Repairing tissue by activating cells

Abstract

The name of the invention is repairing tissue by activating cells. The present invention relates to an activating composition comprising cells, which may be any cell type for use in cell therapy, where the cells are activated by a chemotherapeutic agent. Further, provided is an activation composition comprising a supernatant of the composition, the supernatant comprising cells of any cell type, which may be used in cell therapy, where the cells are activated by a chemotherapeutic agent, and where the supernatant is used as therapy. The invention further provides methods for treating or preventing a disease or condition comprising the use of the activating composition.

Description

[0001] This application is a divisional application. The filing date of the original application is August 29, 2019, the application number is 2019800725242 (PCT / IL2019 / 050972), and the name of the invention is “Tissue repair by activated cells”. Background Art

[0002] Tissue regeneration provides an important ability for organisms to renew damaged tissues and maintain normal organ function in aging, trauma, autoimmune diseases, and malignancies. Tissue repair is a complex multi-parameter process. It involves the initial secretion of proinflammatory cytokines and chemokines at the damaged site, and then recruits various mesenchymal and hematopoietic cells to the damaged tissue to protect it from the destructive inflammatory process. This process leads to an anti-inflammatory cascade, which ultimately restores the integrity of the organ and regenerates the damaged tissue. Therefore, in the context of tissue damage and repair, the main interest of cell therapy research focuses on the use of different cell types with unique anti-inflammatory and regenerative properties to enhance the repair of native tissues to damage and initial acute proinflammatory cascades. Among these cells are mesenchymal stem cells (MSCs) and macrophages.

[0003] Present in various mesenchymal tissues, MSCs obtain strong anti-inflammatory properties by secreting factors such as IL-1RA, PGE2, TSG6 and TGFβ, thereby inducing immunosuppressive activity. In addition, we and others have previously shown that MSCs are specifically located in tumor or damaged tissue sites, and reduce inflammation and improve angiogenesis and tissue repair. In addition, it has been previously shown that in pancreatic cancer, chemotherapy-activated MSCs enrich and retain cancer stem cell (CSC) niches by activating the CXCL10-CXCR3 axis, and enhance resistance to cytotoxic agents, thereby leading to tumor growth and enhanced invasiveness. It is also known that macrophages play a key role in tissue homeostasis and wound healing. As highly plastic cells, macrophages phenotypically change from a pro-inflammatory state to an anti-inflammatory "repair" state, secreting IL-10, transforming growth factor β (TGFβ), insulin-like growth factor-1 (IGF1) and VEGF, inhibiting T cells and promoting tissue repair. In addition, macrophages enhance tissue regeneration and tumor growth by inducing angiogenesis and lymphangiogenesis. Taken together, these abilities make macrophages and MSCs ideal targets for cell-based therapies.

[0004] However, despite the therapeutic potential of certain cell types to repair damaged tissues, cell therapy is still not the first-line treatment method assigned. One of the main reasons is the lack of cell specificity and activity, thus delaying and minimizing tissue regeneration and wound healing. Specifically, current methods use various growth factors and cytokines to activate effector cells, such as TNFα and IFNγ. These factors have been shown to activate stromal cells, thereby contributing to tumor growth inhibition and tissue repair. In other cases, cell activation requires expensive genetic modification, which can also increase the toxicity and immunocompatibility of effector cells, and the efficiency of this method is relatively low. Here, we propose a new, simple and cost-effective method for the specific activation of different cells to induce tissue regeneration and repair. Summary of the invention

[0005] In some embodiments of the present invention, activation compositions are provided that include any cell type activated by a chemotherapeutic agent for use in cell therapy.

[0006] In some embodiments of the invention, an activation composition is provided comprising any cell type activated by a chemotherapeutic agent for cell therapy, wherein the cell type activated by a specific chemotherapeutic agent for cell therapy is separated from conditioned medium, and wherein the conditioned medium is used as a therapy.

[0007] In some embodiments of the present invention, an activation composition is provided, which comprises mesenchymal stem cells and / or macrophages activated by a chemotherapeutic agent or any other cell type that can be used for cell therapy, wherein the mesenchymal stem cells and / or macrophages activated by a specific chemotherapeutic agent and / or the cell type that can be used for cell therapy are separated from conditioned medium, and wherein the conditioned medium is used as a therapy.

[0008] In some embodiments of the present invention, a method for producing activated cells is provided, which activated cells can be any cell type used in cell therapy for tissue repair, the method comprising: activating a cell type for cell therapy by culturing any cell type for cell therapy with a chemotherapeutic agent.

[0009] In some embodiments of the present invention, a method for producing activated mesenchymal stem cells and / or activated macrophages for tissue repair is provided, the method comprising: activating mesenchymal stem cells and / or macrophages by culturing the mesenchymal stem cells and / or macrophages with a chemotherapeutic agent.

[0010] In some embodiments of the present invention, there is provided a method for promoting tissue regeneration or organ repair or for reducing inflammation in a mammal, the method comprising:

[0011] (a) obtaining an activated composition of any cell type activated by a chemotherapeutic agent for use in cell therapy; and

[0012] (b) administering the activated composition to a mammal in need of tissue regeneration, organ repair or reduction of inflammation.

[0013] In some embodiments of the present invention, there is provided a method for promoting tissue regeneration or organ repair or for reducing inflammation in a mammal, the method comprising:

[0014] (a) obtaining an activating composition of mesenchymal stem cells and / or macrophages activated by a chemotherapeutic agent; and

[0015] (b) administering the activated composition to a mammal in need of tissue regeneration, organ repair or reduction of inflammation.

[0016] In some embodiments of the present invention, there is provided a method of inducing angiogenesis in a mammal, the method comprising:

[0017] (a) obtaining an activated composition of any cell type activated by a chemotherapeutic agent for use in cell therapy; and

[0018] (b) administering the activating composition to a mammal in need of tissue regeneration or organ repair, wherein angiogenesis enhances tissue regeneration or organ repair.

[0019] In some embodiments of the present invention, there is provided a method of inducing angiogenesis in a mammal, the method comprising:

[0020] (a) obtaining an activation composition of human mesenchymal stem cells and / or macrophages activated by a chemotherapeutic agent; and

[0021] (b) administering the activating composition to a mammal in need of tissue regeneration or organ repair, wherein angiogenesis enhances tissue regeneration or organ repair.

[0022] In some embodiments of the present invention, an activation composition is provided, which comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or other tissues resident cells activated by anticancer drugs.

[0023] In some embodiments, the anti-cancer drug can be a chemotherapeutic agent.

[0024] In some embodiments of the present invention, an activation composition is provided, which comprises conditioned medium from mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues activated by anti-cancer drugs.

[0025] In some embodiments of the present invention, a method for producing a composition of activated mesenchymal stem cells and / or activated macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues for tissue repair or for treating diseases associated with tissue damage is provided, the method comprising: activating mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues by culturing them with an anticancer drug; Stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or cells derived from adipose tissue and / or other tissue resident cells, and optionally conditioned medium is separated from activated mesenchymal stem cells and / or activated macrophages and / or T cells and / or fibroblasts, activated mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or cells derived from adipose tissue and / or other tissue resident cells or their supernatants are used for tissue repair or for treating diseases associated with tissue damage. In some embodiments of the invention, the step of culturing or cultivating cells with an anticancer agent lasts from about 10 minutes to about 72 hours.

[0026] In some embodiments of the invention, conditioned medium is obtained from at least 10 5 -10 7 Activated cells were collected.

[0027] In some embodiments of the present invention, a method for promoting tissue regeneration or organ repair of a damaged organ and / or for reducing inflammation in a mammal is provided, the method comprising: administering the activating composition of the present invention to a mammal in need of tissue regeneration, organ repair or reducing inflammation.

[0028] In some embodiments of the present invention, a method for inducing angiogenesis and / or inducing immunosuppression in a mammal is provided, the method comprising: administering the activating composition of the present invention to a mammal in need of tissue regeneration or organ repair, wherein angiogenesis enhances tissue regeneration or organ repair of a damaged organ.

[0029] In some embodiments of the present invention, there is provided a method for regenerating tissue in a damaged organ, comprising the step of administering the activating composition of the present invention directly or by peripheral administration to the damaged organ.

[0030] In some embodiments of the invention, the damaged organ is the pancreas, breast, ovary, lung, heart, kidney, lung, GI tract, intestine, skin, liver, or brain.

[0031] In some embodiments of the invention, the anticancer drug is a chemotherapeutic agent selected from the group consisting of gemcitabine, paclitaxel, a combination of paclitaxel and anakinra, anakinra, cisplatin, 5-FU, dacarbazine, temozolomide, and any combination thereof, or wherein the anticancer drug is an antibody, a molecular targeted drug such as bortezomib, radiation, and an immunotherapeutic agent, including immune checkpoint inhibitors. In some embodiments of the invention, wherein if:

[0032] If the damaged organ is the pancreas, the chemotherapeutic agent is gemcitabine;

[0033] If the damaged organ is the breast, ovary, or lung, the chemotherapeutic agent is paclitaxel;

[0034] If the damaged organ is the heart, the chemotherapeutic agent is a combination of paclitaxel and anakinra;

[0035] If the damaged organ is the kidney or lung, the chemotherapeutic agent is cisplatin;

[0036] If the damaged organ is the GI tract or intestine, the chemotherapeutic agent is 5-FU;

[0037] If the damaged organ is the skin, the chemotherapeutic agent is dacarbazine;

[0038] the damaged organ is the brain, then the chemotherapeutic agent is temozolomide; and

[0039] The damaged organ is the liver and the chemotherapeutic agent is gemcitabine.

[0040] In some embodiments of the present invention, a method of treating or preventing a disease associated with tissue damage in a subject in need thereof is provided, comprising the steps of administering an activating composition directly to a degenerated organ or to a subject in need thereof by peripheral administration.

[0041] In some embodiments of the invention, if the disease or disorder is pulmonary fibrosis, the anticancer drug is paclitaxel, cisplatin or gemcitabine; if the disease or disorder is liver fibrosis, the anticancer drug is gemcitabine.

[0042] In some embodiments of the present invention, there is provided a method of healing a wound, comprising the step of applying an activating composition of the present invention to the wound.

[0043] In some embodiments of the invention, the anticancer drug is paclitaxel.

[0044] In some embodiments of the invention, the cells are incubated with the anti-cancer drug for about 10 minutes to about 72 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention is described herein by way of example only, with reference to the accompanying drawings. With specific reference now to the detailed drawings, it is emphasized that the details are shown by way of example only, and are presented only for the purpose of exemplary discussion of the preferred embodiments of the present invention, and for the purpose of providing what is believed to be the most useful and most easily understood description of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention, and the description in conjunction with the drawings will be apparent to those skilled in the art as to how several forms of the present invention may be practiced.

[0046] In the attached picture:

[0047] Figure 1 (A, B and C): Figure 1A This is a photo showing that gemcitabine promotes MSC homing to tumors and induces the enrichment of stem cell populations

[0048] 8-10 week old SCID mice were implanted subcutaneously with PANC1 cells (n=5 mice / group). When tumors reached 500 mm3, mice were treated with gemcitabine (500 mg / kg) or vehicle control. Figure 1A : After 72 hours, tumors were harvested and tumor sections were immunostained using antibodies against CD105 (yellow) and αSMA (green) that recognize MSCs and anti-CD133 (red) that recognize CSCs. Cell nuclei were stained with DAPI (blue). Red arrows indicate CSCs, while white arrows indicate MSCs. Scale bar: 100 μm. Figure 1B : On the right is a graph showing the measured and plotted distances between MSCs and CSCs (n>15 fields / group), Figure 1B On the left is a graph showing the percentage of MSCs in tumor single cell suspensions quantified by flow cytometry. Figure 1C: Figure showing the percentage of TIC in culture assessed by flow cytometry: PANC1 cells were cultured in serum-free medium (control) or serum-free medium supplemented with conditioned medium derived from control MSC cultures (MSC), gemcitabine-cultured MSCs (MSC-GEM) or gemcitabine- and anti-CXCL10-treated MSCs (MSC-GEM+anti-hCXCL10). After 3 days, the percentage of TIC in culture was assessed by flow cytometry.

[0049] Figure 2 (A and B) shows that gemcitabine-activated MSC conditioned medium induces endothelial cell recruitment and promotes immunosuppression

[0050] Matrigel plugs containing 10% conditioned medium (CM) produced by gemcitabine-activated MSCs or untreated MSCs were injected subcutaneously into the ventral flank of 8- to 10-week-old BALB / c mice. Ten days later, the plugs were removed and stained for histological analysis and flow cytometric evaluation. Figure 2A Hematoxylin & eosin staining of Matrigel plugs containing CM from control (left) or gemcitabine-activated MSCs (right) are shown. Figure 2B Matrigel plugs treated as single cell suspensions were immunostained for different cell types and analyzed by flow cytometry as indicated.

[0051] FIG. 3 (A, B, or C) is a graph showing that chemotherapy-activated MSCs inhibit T cell activation

[0052] Splenocytes isolated from 8-10 week old BALB / c mice were cultured with CD3+ / CD28+ T cell activation beads in the absence (baseline) or presence of CM from untreated MSCs or chemotherapy activated MSCs. After 24 hours, splenocytes were harvested and activated cytotoxic T cells (CD8+( Figure 3A )、CD8+ / CD25+( Figure 3B ) or CD8+ / CD107a+( Figure 3C NC-negative control (splenocytes cultured in the absence of activated beads), GEM-gemcitabine, PTX-paclitaxel, CIS-cisplatin.

[0053] Figure 4 (A, B, C and D) shows that chemotherapy activated MSCs induce wound healing

[0054] Boyden chamber assay was used to evaluate PANC1 (2x10 5 The migration properties of individual cells. Figure 4AAs shown in the figure, cells were tested in the presence of CM obtained from MSCs exposed to several types of chemotherapeutic drugs. Figure 4B and 4C As shown, the motility properties of PANC1 cells were analyzed using a scratch wound assay when cultured in the presence of CM from control or chemotherapy-activated MSCs. Figure 4D The wound healing properties were evaluated in vivo in mice injected with chemotherapy activated MSC or its control in the photos in Figure 1. In brief, a small incision was made in the back of 8 to 10 week old BALB / c mice. The next day, the wound was treated externally with the conditioned medium of the MSC cultured with control MSC or gemcitabine. The therapy was performed every other day until the end of the experiment. The healing process was tracked every other day for 2 weeks until the wound was completely closed.

[0055] Figure 5 This is a picture showing the homing of cells to the lungs in mice induced by pulmonary fibrosis.

[0056] 0.04 units of bleomycin (Baxter Oncology GmbH, Germany) were administered intratracheally to 10 week-old Balb / c mice or treated with vehicle control. After 1 week, mice were not treated, or CM of control MSCs or chemotherapy-activated MSCs was administered intratracheally (IT). Treatment was performed every 1 day for 1 week. After 14 days of bleomycin administration, bronchoalveolar lavage (BAL) was performed and cells were counted. The number of cells from BAL was displayed.

[0057] FIG. 6 (A, B, C, and D) are graphs showing that chemotherapy-activated stem cells suppress inflammation in lung tissue.

[0058] like Figure 5 As indicated, cells were immunostained for different surface markers to assess immune cell lineage and their functions, such as inflammatory or anti-inflammatory states. Cells were then analyzed by flow cytometry and the percentage of cells in each sample was tested. Fig. 6A As shown in Figure 3-D, the percentage of pro-inflammatory cells decreased, while the percentage of anti-inflammatory cells increased, thus demonstrating the tissue regeneration process.

[0059] Figure 7 (A and B) are photographs showing that chemotherapy-activated mesenchymal stem cells reduce lung fibrosis.

[0060] 10 week old Balb / c mice were given 0.04 units of bleomycin (Baxter Oncology GmbH, Germany) intratracheally (IT) or treated with vehicle control. After 1 week, mice were not treated, or conditioned medium (CM) of control MSCs or chemotherapy activated MSCs was given IT. Treatment was performed every other day for 1 week. 14 days after bleomycin was given, lungs were harvested and subsequently fixed with PFA. Paraffin-embedded lung tissue was sliced ​​and stained with hematoxylin and eosin (H&E) solution ( Figure 7 A) or Sirius Red staining to assess collagen content ( Figure 7 B). The results demonstrate that in the lungs of bleomycin-treated mice (control or MSC control-administered mice), there was an extensive inflammatory process followed by increased collagen deposition (indicated by white arrows), whereas in the fibrotic lungs of mice administered CM of chemotherapy-activated MSCs, inflammation was completely abolished and collagen deposition was reduced.

[0061] Figure 8 is a graph showing that chemotherapy-activated mesenchymal stem cell therapy does not induce toxicity (measured by total body weight).

[0062] Female C57Bl / 6 mice aged 8 to 10 weeks were intraperitoneally given DNM (5 mg / kg) or treated with vehicle control. Two weeks after the start of treatment, mice were not treated or injected with conditioned medium or exosome fractions of control MSCs or chemotherapy-activated MSCs. Therapy was performed every other day for 2 weeks. Mouse weight was monitored every other day to reflect the severe toxic effects of therapy.

[0063] FIG. 9 (A, B, and C) are graphs showing that chemotherapy-activated MSC therapy restores liver physiology in a liver fibrosis model.

[0064] Female C57Bl / 6 mice aged 8 to 10 weeks were intraperitoneally administered DNM (5 mg / kg) or treated with vehicle control. Two weeks after the start of treatment, mice were not treated or injected with conditioned medium of control MSCs or chemotherapy-activated MSCs or their exosome fractions. Therapy was performed every other day for 2 weeks. At the end point, blood was drawn from treated and control mice to assess liver physiological function. Albumin ( Fig. 9A ), alanine aminotransferase (ALT, Fig. 9B ) and aspartate aminotransferase (AST, Fig. 9C ) levels indicated that chemotherapy-activated MSC therapy with administration of ac-conditioned medium or exosomes improved liver physiological parameters in fibrotic liver.

[0065] FIG. 10 (A, B, C, and D) are graphs showing that chemotherapy-activated mesenchymal stem cells suppress liver inflammation.

[0066] Single cell suspensions from livers treated with DNMs to produce fibrotic liver disease were immunostained for different surface markers to assess fibroblast and immune cell lineages. The cells were then analyzed by flow cytometry and the percentage of cells in each sample was tested. Activated MSC therapy inhibited pro-inflammatory cells (T helper cells, cytotoxic T cells ( Fig. 10A ), NK cells, B cells ( Fig. 10B )、MDSC、Macrophages( Fig. 10C ), fibroblasts and Kupffer cells ( Fig. 10D ) and the percentage of stimulated anti-inflammatory populations, including Kupffer cells and myeloid-derived suppressor cells (MDSCs), thus indicating that our therapy promotes increased tissue regeneration in the liver.

[0067] FIG. 11 (A, B, C, and D) shows that chemotherapy-activated mesenchymal stem cells reduce liver fibrosis.

[0068] Female C57Bl / 6 mice aged 8 to 10 weeks were given DNM (5 mg / kg) intraperitoneally or treated with vehicle control. Two weeks after the start of treatment, mice were not treated or injected with conditioned medium or exosome fractions of control MSCs or chemotherapy-activated MSCs. Therapy was performed every other day for 2 weeks. Four weeks after the start of DNM treatment, mice were killed, livers were harvested, and processed for fibrosis assessment. When compared with other groups, soluble collagen levels were significantly decreased in mice treated with CM of chemotherapy-activated MSCs, indicating reduced liver fibrosis ( Fig.11A Meanwhile, paraffin-embedded liver tissues were sliced ​​and stained with hematoxylin and eosin (H&E) solution ( Fig. 11B ) or Sirius Red staining to assess collagen content ( Fig. 11C and Fig.11D ). The results showed that collagen deposition was increased in the livers of DNM-treated mice (indicated by white arrows), whereas collagen deposition was significantly reduced to the level of untreated controls in the fibrotic livers of mice given CM of chemotherapy-activated MSCs or their produced exosomes. DETAILED DESCRIPTION

[0069] Surprisingly, it is found that anticancer agents, such as, for example, chemotherapeutic agents, can highly effectively activate mesenchymal stem cells (MSC), macrophages, fibroblasts and T cells in vivo and in vitro, wherein it is found that activated MSC, macrophages, fibroblasts and T cells or their conditioned medium effectively regenerate tissues. More surprisingly, it is found that specific chemotherapeutic agents have a significant effect in certain tissues and for specific clinical applications. Therefore, the present invention provides a method for specifically activating various cells by specific anticancer agents (such as chemotherapeutic agents). Activated cells can be used to induce tissue regeneration, repair specific organs and treat various conditions, including lung or liver fibrosis. The present invention further provides a method for tissue repair (including wound healing and fibrosis healing) by locally treating physiological or damaged tissues or organs with activated products of mesenchymal stem cells and / or macrophages and / or fibroblasts activated by anticancer agents (such as chemotherapeutic drugs) and / or any other cell types that can be used for cell therapy. Further provided are methods of treating wounds using CMs of mesenchymal stem cells and / or macrophages and / or fibroblasts and / or T cells, or any other cell type useful for cell therapy, specifically activated by an anti-cancer agent (eg, a chemotherapeutic agent).

[0070] In some embodiments of the present invention, there is provided a method for treating or preventing a disease associated with tissue damage in a subject in need thereof, the method comprising:

[0071] Contacting activated mesenchymal stem cells (MSCs) and / or any other activated cells described above and / or contacting conditioned medium of activated MSCs and / or any other activated cells with a tissue that has undergone tissue damage in a subject in need thereof, wherein the MSCs are autologous or allogeneic non-transfected MSCs.

[0072] As used herein, "preventing" or "prevention" or "inhibit" or "inhibition" interchangeably refers to the possibility of reducing the risk of developing a disease or disorder (which may be a disease associated with tissue damage) (e.g., when the subject has not yet experienced or does not show all symptoms of the disease or the symptoms are milder, the onset of the disease will be delayed or inhibited). The biological or physiological parameters used to identify such patients are well known to physicians and depend on the disease or disorder.

[0073] The term "treatment" or "treating" of a subject includes applying or administering the secreted cell products of activated MSCs and / or other cells useful for the present invention to a subject (or applying or administering the compounds of the present invention to cells or tissues of a subject) to stabilize, cure, heal, reduce, alleviate, alter, remedy, reduce deterioration, mitigate, improve, or affect a disease or condition, a symptom of a disease or condition, or the risk (or susceptibility) of a disease or condition. The term "treat" refers to any indicator of successful treatment or mitigation of an injury, pathology, or condition, including any objective or subjective parameter, such as reduction; alleviation; reduction in the rate of deterioration; reduction in severity of a disease; stabilization, reduction in symptoms, or making a patient more tolerant to an injury, pathology, or condition; slowing the rate of degeneration or decline; making the endpoint of degeneration less debilitating; or improving the physical and mental health of a subject. In some embodiments, the term "treat" may include extending the life expectancy of a subject and / or delaying before additional treatment is required.

[0074] The term "tissue damage" herein refers to pathological conditions including structural changes in the extracellular matrix (ECM) or cells within a specific tissue (including burns, apoptotic cells, or necrotic cells). Such damaged tissue may be due to immune or autoimmune activity, internal or external forces, inflammatory processes, trauma, etc.

[0075] In some embodiments of the present invention, methods for treating or preventing diseases associated with tissue damage are provided. Diseases associated with tissue damage are one or more of the following diseases, conditions or disorders:

[0076] Osteoarthritis, avascular necrosis or severe degenerative joint disease;

[0077] Stroke or degenerative brain disease selected from dementia, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's chorea, Pick's disease, Creutzfeldt-Jakob disease, and combinations thereof;

[0078] Muscular dystrophy, such as, for example, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emory-Dreyfuss muscular dystrophy, Langerhans-Dreyfuss muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), Limb-Girdle muscular dystrophies, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (Steinert's disease), and congenital muscular dystrophy;

[0079] Fibrotic or degenerative liver disease, inflammatory conditions, cirrhosis;

[0080] Fibrotic lung disease, chronic obstructive pulmonary disease;

[0081] Autoimmune diseases, such as lupus, type 1 diabetes, psoriasis, and skin eczema;

[0082] Osteoarthritis - the most common form of arthritis - is a disease characterized by a slow degenerative process of the articular cartilage, subchondral bone and low-grade inflammation associated with the formation of marginal osteophytes. Osteoarthritis in its chronic form is thought to affect 15% of the population. Of these, one quarter are severely disabled. Most cases of osteoarthritis have no known cause and are known as primary osteoarthritis. When the cause of osteoarthritis is known, the condition is known as secondary osteoarthritis. Secondary osteoarthritis is caused by another disease or condition. Conditions that may lead to secondary osteoarthritis include repeated injuries or surgery to joint structures, abnormalities of the joints at birth (congenital anomalies), gout, diabetes and other hormonal disorders. Other forms of arthritis are systemic diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE);

[0083] Osteoarthritis primarily involves the hips, knees, spine, and interphalangeal joints. In severe osteoarthritis, complete loss of the cartilage cushion causes friction between bones, resulting in pain at rest or pain with limited movement. Osteoarthritis is characterized by the slow degradation of cartilage over several years;

[0084] Retinal degenerative disorders, Refsum's disease, Stevens-Lemme-Osborn syndrome, Schnyder crystalline corneal dystrophy, drusen, age-related macular degeneration, and diabetic retinopathy;

[0085] Some other examples: Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS, Lou Gehrig's Disease), Charcot-Marie-Tooth disease (CMT), chronic traumatic encephalopathy, cystic fibrosis, some cytochrome C oxidase deficiencies (often the cause of the degenerative Leigh syndrome), Ehrlich-Danlos syndrome, fibrodysplasia ossificans progressiva, Friedreich's ataxia, frontotemporal dementia (FTD), cardiovascular disease (e.g., atherosclerosis, such as coronary artery disease, aortic stenosis, etc.), Huntington's chorea, infantile neuroaxonal dystrophy, keratoconus (KC), keratoglobose, leukoencephalopathy, macular degeneration (AMD), Marfan syndrome (MFS), some mitochondrial myopathies, mitochondrial DNA depletion syndrome, multiple sclerosis (MS), multiple system atrophy (MS). atrophy), muscular dystrophy (MD), neuronal ceroid lipofuscinosis, Nieto-Pitt disease, osteoarthritis, osteoporosis, Parkinson's disease, pulmonary hypertension, prion diseases (CJD, fatal familial insomnia, etc.), progressive supranuclear palsy, retinitis pigmentosa (RP), rheumatoid arthritis, Sandhoff disease, spinal muscular atrophy (SMA, motor neuron disease), subacute sclerosing panencephalitis, Tay-Sachs disease, vascular dementia (may not be neurodegenerative in itself, but often occurs with other forms of degenerative dementia);

[0086] Other examples are burns, broken bones, wounds, diabetic wounds, as in type 1 diabetes;

[0087] Further examples are autoimmune diseases such as, for example, rheumatoid arthritis, psoriasis, sarcoidosis, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus;

[0088] "Activated cells" or "activated mesenchymal stem cells" or "activated macrophages" or "activated fibroblasts" etc. herein refer to cells, mesenchymal stem cells or macrophages or other cells that have been previously cultured with an anticancer agent using standard serum-free medium for at least 10, 20, 30, 40, 50 minutes or 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 48 or 72 hours or longer. It should be noted that the cultivation should be terminated before the cells undergo apoptosis and / or death. These activated cells or the conditioned medium produced therefrom can be used in the methods described herein. Examples of such anticancer agents are gemcitabine, paclitaxel, paclitaxel and anakinra, cisplatin, 5-FU, dacarbazine, temozolomide targeted drugs (e.g., bortezomib), radiation, anti-angiogenic drugs, and immune checkpoint inhibitory antibodies. The term "activated composition" includes cells activated by anticancer therapy or its conditioned medium or both. The conditioned medium may be diluted, concentrated, or lyophilized. The activated cells are any of the following: activated mesenchymal stem cells and / or activated macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or other tissue-resident cells or any other activated cells that can be used for tissue repair.

[0089] The amount of activated MSCs or any other activated cells of the present invention, or the conditioned medium produced therefrom, the dosage form and dosage regimen, and the location in the case of local administration depend on the type of disease or disorder, the location of the damaged tissue or organ, and the severity of the disease. In most cases, but not exclusively, the conditioned medium is at least 10 5 -10 6 In some embodiments, treatment is performed using at least 1-10×10 6 cells.

[0090] It should be noted that in some embodiments of the invention, the conditioned medium can be concentrated by, for example, centrifugation at 1100,000 g to obtain exosomes and other products. In some cases, the conditioned medium can be lyophilized to maintain it as a dry product, for example, for later reconstitution. Note that in the treatment methods of the present invention, the term "conditioned medium" or "conditioned media" or "CM" also refers to the secretome, the dry product, the supernatant, and the uncontacted conditioned medium.

[0091] For example, for a method of treating intervertebral disc degeneration in a patient, the method comprises: directly injecting activated mesenchymal stem cells (MSCs) or conditioned medium of activated MSCs into the lumbar-related muscles proximal to the lumbar intervertebral disc of the patient, wherein the MSCs are autologous or allogeneic non-transfected MSCs; wherein the lumbar-related muscles are selected from the group consisting of the psoas major, multifidus, transverse spinous and sacrospinal muscles. The treatment dosage regimen is 1-10 times, and the number of cells after cell activation is 1×10 6 In some cases, the activated cell-conditioned medium specified above may be injected 1-10 times or more in a volume of 100 μl, depending on the healing process monitoring.

[0092] Chemotherapy-activated MSC-enriched stem cells

[0093] MSC is one of the main candidates for tissue regeneration and cell therapy due to its high proliferation potential and the ability to differentiate into main mesenchymal tissue. In addition, it has been shown that MSC promotes the systemic resistance of chemotherapy in solid tumors by secreting two specific fatty acids after being exposed to platinum cytotoxic agents. These fatty acids indirectly protect colon and lung cancer cells from the cytotoxic effects of chemotherapy. However, the interaction of MSC and tissue-resident stem cells is unclear, especially after chemotherapy. For this reason, as seen in the embodiments, subcutaneous pancreatic tumors are analyzed and generated by injecting PANC1 cells into the flanks (flanks) of 8 to 10 week-old SCID mice. When the tumor reaches certain size, mice are treated with gemcitabine, and mice are taken out and killed after 3 days. After gemcitabine therapy, the tumor of MSC and cancer stem cells (CSC) is immunostained, and it is proved that there is a rare tumor cell group of increased drug resistance and increased tumorigenicity. Specifically, compared with untreated control, MSC home to tumor, and close to CSC positioning ( Figure 1A ).in addition, Figure 1BIt was demonstrated that chemotherapy induces a large number of MSCs to be recruited to pancreatic tumors, which is similar to the infiltration of MSCs to damaged tissue sites. In addition, in order to study whether MSCs contribute to CSC repopulation, conditioned medium (CM) of untreated MSCs or gemcitabine-cultured MSCs was administered, and CSC enrichment was evaluated using phenotypic characterization by flow cytometry. Compared with the effect of untreated MSCs, activation of MSCs with chemotherapy resulted in significant enrichment of CSCs. Secretome analysis of gemcitabine-activated MSCs showed that, relative to untreated MSCs, after chemotherapy treatment, multiple other factors such as CXCL10 that affect tissue remodeling and / or regeneration were highly elevated in the conditioned medium, as shown in Table 2. It was shown that CXCL10 promotes CSC enrichment by binding to the CXCR3 receptor and continuously inducing STAT3 signaling. Neutralizing CXCL10 with anti-CXCL10 antibodies eliminated the enrichment effect of chemotherapy-activated MSCs on CSCs ( Figure 1C ).

[0094] Chemotherapy-activated MSCs promote immunosuppression and induce angiogenesis

[0095] MSC is known for its ability to home to damaged parts and promote tissue regeneration. In addition, MSC secretes cells of the entire environment and chemokines, including TGFβ, IL-6, PGE2, IDO, LIF, to regulate the recruitment of various hematopoietic cell types (it regulates tissue structure). Carry out the matrix glue plug assay to test these abilities. For this reason, the matrix glue plug containing the conditioned medium (CM) generated by the MSCs cultured by untreated or gemcitabine is implanted into the flank of Balb / c mice. After 10 days, the plug is taken out and prepared as a single cell suspension. Phenotypic analysis is performed on the cells from the plug by flow cytometry to identify the cells recruited to the plug. The CM of the MSCs processed from gemcitabine is shown to induce the recruitment of endothelial cells, indicating that the angiogenesis potential increases after gemcitabine therapy. In addition, when compared with the plug containing the CM of the control MSCs, in the matrix glue plug with the CM of the MSCs activated from gemcitabine, most immune populations, including the colonization of macrophages, NK cells, T cells and B cells, are significantly reduced ( Figure 2A -B). To further elucidate the immunomodulatory effects of chemotherapy-exposed MSCs, T cell activation assays were performed by administering CM of naive or chemotherapy-activated MSCs to splenocytes extracted from naive Balb / c mice. As a result, chemotherapy-activated MSCs significantly reduced the total amount of CD8 T cells and inhibited the activation of cytotoxic T cells (Figure 3). Overall, the results provide additional support that in tumors, similar to damaged tissues, MSCs acquire a powerful regenerative capacity promoted by increasing angiogenesis and inducing immunosuppression to increase overall tissue repair.

[0096] Chemotherapy-activated MSCs increase effective tissue repair

[0097] Based on the enhanced pro-angiogenesis and immunosuppressive properties of chemotherapy-activated MSC, the ability of MSC to heal wounds was tested. For this reason, as described in Materials and Methods, the influence of MSC on the migration properties of other cells was tested by performing a modified Boyden box assay. In doing so, PANC1 was cultivated in the upper box (upper chamber) of the hole, and the CM of chemotherapy-activated MSC or the CM of untreated MSC was loaded in the lower box (lower chamber). After 24 hours, the cells that migrated to the lower side of the box were fixed and subsequently stained with crystal violet. It was found that compared with the control group, the CM of gemcitabine-activated MSC significantly increased the migration properties of PANC1 cells. Interestingly, the CM generated by the MSC exposed to other cytotoxic agents (such as paclitaxel and cisplatin) did not affect the migration ability of cells ( Figure 4A In addition, a scrape wound assay was performed to further demonstrate the regenerative potential of chemotherapy-activated MSCs. Cells treated with CM from gemcitabine-cultured MSCs closed gaps faster than untreated control cells or cells exposed to CM from untreated MSCs ( Figure 4B and 4C In parallel with the migration assay results, the wound closure time of cells treated with CM from MSCs exposed to PTX or cisplatin was similar to that of the control group.

[0098] Next, the wound healing properties of chemotherapy-activated MSCs were tested in vivo. For this purpose, a 0.5 cm incision was made on the back of SCID mice. After 24 hours, the wounds were treated externally with conditioned medium of control MSCs or MSCs pre-exposed to 10 nM gemcitabine. Control mice were not treated. Wound healing was monitored until the wound was completely closed. The results showed that compared with the control group, the wound healing of mice treated with gemcitabine-activated MSCs was the fastest ( Figure 4D ). Taken together, our results demonstrate that chemotherapy-cultured MSCs greatly induce MSC regeneration and wound healing characteristics, thus contributing to injury repair.

[0099] In embodiments of the present invention, it is shown that chemotherapeutic agents can be used to activate cells that can be used for various indications that require cell regeneration, such as macrophages and / or MSCs and / or T cells and / or fibroblasts. Some examples are shown in Table 1 below. In some embodiments, the therapeutic effect of a specific chemotherapeutic agent, such as reflected in cell regeneration, is specific to certain organs, as exemplified in Table 1.

[0100] In an embodiment of the present invention, an activation composition defined below is provided. According to some embodiments of the present invention, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts activated by anticancer agents (such as chemotherapeutic agents). In other embodiments, the activation composition comprises the supernatant or conditioned medium of a preparation, which comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts activated by a specific anticancer agent (such as a chemotherapeutic agent) and, for example, using the secretory group of such activated cells by centrifugation. The term "secretome" refers to proteins, metabolites, enzymes, lipids, sugar molecules, and extracellular vesicles, such as exosomes secreted by cells into the extracellular space.

[0101] "Extracellular vesicles", "exosomes" are small vesicles that are blubbing or secreted from cells that contain proteins, RNA, DNA, and other biological materials that can be transferred between cells. These vesicles have been shown to play a role in physiological and pathological conditions.

[0102] In some embodiments of the present invention, an activation composition is provided, which comprises mesenchymal stem cells and / or macrophages activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts activated by a chemotherapeutic agent are separated from conditioned medium, and wherein the conditioned medium is used as a therapy.

[0103] In some embodiments of the present invention, a method for producing activated mesenchymal stem cells and / or activated macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues for tissue repair is provided, comprising: activating mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues by culturing mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues with suitable chemotherapeutic agents.

[0104] In some embodiments of the present invention, there is provided a method for promoting tissue regeneration, organ repair or reducing inflammation in a mammal, the method comprising:

[0105] (a) obtaining an activated composition of mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues activated by a chemotherapeutic agent; and

[0106] (b) administering the activated composition to a mammal in need of tissue regeneration, organ repair or reduction of inflammation.

[0107] In some embodiments of the present invention, there is provided a method of inducing angiogenesis in a mammal, the method comprising:

[0108] (a) obtaining an activation composition of human mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues activated by a chemotherapeutic agent; and

[0109] (b) administering the activating composition to a mammal in need of tissue regeneration or organ repair, wherein angiogenesis enhances tissue regeneration or organ repair.

[0110] In an embodiment of the present invention, a method for regenerating tissue in a damaged organ is provided, comprising the following steps: contacting macrophages and / or mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues with a chemotherapeutic agent to provide activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues, and administering the activated cells to the damaged organ.

[0111] In some embodiments of the invention, the damaged organ is pancreas, breast, ovary, lung, heart, kidney, lung, GI tract, intestine, skin, liver or brain. In some embodiments, the chemotherapeutic agent is gemcitabine, paclitaxel, a combination of paclitaxel and anakinra, anakinra, cisplatin, 5-FU, dacarbazine or temozolomide, or any combination thereof.

[0112] In some embodiments of the invention, if the damaged organ is the pancreas, the chemotherapeutic agent used for cell activation is gemcitabine.

[0113] In some embodiments of the invention, if the damaged organ is the breast, ovary or lung, the chemotherapeutic agent used for cell activation is paclitaxel.

[0114] In some embodiments of the invention, if the damaged organ is the heart, the chemotherapeutic agent that can be used for cell activation is a combination of paclitaxel and anakinra or anakinra alone.

[0115] In some embodiments of the invention, if the damaged organ is the kidney, ovary or lung, the chemotherapeutic agent used for cell activation is cisplatin.

[0116] In some embodiments of the invention, if the damaged organ is the GI tract, such as the intestine, the chemotherapeutic agent used for cell activation is 5-FU.

[0117] In some embodiments of the invention, if the damaged organ is the skin, the chemotherapeutic agent used for activation is dacarbazine.

[0118] In some embodiments of the invention, if the damaged organ is the brain, the chemotherapeutic agent used for activation is temozolomide.

[0119] In some embodiments of the present invention, a method for treating pancreatitis is provided, comprising the steps of contacting mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type used for cell therapy with a chemotherapeutic agent to provide activated mesenchymal stem cells, macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type used for cell therapy, and administering the activated mesenchymal stem cells, macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or activated cell types used for cell therapy to the pancreas by direct injection or by peripheral administration. In some embodiments, the chemotherapeutic agent is gemcitabine. In some embodiments, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell types that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or other cell types that can be used for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, and wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0120] In some embodiments of the present invention, a method for treating breast, lung or ovarian damage is provided, comprising the steps of contacting macrophages or mesenchymal stem cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues or any other cell type used for cell therapy with a chemotherapeutic agent to provide activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or other activated cell types for cell therapy, and administering the activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues or activated cell types for cell therapy to the breast, lung or ovary. In some embodiments, the chemotherapeutic agent is paclitaxel.

[0121] In some embodiments, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell types that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or other cell types that can be used for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0122] In some embodiments of the present invention, a method for treating myocardial infarction (MI) or heart failure is provided, comprising the steps of contacting macrophages or mesenchymal stem cells and / or any other cell type used for cell therapy with a chemotherapeutic agent to provide activated macrophages or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or activated cell types for cell therapy, and administering the activated macrophages or activated mesenchymal stem cells or activated cell types for cell therapy to the heart. In some embodiments, the chemotherapeutic agent is a combination of paclitaxel and anakinra or anakinra alone. In some embodiments, the activated composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or other tissue-resident cells and / or any other cell type that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or other tissue-resident cells and / or cell types for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, and wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0123] In some embodiments of the present invention, a method for treating kidney injury or kidney disease is provided, comprising the steps of contacting macrophages or mesenchymal stem cells or any other cell type for cell therapy with a chemotherapeutic agent to provide activated macrophages or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or activated cell types for cell therapy, and applying activated macrophages or activated mesenchymal stem cells and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or activated cell types for cell therapy to the kidney. In some embodiments, the chemotherapeutic agent is cisplatin.

[0124] In some embodiments, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or cell types used for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, and wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0125] In some embodiments of the present invention, there is provided a method for treating inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, comprising the steps of contacting macrophages and / or mesenchymal stem cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type used for cell therapy with a chemotherapeutic agent to provide activated macrophages and / or activated mesenchymal stem cells, and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or activated cell types for cell therapy, and administering activated macrophages or activated mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues or activated cell types for cell therapy to the GI tract or intestine. In some embodiments, the chemotherapeutic agent is 5-FU.

[0126] In some embodiments, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or cell types for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, and wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0127] In some embodiments of the present invention, a method for treating skin wounds, such as burns, diabetic lesions, etc., is provided, comprising the following steps: contacting macrophages and / or mesenchymal stem cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type used for cell therapy with a chemotherapeutic agent to provide activated macrophages or activated mesenchymal cells and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or activated cell types for cell therapy, and applying the activated macrophages and / or T cells and / or fibroblasts and / or activated mesenchymal stem cells and / or adipose tissue-derived cells and / or resident cells of other tissues or activated cell types for cell therapy to the skin at a location where the wound can be repaired. In some embodiments, the chemotherapeutic agent is dacarbazine. In some embodiments, the activating compound comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or adipose tissue-derived cells and / or resident cells of other tissues and / or cell types used for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, and wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0128] In some embodiments of the present invention, a method for treating neurodegenerative diseases, such as ALS, Parkinson's disease, Alzheimer's disease, etc., is provided, comprising the following steps: contacting macrophages and / or mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell types used for cell therapy with a chemotherapeutic agent to provide activated macrophages and / or activated mesenchymal stem cells and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or other cell types used for cell therapy, and administering the activated macrophages and / or T cells and / or fibroblasts and / or activated mesenchymal stem cells and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or other activated cell types used for cell therapy to damaged areas in the brain. In some embodiments, the chemotherapeutic agent is temozolomide.

[0129] In some embodiments, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or cell types for cell therapy activated by a specific chemotherapeutic agent are separated from the supernatant, and wherein the supernatant is used as a therapy that can be used in the above-mentioned treatment methods.

[0130] In some embodiments of the present invention, a method for treating aging-related conditions is provided, comprising the steps of contacting macrophages and / or mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or any other cell types used for cell therapy with a chemotherapeutic agent to provide activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or other activated cell types used for cell therapy, and administering the activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or other activated cell types used for cell therapy to damaged areas in the brain. In some embodiments, the chemotherapeutic agent is temozolomide.

[0131] In some embodiments, the activation composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or any other cell type that can be used for cell therapy activated by a chemotherapeutic agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues and / or cell types for cell therapy activated by a specific chemotherapeutic agent are separated from conditioned medium, wherein the conditioned medium is used as a therapy that can be used in the above-mentioned treatment methods.

[0132] In some embodiments of the present invention, a method for treating autoimmune-related conditions is provided, comprising the steps of contacting macrophages and / or mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or any other cell types used for cell therapy with a chemotherapeutic agent to provide activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or other activated cell types used for cell therapy, and administering the activated macrophages and / or activated mesenchymal stem cells and / or T cells and / or fibroblasts and / or neural tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or resident cells of other tissues or other activated cell types used for cell therapy to damaged areas in the brain. In some embodiments, the chemotherapeutic agent is methotrexate.

[0133] Table 1: Different chemotherapies that can be used to activate cells for various indications.

[0134]

[0135] Table 2: Secretome of mesenchymal stem cells activated with gemcitabine chemotherapy (Protein expression in conditioned medium of MSCs expressed as fold increase between naive and gemcitabine-activated MSCs)

[0136]

[0138]

[0139]

[0140] In some embodiments, there is provided a method for preparing a medicament for treating damaged tissue, comprising the steps of:

[0141] analyzing the secretome of conditioned medium from mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural cells and / or adipocytes activated by anticancer drugs;

[0142] preparing a composition comprising a combination of two or more active ingredients identified in the secretome;

[0143] The compositions were evaluated for their efficiency.

[0144] In some embodiments, the ratio between the two or more active ingredients is about the same as the ratio determined in the secretome.

[0145] The method can be used at an individual level, i.e. different cells activated by different anticancer agents will result in different active ingredients and / or different ratios therebetween. Thus, an individual composition can be prepared for each person.

[0146] Therefore, diseases or disorders or conditions associated with tissue damage can be treated by using conditioned medium from mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural cells and / or adipocytes activated by anti-cancer drugs, or by determining the components in their secretomes, determining the ratios therebetween and preparing such compositions for use as drugs.

[0147] Example

[0148] Materials and methods

[0149] Cell culture

[0150] Human pancreatic adenocarcinoma cell line (PANC1) was purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium (DMEM). After thawing in the original stock solution, the medium was supplemented with 10% FCS in the presence of 1% penicillin-streptomycin, 1% sodium pyruvate and 1% L-glutamine at 37°C in a 5% CO2 environment for no more than 6 months.

[0151] To analyze CSC enrichment, PANC1 cells were cultured in serum-free medium (control), CM derived from untreated or chemotherapy-cultured MSCs, or co-cultured with untreated or chemotherapy-cultured MSCs in serum-free medium at a ratio of 10: 1. In some groups, serum-free medium was supplemented with anti-hCXCL10 (1 μg / ml, R&D Systems). After 3 days, the CSC enrichment of the culture was assessed using immunophenotyping as described below.

[0152] Generation of chemotherapy educated MSC or MSC-derived conditioned medium

[0153] Human bone marrow MSCs (LONZA, Switzerland) were cultured in minimal essential medium-α (αMEM) supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, and 1% streptomycin. The medium was changed every 3 days, and the cells were maintained in culture up to passage 7.

[0154] In some cases, human MSCs isolated from bone marrow aspirates of healthy patients were used and cultured in culture dishes supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate and 1% streptomycin in minimal essential medium-α (αMEM). MSCs were purified based on their ability to adhere to plastic culture dishes. The culture medium was changed every 3 days until the hematopoietic cells were washed away, leaving a homogenous culture of adhered MSCs. The MSCs obtained were expanded and passaged, and cells up to the 7th generation were used for experiments.

[0155] In some cases, mouse MSCs were isolated from bone marrow aspirates and cultured in culture dishes supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, and 1% streptomycin in minimal essential medium-α (αMEM). MSCs were purified based on their ability to adhere to plastic culture dishes. The culture medium was changed every 3 days until the hematopoietic cells were washed away, leaving a homogenous culture of adhered MSCs. The mouse MSCs obtained were expanded and passaged, and cells up to 10 generations were used for experiments.

[0156] To generate chemotherapy cultured MSCs, cultured MSCs were exposed to paclitaxel (100 nM), gemcitabine (10 nM), cisplatin (10 μM) or vehicle control for 24 h. To generate MSC-derived CMs, chemotherapy cultured MSCs (described above) were re-seeded in serum-free medium at a concentration of 1×105 cells / ml. After 72 h, CMs were collected.

[0157] Animal models, treatments, and live imaging

[0158] The use of animals and experimental protocols were approved by the Animal Care and Use Committee of the Technion. 6 The tumor size was assessed regularly by caliper using the formula width 2×length×0.5. Mice were treated with 500 mg / kg gemcitabine (Eli Lilly Ltd.) or vehicle control.

[0159] For wound healing assay in vivo, small (0.5 cm) incisions were made in the backs of 8 to 10 week-old BALB / c mice. On the day of surgery, mice were treated externally or not treated with the conditioned medium of untreated or gemcitabine-activated MSCs. Wound size was monitored every day and assessed with a vernier caliper. Mice were randomly divided before surgery.

[0160] Matrigel plug assay

[0161] Matrigel (0.5 ml) containing pelleted conditioned medium from untreated MSCs or MSCs pre-exposed to gemcitabine, paclitaxel, or cisplatin was injected subcutaneously into each flank of BALB / c female mice (8-10 weeks old) (n=5 mice / group). The plugs were removed 10 days later and single cell suspensions were subsequently prepared for histological evaluation or flow cytometric analysis as previously described (13). Cell suspensions were immunostained with an antibody cocktail to detect cell types defined by their surface markers: EC: CD31+ / VEGFR2+ / CD45-; MDSC: Gr-1+ / CD11b+ / CD45+; M1 macrophages: F4 / 80+, CD11b+, CD11c+, CD206-; M2 macrophages: F4 / 80+, CD11b+, CD11c+, CD206+; NK cells NKp46+, CD45+; B cells CD45R(B220)+, CD45+; and T cells CD3ε+, CD45+. All monoclonal antibodies were purchased from Biolegend, BD Biosciences, R&D Systems, and Macs Militenyi Biotec and used according to the manufacturer's instructions. Monoclonal antibodies conjugated to the following fluorescent dyes were purchased: Gr-1-fluorescein isothiocyanate (FITC), VEGFR2-red phycobilin (PE), CD11b-peridin-chlorophyll protein (PerCP), CD31-FITC and CD45-APC-Cy7, F4 / 80-PE, CD11c-APC-Cy7, CD206-BV421, NKp46-APC, CD45R (B220)-BV605, CD3ε-Pacific blue. When necessary, after erythrocyte lysis, cell suspensions were analyzed by CyAn ADP flow cytometer and Summit v4.3 software (Beckman Coulter). Acquisition of at least 100,000 cells per sample was performed. The analysis is considered informative when a sufficient number of events (usually 50–150) are collected in the EC, MDSC, macrophage, B cell, or T cell enumeration gates in untreated control animals. The percentage of stained cells is determined and compared to the appropriate negative control. Positive staining is defined as greater than nonspecific background staining and 7-aminoactinomycin D (7AAD) is used to distinguish apoptotic cells and dead cells from live cells.

[0162] Evaluation of the effect of MSCs on cytotoxic T cell activity

[0163] Cytotoxic T cell activity was assessed using a mouse T cell activation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. Briefly, conditioned medium from naive MSCs or MSCs pre-exposed to different chemotherapeutic agents (gemcitabine 10 nM, paclitaxel 100 nM, or cisplatin 10 μM) was used to activate cytotoxic T cells in the presence or absence of anti-mouse CD3ε and CD28 biotinylated magnetic beads (10 μM). 6 Beads / sample) were added to spleen cells from naive BALB / c mice (10 7 ) were cultured at 37°C for 24 hours. The beads simulated antigen presenting cells and activated resting T cells. Subsequently, the cells were centrifuged at 470×g for 5 min at room temperature. The cell pellet was resuspended in PBS, and the levels of total CD8+ T cells and activated T cells (CD8+ / CD25+ or CD8+ / CD107a+) were quantified by flow cytometry.

[0164] Scratch wound assay

[0165] In vitro scraping assay was performed using PANC1 cells cultured in 96-well flat transparent plates (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FCS DMEM. Before assaying the cells, they were starved for 16 hours in serum-free DMEM. Before scraping, cells were incubated with CM of untreated MSCs or MSCs exposed to paclitaxel (100 nM), gemcitabine (10 nM), cisplatin (10 μM). Time-lapse images of cell migration were captured using the IncuCyte ZOOM HD / 2CLR system by a × 100 objective lens and analyzed by IncuCyte ZOOM 2016B software (Essenbioscience, Ann Arbor, MI).

[0166] Improved Boyden chamber assay

[0167] The effects of CM from untreated MSCs or MSCs exposed to paclitaxel, gemcitabine, or cisplatin on the migratory properties of PANC1 cell cultures were evaluated in fibronectin-coated Boyden boxes using a previously described protocol (14, 15). Briefly, serum-starved cells (2 × 10 cells / mL in 0.2 ml serum-free DMEM) were cultured in 1% paraformaldehyde (pH 7.0) for 24 h. 5Cells) were added to the upper compartment of a box coated with 50 μl of fibronectin (10 μg / ml). The lower compartment was filled with CM from MSCs that were not treated or pretreated with different chemotherapeutic agents as described above. After 24 hours, cells that migrated to the bottom filter were stained with crystal violet and counted per ×100 objective under an inverted microscope (Leica DMIL LED). All experiments were performed 3 times.

[0168] Immunostaining

[0169] Tumor or matrix gel is embedded in OCT (Sakura, Japan) and then frozen at -80 ° C. Tissue sections (10 μm thick) are prepared using a Leica CM 1950 slicer (Leica, Germany). The sections are fixed in cold acetone for 15 minutes. CSCs are stained with PE-conjugated antibodies against human prominin-1 (CD133, 1:250, Macs MiltenyiBiotec). To identify MSCs, sections are stained with FITC-conjugated antibodies (CD105) against endoglin and APC-conjugated antibodies (1:200, BD Biosciences) against CD44. Tumor sections are analyzed by a camera attached to an inverted microscope (Leica CTR 6000) using Leica Application suite version 3.4.0 software or LSM 700Zeis confocal microscope (Zeiss, Germany). Cell nuclei were stained with DAPI (Electron Microscopy Sciences, PA, USA).

[0170] Cytokine Array

[0171] According to the manufacturer's instructions, CM from gemcitabine-cultured MSCs or control MSCs were applied to a proteome profiler human XL cytokine array (ARY022B, R&D Systems, MN). The signals corresponding to each factor in the quantitative array were analyzed by densitometry. The ratio between the expression levels of various factors secreted by gemcitabine-cultured MSCs and untreated MSCs was calculated.

[0172] Statistical analysis

[0173] Data are expressed as mean ± standard deviation (SD). The statistical significance of the differences was assessed by one-way ANOVA followed by Tukey adhoc statistical test using GraphPad Prism 5 software (La Jolla, CA). Student t test was used in some experiments when only two groups were compared. Differences between all groups were compared with each other and were considered significant when p values ​​were below 0.05.

[0174] result

[0175] Chemotherapy-activated MSC-enriched stem cells

[0176] MSCs are one of the leading candidates for tissue regeneration and cell therapy due to their high proliferation potential and ability to differentiate into predominantly mesenchymal tissues. Furthermore, MSCs have been shown to promote systemic resistance to chemotherapy in solid tumors by secreting two specific fatty acids after exposure to platinum-based cytotoxic agents. These fatty acids indirectly protect colon and lung cancer cells from the cytotoxic effects of chemotherapy. However, the interaction of MSCs with tissue-resident stem cells is still unclear, especially after chemotherapy. To this end, we analyzed subcutaneous pancreatic tumors that were injected into the flanks of 8- to 10-week-old SCID mice with 5 × 10 6 When the tumor reached 500 mm3, mice were treated with 500 mg / kg gemcitabine and removed and sacrificed 3 days later. After gemcitabine therapy, tumor immunostaining for MSCs and CSCs was shown. Specifically, when compared to untreated controls, MSCs homed to the tumor and were in close proximity to CSCs ( Figure 1A ).in addition, Figure 1B It was shown that chemotherapy induces a large recruitment of MSCs to pancreatic tumors, which is similar to the infiltration of MSCs to damaged tissue sites, as shown previously. In addition, to investigate whether MSCs contribute to CSC repopulation, we applied CM of untreated MSCs or gemcitabine-cultured MSCs and evaluated CSC enrichment using phenotypic characterization by flow cytometry as described in Materials and Methods. We show that chemotherapy-activated MSCs lead to significant enrichment of CSCs when compared to the effect of untreated MSCs. Secretome analysis of gemcitabine-activated MSCs showed that CXCL10 secretion was highly elevated after chemotherapy treatment relative to untreated MSCs, as shown in Table 1. We show that CXCL10 promotes CSC enrichment by binding to the CXCR3 receptor and continuously inducing STAT3 signaling. Neutralizing CXCL10 with anti-CXCL10 antibodies counteracted the enrichment effect of chemotherapy-activated MSCs on CSCs ( Figure 1CThese results were published in the January issue of Cancer Research (6). In summary, our results suggest that MSCs contribute to tissue-resident CSCs following activation by chemotherapy.

[0177] Chemotherapy-activated MSCs promote immunosuppression and induce angiogenesis

[0178] MSC is known for its ability to home to the injured site and promote tissue regeneration. In addition, MSC secretes cytokines and chemokines of the entire environment, including TGFβ, IL-6, PGE2, IDO, LIF, to regulate the recruitment of various hematopoietic cell types that regulate tissue structure. In order to test these abilities under our experimental conditions, matrix gel plugs were measured. For this reason, matrix gel plugs containing CM produced by untreated MSC or gemcitabine-cultured MSC were implanted into the flanks of 8 to 10 week-old Balb / c mice. After 10 days, the plugs were removed and prepared as single cell suspensions. Phenotypic analysis was performed on cells from the plugs by flow cytometry to identify cells recruited to the plugs. We found that the CM from gemcitabine-treated MSCs induced the recruitment of endothelial cells, indicating that the potential for angiogenesis increased after gemcitabine therapy. In addition, when compared with the plugs containing CM of control MSCs, in the matrix gel plugs with CM from gemcitabine-activated MSCs, the colonization of most immune species populations (including macrophages, NK cells, T cells and B cells) was significantly reduced ( Figure 2A -B). To further elucidate the immunomodulatory effects of chemotherapy-exposed MSCs, T cell activation was performed by administering CM of naive or chemotherapy-activated MSCs to splenocytes extracted from naive Balb / c mice. As a result, chemotherapy-activated MSCs significantly reduced the total amount of CD8 T cells and inhibited the activation of cytotoxic T cells (Figure 3). In summary, this result provides additional support that in tumors, similar to damaged tissues, MSCs acquire a powerful regenerative capacity that increases angiogenesis and induces immunosuppression, thereby improving overall tissue repair.

[0179] Chemotherapy-activated MSCs increase effective tissue repair

[0180] Based on the enhanced pro-angiogenic and immunosuppressive properties of chemotherapy-activated MSCs, the ability of MSCs to heal wounds was evaluated. To this end, the effect of MSCs on the migratory properties of other cells was tested by performing a modified Boyden box assay as described in Materials and Methods. To do so, PANC1 (2×10 6The lower box was loaded with CM of chemotherapy-activated MSCs or CM of untreated MSCs. After 24 hours, the cells that migrated to the lower side of the box were fixed and then stained with crystal violet. It was found that CM of gemcitabine-activated MSCs significantly increased the migration properties of PANC1 cells compared with the control group. Interestingly, CM produced by MSCs exposed to other cytotoxic agents (such as paclitaxel and cisplatin) did not affect the migration ability of the cells ( Figure 4A In addition, a scratch wound assay was performed to further demonstrate the regenerative potential of chemotherapy-activated MSCs. Cells treated with CM from gemcitabine-cultured MSCs closed the gap faster than untreated control cells or cells exposed to CM from untreated MSCs ( Figure 4B -C). In parallel with the migration assay results, the wound closure time of cells treated with CM of MSCs exposed to PTX or cisplatin was similar to that of the control group.

[0181] Next, the wound healing properties of chemotherapy-activated MSCs were tested in vivo. For this purpose, a 0.5 cm incision was made on the back of BALB / c mice. After 24 hours, the mice were treated externally with conditioned medium of untreated MSCs or MSCs pre-exposed to 10 nM gemcitabine. Control mice were not treated. Wound healing was monitored until the wound was completely closed. The results showed that compared with the control group, mice treated with gemcitabine-activated MSCs had the fastest wound healing ( Figure 4D ). Taken together, our results suggest that chemotherapy-cultured MSCs greatly induce MSC regeneration and wound healing properties, thus contributing to injury repair.

[0182] Example 2

[0183] Chemotherapy-activated MSCs for the treatment of pulmonary fibrosis

[0184] Bronchoalveolar lavage

[0185] All animal studies and animal experimental protocols were approved by the Animal Care and Use Committee of the Technion. A mouse pulmonary fibrosis model was established by intratracheal (IT) administration of a single dose of bleomycin as shown in Van Hoecke, L., et al., Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration. J Vis Exp, 2017 (123) and Cox, TR, et al., LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis. Cancer Res, 2013. 73 (6): p. 1721-32.

[0186] In brief, 0.04 units of bleomycin were intratracheally (IT) administered to 10 week-old female BALB / c mice, or treated with vehicle control. After 1 week, mice were not treated, or IT was administered with the conditioned medium of control MSC or chemotherapy activated MSC. This treatment (conditioned medium of 70 μl activated MSC, as described in the method section above) was carried out every 1 day for 1 week. After 14 days of bleomycin administration, bronchoalveolar lavage (BAL) was performed by washing lungs with Hank's balanced salt solution (HBSS) supplemented with 100uM ethylenediaminetetraacetic acid (EDTA, all from Biological industries ltd, Israel), and flow cytometry was used to analyze pulmonary inflammatory cell infiltration. Then, lungs were harvested, fixed and processed for histology.

[0187] Evaluation of inflammatory cell types in bronchoalveolar lavage (BAL) using flow cytometry

[0188] To evaluate various immune cell types after the BAL procedure, cells were extracted from bronchoalveolar lavage fluid of mice treated with bleomycin or control mice. The cells were then immunostained and analyzed to define various innate and adaptive immune cell populations, including T cells (CD4+ and CD8+), B cells (B220), macrophages (CD11b+F4 / 80+, CD11c±, CD206±), NK cells (NKp46), and myeloid-derived suppressor cells (MDSC, CD11b+, Ly6G±, Ly6C±). All monoclonal antibodies were purchased from BioLegend (San Diego, CA, USA) and used according to the manufacturer's instructions. Samples were collected using the LSR Fortessa flow analyzing system (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo X software (FlowJo, LLC, Ashland, OR, USA).

[0189] Histology

[0190] Lungs from control or treated mice were harvested at the end point and subsequently fixed with PFA. Paraffin-embedded lung tissues were sectioned and stained with hematoxylin and eosin (H&E) solution. To assess collagen content, which represents fibrosis, lung sections were stained with SiriusRed solution (marked in red).

[0191] result

[0192] When functional tissue is replaced by rough connective tissue in large quantities and physiological function is impaired, tissue damage usually leads to the development of scar tissue and fibrosis. Specifically, pulmonary fibrosis (PF) is an interstitial lung disease, and its cause is unknown in most cases. Lung injury, inflammatory process, lung disease, exposure to radiation and chemicals may induce PF, and its mortality rate rises. In the case of experimental animals, bleomycin is a widely used reagent for inducing pulmonary fibrosis models. Therefore, a bleomycin-induced fibrosis model was implemented to evaluate the efficacy of the proposed stem cell-based therapy to prevent and cure fibrosis. Specifically, the evaluation of the number of cells in BAL samples showed that, as described in the method section above, when compared to the control group, the conditioned medium (CM) of chemotherapy-activated MSCs administered in the lungs treated with bleomycin enhanced cell recruitment and homing to the lungs ( Figure 5). Specifically, mice induced with pulmonary fibrosis treated with CM of chemotherapy-activated MSCs resulted in an increase in CD45-positive cells compared to all other groups. These cells consisted of increased percentages of anti-inflammatory macrophages and bone marrow-derived suppressor cells (both of which represent tissue regeneration and growth) and decreased percentages of pro-inflammatory macrophages, T cells, B cells, and NK cells (all of which represent tissue inflammation that inhibits tissue regeneration) (Figure 6).

[0193] Histological analysis of bleomycin-treated mice or control mice treated with CM of chemotherapy-activated MSCs supported the flow cytometry data of BAL samples. Specifically, extensive inflammation (indicated by white arrows) was observed in the lungs of bleomycin-treated mice or mice treated with CM of bleomycin and non-activated control MSCs. In contrast, lungs treated with chemotherapy-activated MSCs showed minimal inflammation levels, similar to those of lungs of untreated controls ( Figure 7 A). In addition, Sirius red staining of lungs from control bleomycin-treated mice or mice treated with CM from non-activated MSCs showed increased collagen deposition (indicated by white arrows), indicating peribronchial thickening ( Figure 7 B).

[0194] Overall, these results indicate that in mice treated with CM of chemotherapy-activated MSCs, the inflammatory process in the lungs was suppressed and, therefore, tissue fibrosis was also suppressed and, therefore, the amount of lung tissue fibrosis was reduced.

[0195] Example 3

[0196] Chemotherapy-activated MSCs for the treatment of liver fibrosis

[0197] Hepatocellular carcinoma, cirrhosis and other liver pathologies remain one of the leading causes of death. In most cases, alcohol abuse, unhealthy diet, viral infections and recurrent inflammation lead to the spread of scar tissue and the development of extensive liver fibrosis, which exacerbates and promotes liver failure. This occurs because functional liver tissue is replaced by fibrotic spikes, which destroy the effective blood supply, induce bile duct strictures and lead to impaired liver function. However, effective drugs have not yet been developed to treat and reverse the damage associated with liver fibrosis. Therefore, effective treatment strategies are needed to maintain liver tissue structure and function and prevent fibrotic tissue formation.

[0198] The methods described in the current examples suggest additional applications for activated cell-based therapies by inducing liver tissue regeneration and preventing liver diseases including liver fibrosis, which can further develop into cirrhosis and liver cancer.

[0199] method

[0200] Generation of chemotherapy-activated MSCs or MSC-derived conditioned medium or activated MSC-derived exosomes

[0201] Mouse MSCs were isolated from bone marrow aspirates and cultured in culture dishes supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, and 1% streptomycin in minimal essential medium α (αMEM). MSCs were purified based on their ability to adhere to plastic culture dishes. The culture medium was changed every 3 days until the hematopoietic cells were washed away, leaving a homogenous culture of adherent MSCs. The obtained mouse MSCs were expanded and passaged, and cells up to the 10th generation were used for experiments.

[0202] To generate chemotherapy-cultured MSCs, cultured MSCs were exposed to gemcitabine (0-10 nM), or vehicle control for 24 h. To generate MSC-derived CMs, chemotherapy-cultured MSCs (described above) were incubated at 1×10 5 The cells were replated in serum-free medium at a concentration of 10 cells / ml. After 72 hours, CM was collected.

[0203] To extract the exosome fraction from activated MSCs, the CM of chemotherapy-activated MSCs was centrifuged at 20,000 g for 1 h at 4 °C to separate microparticles and apoptotic bodies. Therefore, the obtained supernatant was ultracentrifuged at 110,000 g for 2 h at 4 °C. The pellet was resuspended in PBS, aliquoted and stored at -80 °C.

[0204] Induced mouse liver fibrosis model

[0205] All animal studies and animal experimental protocols have been approved by the Animal Care and Use Committee of the Technion. A mouse liver fibrosis model was established by continuous injection of dimethylnitrosamine (DNM) for 4 weeks by intraperitoneal (IP) administration for 3 consecutive days, as previously described in Chooi, KF, et al., The Dimethylnitrosamine or N-Nitrosodimetilamine (DNM or NDMA) Induced Liver Fibrosis Model in the Rat. J Vis Exp, 2016 (112) and Cox, TR, et al., LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis. Cancer Res, 2013. 73 (6): p. 1721-32. Briefly, 8 to 10 week old female C57 / Bl mice were IP administered DNM (5 mg / kg; Sigma Aldrich, Israel) or treated with vehicle control. Two weeks after the start of treatment, mice were left untreated or injected with conditioned medium of control MSCs or chemotherapy-activated-MSCs or exosomes generated by chemotherapy-activated MSCs. Therapy was given every other day for 2 weeks. When mice were sacrificed, livers were removed and processed for histological analysis and assessment of fibrosis grade. In parallel, single-cell suspensions were processed for individual liver lobes and subpopulations of inflammatory cells and fibroblasts were analyzed by flow cytometry. Additional liver lobes were processed to assess soluble collagen content.

[0206] Assessment of liver fibrosis based on liver physiology

[0207] To test the physiological function of the liver in DNM-induced liver fibrosis mice, mice were not treated or injected intraperitoneally (IP, every other day for 2 weeks) with conditioned medium of chemotherapy-activated MSCs or control MSCs or exosomes derived from chemotherapy-activated MSCs. Blood was drawn from treated and control mice at the end point, and liver function parameters such as total protein, albumin, bilirubin and specific liver enzymes, including alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were assessed.

[0208] Assessment of cell types colonizing the liver using flow cytometry

[0209] To evaluate the various cell types infiltrating the liver, cells were extracted from the liver of DNM-treated mice or control mice. The cells were then immunostained and analyzed to define liver fibroblasts (CD90+) and various innate and adaptive immune cell populations, including T cells (CD4+ and CD8+), B cells (B220+), macrophages (CD11b+F4 / 80+, CD11c±, CD206±), NK cells (NKp46+), and bone marrow-derived suppressor cells (MDSC; CD11b+, Ly6G±, Ly6C±). All monoclonal antibodies were purchased from BioLegend (San Diego, CA, USA) and used according to the manufacturer's instructions. Samples were collected using the LSRFortessa flow analysis system (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo X software (FlowJo, LLC, Ashland, OR, USA).

[0210] Histology

[0211] To evaluate the effects of activated cell-based therapy on the progression of liver fibrosis, its tissue composition and structure, livers were harvested from control and treated mice and fixed with 4% paraformaldehyde (PFA) solution. Paraffin-embedded liver tissue sections were sliced ​​and stained with hematoxylin and eosin (H&E). To evaluate the expression of collagen aggregation in the liver and to evaluate the degree of fibrosis in DNM-treated or untreated mice, the amount of soluble collagen levels was assessed using SircolTM soluble collagen assay (Biocolor, UK). In addition, liver sections were stained with Sirius Red and collagen deposition was analyzed.

[0212] result

[0213] Studying the liver function of MSCs activated in response to chemotherapy

[0214] When functional tissue is replaced by rough connective tissue in large quantities and physiological function is impaired, tissue damage usually leads to the development of scar tissue and fibrosis. Specifically, liver fibrosis is a key stage of liver pathology, which develops into cirrhosis and cancer in most cases. Although several factors can promote liver fibrosis, such as viral infection (HCV), diet, increased drinking and genetic aberrations, the exact cause of the development of fibrotic tissue in the liver is still unclear. In the case of experimental animals, DNM (also known as NDMA) and other chemicals are widely used reagents for inducing liver fibrosis models. Therefore, we implemented a DNM-induced fibrosis model to evaluate the efficacy of our proposed activated cell-based therapy for preventing and curing liver fibrosis. Specifically, 8 to 10-week-old BALB / c mice were induced to suffer from liver fibrosis by continuous weekly injection of DNM. After 2 weeks, mice were treated with IP (every other day for 2 weeks) with the conditioned medium of control MSCs or chemotherapy-activated MSCs or exosomes generated by chemotherapy-activated MSCs. As a control, mice were injected with PBS. Notably, neither DNM injection nor treatment with CM from MSCs or their exosomes resulted in severe toxicity as measured by animal body weight. The body weight of all experimental subgroups remained normal and similar to that of untreated mice ( Figure 8 ). At the endpoint, blood was analyzed for physiological function of the liver of DNM-treated mice control or mice injected with CM of chemotherapy-activated MSCs. As expected, liver function was impaired in DNM-treated mice, as evidenced by decreased albumin levels and increased levels of ALT and AST enzymes. However, mice treated with conditioned medium of gemcitabine-activated MSCs or their exosome fractions showed significant improvement in liver function. That is, mice treated with CM of chemotherapy-activated MSCs exhibited increased albumin levels and decreased liver enzyme levels, which were similar to the parameters of control untreated naive mice ( Fig. 9A -C). In addition, DNM-induced liver fibrosis reduced the level of liver-resident Kupffer cells and promoted an increased influx of fibroblasts and immune cells (including T cells, B cells, NK cells, myeloid cells, and macrophages) into the liver. In summary, elevated levels of these cells indicate increased liver inflammation, which attenuates tissue regeneration and promotes fibrosis. However, treatment with CM of activated MSCs or their exosome fractions reduced the percentage of inflammatory cells in the liver and increased immunosuppressive bone marrow-derived suppressor cells, thereby promoting tissue regeneration ( Fig. 10A -D).

[0215] To assess the grade of fibrosis and collagen deposition in the livers of mice treated with CM of chemotherapy-activated MSCs or control mice, we assessed soluble collagen levels in treated livers. Treatment with CM of activated MSCs resulted in a significant decrease in soluble collagen ( Fig.11A ). Histological analysis of the liver supports the above data for treated livers. Specifically, H&E and Sirius red staining demonstrated extensive fibrosis around the central canals and inlet areas of DNM-treated livers (indicated by white arrows). In contrast, the liver histology of mice treated with conditioned medium of gemcitabine-activated MSCs was similar to that of the livers of untreated healthy (naive) control mice, indicating complete regeneration of fibrotic tissue. As expected, treatment with our exosome therapy resulted in a similar reduction in liver fibrosis, while CM of control MSCs did not show any significant improvement in liver fibrosis in DNM-treated mice ( Fig. 11B -D).

[0216] Overall, these results indicate that in mice treated with CM of gemcitabine-activated MSCs, hepatic inflammation was suppressed, which improved tissue regeneration and thus prevented and reduced liver fibrosis and restored healthy liver function.

[0217] Although the present invention has been described in conjunction with the specific embodiments of the present invention, it is apparent that various substitutions, modifications and changes will be apparent to those skilled in the art. Therefore, it is intended to cover all such substitutions, modifications and changes that fall within the spirit and broad scope of the appended claims.

[0218] All publications, patents and patent applications mentioned in this description are incorporated herein by reference in their entirety to the same extent as if each publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, the reference or identification of any reference in this application should not be construed as an admission that the reference can be used as prior art of the present invention. With respect to the use of this section title, it should not be construed as necessarily limiting.

Claims

1. An activation composition comprising mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells and / or adipose tissue-derived cells and / or resident cells of other tissues activated by anticancer drugs.

2. The activating composition of claim 1, wherein the anticancer drug is a chemotherapeutic agent.

3. An activation composition comprising conditioned medium from mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts and / or neural tissue cells and / or adipose tissue-derived cells and / or resident cells of other tissues activated by anticancer drugs.

4. A method for producing an activated mesenchymal stem cell and / or activated macrophage and / or T cell and / or fibroblast composition for tissue repair or for treating a disease associated with tissue damage, the method comprising: The mesenchymal stem cells and / or the macrophages and / or the T cells and / or the fibroblasts and / or the neural tissue cells and / or the adipose tissue-derived cells and / or the resident cells of other tissues are activated by culturing the mesenchymal stem cells and / or the macrophages and / or the T cells and / or the fibroblasts and / or the neural tissue cells and / or the adipose tissue-derived cells and / or the resident cells of other tissues with anti-cancer drugs, and optionally separating the conditioned medium from the activated mesenchymal stem cells and / or the activated macrophages and / or the T cells and / or the fibroblasts, and the activated mesenchymal stem cells and / or the macrophages and / or the T cells and / or the fibroblasts and / or the neural tissue cells and / or the adipose tissue-derived cells and / or the resident cells of other tissues or the conditioned medium thereof are used for tissue repair or for treating diseases associated with tissue damage.

5. The method of claim 4, wherein the culturing step lasts from about 10 minutes to about 72 hours.

6. The method of claim 5, wherein the conditioned medium is from at least 10 5 -10 6 Activated cells were collected.

7. A method for promoting tissue regeneration or organ repair of a damaged organ and / or for reducing inflammation in a mammal, the method comprising: The activating composition of any one of claims 1 to 3 is administered to a mammal in need of tissue regeneration, organ repair or reduced inflammation.

8. A method for inducing angiogenesis and / or inducing immunosuppression in a mammal, the method comprising: The activating composition of any one of claims 1 to 3 is administered to a mammal in need of tissue regeneration or organ repair, wherein the angiogenesis enhances tissue regeneration or organ repair of a damaged organ.

9. A method for regenerating tissue in a damaged organ, comprising the steps of: The activating composition according to any one of claims 1 to 3 is administered to the damaged organ directly or by peripheral administration.

10. The method of any one of claims 7 or 9, wherein the damaged organ is pancreas, breast, ovary, lung, heart, kidney, lung, GI tract, intestine, skin, liver, or brain.

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