular según la reivindicación 6, o la composición según cualquiera de las reivindicaciones 7-9, para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de un tejido o un órgano enfermo o dañado. 11. The activated mesenchymal cell according to any of claims 1-5, the cell population according to claim 6, or the composition according to any of claims 7-9, to increase, restore or partially or totally replace the functional activity of a diseased or damaged tissue or organ.
12.- La célula mesenquimal activada según cualquiera de las reivindicaciones 1-5, la población celular según la reivindicación 6, o la composición según cualquiera de las reivindicaciones 7-9, para el tratamiento del cáncer. The activated mesenchymal cell according to any of claims 1-5, the cell population according to claim 6, or the composition according to any of claims 7-9, for the treatment of cancer.
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AMIN SHAIMAA NASR ET AL.: "Melatonin-Pretreated Mesenchymal Stem Cells Improved Cognition in a Diabetic Murine Model", FRONTIERS IN PHYSIOLOGY, vol. 12, no. 628107, 18 March 2021 (2021-03-18), XP093012985, ISSN: 1664-042X, [retrieved on 20220309], DOI: 10.3389/fphys.2021.628107 *
EL-MAGD MOHAMMED A ET AL.: "Melatonin maximizes the therapeutic potential of non-preconditioned MSCs in a DEN-induced rat model of HCC", BIOMEDICINE & PHARMACOTHERAPY, vol. 114, no. 108732, 31 May 2019 (2019-05-31), XP093012985, ISSN: 0753-3322, [retrieved on 20220309], DOI: 10.1016/j.biopha.2019.108732 *
MOHAMED YASSER, BASYONY MOHAMED A., EL-DESOUKI NABILA I., ABDO WALIED S., EL-MAGD MOHAMMED A.: "The potential therapeutic effect for melatonin and mesenchymal stem cells on hepatocellular carcinoma", BIOMEDICINE, vol. 9, no. 4, 1 December 2019 (2019-12-01), pages 24, XP093012969, DOI: 10.1051/bmdcn/2019090424 *

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