Enhancement of fibroblast therapeutic activity by RNA

Abstract

To provide a method for enhancing one or more therapeutic properties of a fibroblast population.SOLUTION: A method comprises the step of treating a fibroblast population with an effective amount of exogenous RNA sufficient to enhance one or more therapeutic properties of the fibroblast population.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application was filed on June 12, 2019, and is incorporated herein by reference in its entirety. Priority is claimed to filed U.S. Provisional Patent Application No. 62 / 860,252.

[0002] (Technical field) The technical field of the disclosure includes at least the fields of cell biology, molecular biology, cell therapy, and medicine. include. [Background technology]

[0003] Fibroblasts constitute the main cell type of connective tissue with a spindle-shaped morphology and their classical function is Historically, it was believed that the extracellular matrix was responsible for maintaining the structural integrity of tissues. Fibroblasts also play an important role in the proliferation phase of wound healing, resulting in As a result, extracellular matrix is deposited [1,2]. During wound healing, scar tissue undergoes proliferation. It is formed by fibroblasts. It is currently the subject of intensive research in embryos and in some amphibians. This process allows for scarless healing after injury [3,4]. Many types of tissues and organs gradually undergo fibrosis, including fibrosis of the liver, kidneys, and heart. The process of scar tissue formation is caused by the hyperproliferation of fibroblasts and the During proliferation, cells produce abnormally large amounts of extracellular matrix and collagen, thereby Normal organ structure (parenchyma) is replaced, leading to dysfunction and scarring, and persistent fibrosis. This may further induce

[0004] The present disclosure addresses a long felt need in the art for providing therapeutic compositions for cell therapy. provides a solution to a long-standing need. Summary of the Invention

[0005] Embodiments of the present disclosure include methods and compositions for enhancing fibroblast transplant efficacy. More specifically, in specific embodiments, the present invention provides a method for producing a medicament for the treatment of rhesus mast cells that have enhanced therapeutic activity after transplantation. In a specific embodiment, the present invention provides a method for producing fibroblasts. The present invention relates to a method for increasing the therapeutic activity of cells by utilizing exposure to effective amounts of RNA. do.

[0006] The present disclosure is based on the previously unknown ability of RNA to enhance the therapeutic activity of fibroblasts. Compositions of matter, treatment protocols, and methods of use are provided based on the present invention. In this study, administration of double-stranded RNA was performed using polyinosinic-polycytidylic acid (poly(I:C)) or functionally active derivatives thereof to induce one or more therapeutic properties and / or to inhibit fibroblast growth. This is done by providing it in a sufficient concentration to enhance its therapeutic properties on the cells. In one embodiment, the enhanced therapeutic activity comprises increased fibroblast migration activity. In this study, therapeutic activity was assessed by a) angiogenesis; b) immunomodulation; c) differentiation potential; and d) production of trophic factors. e) the ability to resist apoptosis; f) migratory activity; and g) combinations thereof. The compound is selected from the group consisting of:

[0007] Any limitations discussed with respect to one embodiment of the present invention may also be applied to any other embodiment of the present disclosure. It is specifically contemplated that any of the compositions of the present invention may be applied to any of the compositions of the present disclosure. Any method of the present disclosure may be used in a method for producing or administering any of the compositions of the present invention. Aspects of the embodiments described in the examples may also be used to Elsewhere in the Examples, or in the Outline of the Drawings, Detailed Description, Claims, and Brief Description, etc. It is an embodiment that may be implemented in the context of embodiments described elsewhere in this application. do.

[0008] The foregoing has outlined the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Those points have been outlined rather broadly. Additional features and advantages which form the subject of the claims of this specification. The disclosed concepts and specific embodiments are intended to carry out the same objectives of the present design. It is understood that the invention can be readily used as a basis for modifying or designing other structures in order to It should be understood by those skilled in the art that such equivalent constructions are within the scope of the appended claims. It should be understood by those skilled in the art that the scope of the invention is not to be deviated from the spirit and scope of the invention as set forth in the appended claims. The novel features believed characteristic of the designs disclosed herein are intended to provide additional objectives and The present invention, both as to construction and method of operation, together with its advantages, should be considered in connection with the accompanying drawings. However, the figures are provided for purposes of illustration and description only and are not to be construed as limiting the present invention, as may be better understood from the following description. It is expressly understood that these are provided for purposes of illustration only and are not intended as a definition of the limits of the present disclosure. Further objects, features, aspects, and advantages of the present invention are set forth in part in the following description. These and other features will be apparent in part from the description or may be learned by practice of the invention. The various embodiments disclosed are provided in sufficient detail to enable those skilled in the art to practice the invention. Although other embodiments may be utilized and changes may be made without departing from the scope of the present invention. It should be understood that the following detailed description is not intended to be limiting. The scope of the present invention is best defined by the appended claims, and is not intended to be limiting. Well defined.

[0009] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the invention and its advantages will be provided below by way of a description of an illustrative embodiment, in which the principles of the invention are utilized. Reference is made to the detailed description below and the accompanying drawings (also referred to herein as "FIG" and "FIG.") and obtained by: [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 provides an example of a cell migration assay. Triangles represent tp Poly I:C, "X" represents CpB, and squares represent scrambled RNA; and

[0011] [Figure 2] Figure 2 shows HGF production from fibroblasts after exposure to poly(I:C). Bars from left to right are control, low molecular weight poly(I:C), and high molecular weight poly(I:C). DETAILED DESCRIPTION OF THE INVENTION

[0012] Although various embodiments of the present disclosure have been shown and described herein, such embodiments are by way of example only. It will be apparent to those skilled in the art that the present invention is provided solely as a Numerous variations, modifications, and substitutions will occur to those skilled in the art. It will be appreciated that various alternatives to the configuration may be used.

[0013] As used herein, the terms "or" and "and / or" refer to two or more members in any combination. Used to describe something in combination or exclusively, e.g., "x, y, and / or "z" is "x" only, "y" only, "z" only, "x, y, and z", "(x and y) or z," "x or (y and z)," or "x or y or z." x, It is specifically contemplated that y or z may be specifically excluded from an embodiment.

[0014] Throughout this application, the term "about" is used to indicate that a value is below the range of the device or method being used to determine the value. In order to show that the standard deviation of the error for the method is included, It is used according to its simple and ordinary meaning.

[0015] The term "comprises", which is synonymous with "includes", "including", or "featuring", is inclusive or "Consisting of" is open-ended and does not exclude additional, unrecited elements or method steps. The phrase "essentially from" excludes any unspecified element, step, or ingredient. The phrase "comprises" or "includes" the term "to extend the scope of the described subject matter beyond the specified materials or steps and their basis. and that do not substantially affect the novel features. It is believed that the term "consisting of" or "consisting essentially of" may also be used. can be.

[0016] In accordance with long-standing patent law conventions, the words "a" and "an" refer to the invention inclusive of the claims. As used herein, "one or more" means "one or more" and some embodiments of the disclosure include: consisting of or essentially of one or more elements, method steps, and / or methods of the disclosure Any of the methods or compositions described herein can be used in combination with any other method or composition described herein. It can be implemented in terms of methods or compositions, and different embodiments can be combined. It is intended that

[0017] Throughout this specification, unless the context otherwise requires, the term "comprises" "," "comprises," and "consisting" mean the inclusion of specified steps or elements, but not others. It will be understood that the exclusion of any step or element or group of elements is not intended. It does not imply the exclusion of any element or group of elements. "Consisting of" does not imply "consisting of": Therefore, "consisting of" means that the listed elements are necessary or mandatory. "Consists of" indicates that other elements may be present. "Inherently consists of" means that any element included in the "shall" means that the listed elements are required or essential, but other elements are optional, and may or may not be present depending on whether it affects the activity or action of the specified element This shows:

[0018] Throughout this specification, the terms "one embodiment," "embodiment," "particular embodiment," "related embodiment," "related" and "related" are used interchangeably. "some embodiments," "particular embodiments," "additional embodiments," or "further embodiments" Reference to a particular feature, configuration, or combination thereof described in connection with an embodiment is not intended to be limiting. or features are included in at least one embodiment of the present invention. The appearances of the foregoing phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic may be incorporated into one or more embodiments. can be combined in any suitable manner.

[0019] As used herein, the term "exogenous" refers to RNA that originates outside the fibroblast.

[0020] The term "subject" as used herein is interchangeable with the terms "patient" or "individual." and to treat or otherwise address medical conditions that utilize cell therapy, particularly fibroblast therapy. The subject may be a mammal, e.g., a human, a laboratory animal (e.g., a mammalian animal), or a human subject. primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, birds, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, etc. any organism or organism that is the subject of a method or material, including transgenic non-human animals The subject may be an animal subject. The subject may be, for example, a subject suffering from one or more infectious diseases, one or more genetic disorders, one or more one or more cancers, one or more chronic medical conditions, one or more injuries, or any combination thereof Patients who have or are suspected of having a disease (which may be called a medical condition) such as The disease may or may not be pathogenic. Subjects may be receiving or may be receiving quality treatment. Subjects may be asymptomatic. A subject may be a healthy individual. As used herein, a "subject" or "individual" refers to a person who is They may or may not be institutionalized and may be treated as outpatients in a medical facility. An individual may receive one or more medical compositions via the internet. The present invention may include any age and any sex of human or non-human animals, and therefore includes adults and infants (including This term includes both infants (i.e., children) and young children, including those in utero. Therefore, it does not imply a need for clinical or basic science research. Whatever the purpose of the experiment, individuals may be voluntarily or involuntarily part of it.

[0021] An embodiment of the present disclosure includes the steps of: a) optionally selecting a population of fibroblasts; and b) performing fibroblast cell culture. Treating a population of fibroblasts with a sufficient concentration of RNA to enhance one or more therapeutic properties of the fibroblasts. and treating a population of fibroblasts with a therapeutic agent. RNA is a double-stranded RNA made of polyinosinic-polycytidylic acid (poly(I:C)) The double-stranded RNA may or may not be a double-stranded RNA consisting of polylysine and carboxymethylcellulose. Polyinosinic-polycytidylic acid stabilized with cellulose (Poly ICLC) In certain cases, the therapeutic activity of fibroblasts may be due to the expression of one or more angiogenic factors. The therapeutic activity of fibroblasts may include the production of one or more regenerative factors. The therapeutic activity of the cells may include migratory activity in response to damage-associated signals. The therapeutic activity of fibroblasts may involve the reduction of apoptosis. It can include a combination of

[0022] Fibroblasts are found in a) adipose tissue; b) skin tissue; c) placental tissue; d) hair follicles; and e) keloids. f) bone marrow; g) peripheral blood; h) umbilical cord; i) foreskin; and j) combinations thereof. It may be derived from a source selected from:

[0023] The pharmaceutical preparation of cells may include any of the fibroblasts encompassed herein. The product may or may not also contain RNA, such as poly(I:C).

[0024] Methods for producing fibroblasts are encompassed herein. In certain embodiments, any The method involves, for example, the interaction of a fibroblast with a ligand capable of inducing an interferon response. For example, the step of inducing activation of toll-like receptor 3 via contact may not be included. The method includes administering a therapeutically effective amount of the cells to an individual at risk of having a medical condition or to a patient for whom the cells are therapeutic. an individual having a medical condition (e.g., eliminating or reducing the severity of at least one symptom) The method may further comprise the step of delivering the

[0025] The present disclosure provides a previously unknown discovery of the use of RNA molecules to enhance the therapeutic activity of fibroblasts. In one embodiment, the RNA molecule contains one or more To activate a molecular pathway within fibroblasts that can induce the production of interferon, In one embodiment, the present disclosure is utilized in a non-specific and / or sequence-semispecific manner. The production of one or more interferons may be directed toward the area of injury, e.g., injury-associated. These findings are associated with the enhanced ability of fibroblasts to migrate toward an SDF-1 gradient. In other embodiments, the present disclosure provides a method for enhancing therapeutic activity. , fibrillation with one or more activators of the PKR pathway (including toll-like receptor 3). Therapeutic activity may include enhanced migration, increased cytokine production, and / or stimulation of blast cells. Enhanced production and / or production of new blood vessels (angiogenesis, arteriogenesis and / or neovascularization) This includes increased ability to stimulate

[0026] In one embodiment, the present disclosure provides a method for stimulating and / or enhancing the therapeutic properties of fibroblasts. Without being bound by theory, the present invention provides the use of poly(I:C) as a source of double-stranded RNA. Although not a nucleotide sequence, poly(I:C) in certain embodiments mimics viral RNA and Characterization of Toll-like receptor 3 (TLR3) ligands, known stimulators of the innate immune response When Poly(I:C) comes into contact with fibroblasts, it induces the production of interferon α and / or For the purposes of this disclosure, poly(I) induces the expression of antiviral proteins such as poly(I). :C) consists of antiparallel polynucleotide strands of inosinic acid and cytidylic acid sodium salts It is a synthetic double-stranded RNA. The strands are separated by hydrogen bonds formed between the inosine and cytosine bases. The average chain length of poly(I:C) is 300 to 6,000. The molecular weight ranges from about 180,000 to 3,600,000 base pairs, which corresponds to approximately 180,000 to 3,600,000 daltons. The formula is (C 10 H 10 N4NaO7P) x (C9H1)1NaN3O7P) x .is. In some embodiments, poly(I:C) is an unstable molecule in aqueous solution. Considering this, to achieve an effective fibroblast-enhancing effect, poly(I:C) should be added immediately before use. In some situations, multiple in vitro administrations may be required. In some embodiments, poly(I:C) is added to tissue culture media to maintain fibroblast activation. It is formulated with one or several bioadhesive polymers that can extend its residence time in the nutrient obtain.

[0027] In some embodiments, the present disclosure provides polyinosinic-polycytidylic acid (poly(I:C)) and starch, alginate, polysaccharide or DPPC (dipalmitoyl phosphate) Activated fibroblasts are prepared using a carrier polymer selected from the group consisting of hydroxylase, hydroxypropyl methylcellulose ... Microparticles refer to particles with an average particle size of 0.1 μm to 100 μm. In certain cases, the carrier polymer may be derived from corn, potato, or cassava. In another embodiment, the nanoparticles are starches obtained by in vitro polymerization. It can be utilized for the delivery of poly(I:C) to fibroblasts. A mixture of Poly(I:C) and starch is prepared according to the present disclosure in a ratio of Poly(I:C) / Starch. The ratio is 1 / 200(w / w) to 1 / 0.1(w / w), especially 1 / 100(w / w) to 1 / 1 (w / w), and even more preferably in the range of 1 / 100 (w / w) to 1 / 5 (w / w). Meanwhile, the ratio Poly(I:C) / starch is used between 1 / 12 and 1 / 9 (w / w). can be.

[0028] The fibroblasts may be cultured fibroblasts, and when referring to cultured fibroblasts, they may be referred to as senescent fibroblasts. The term (replicative senescence or cellular senescence) refers to the properties resulting from finite cell culture, i.e., finite The inability to multiply beyond population doublings (sometimes called the Hayflick limit) The in vitro lifespan of different cell types varies, but the maximum lifespan is typically 1 less than 0.00 population doublings (this indicates that all cells in the culture are senescent and therefore (The number of doublings required to make something indivisible.) Aging does not depend on chronological time, but rather, It is measured by the number of cell divisions or population doublings that a culture undergoes. Cells rendered quiescent by the removal of growth factors will grow and divide when the growth factors are reintroduced. The cells can then be repopulated and undergo the same number of doublings as the equivalent cells grown continuously. As used herein, the term "growth medium" generally refers to a medium sufficient for the cultivation of umbilicus-derived cells. In particular, one particular medium for culturing the cells disclosed herein is Dulbecco's medium. Modified Essential Medium (also abbreviated herein as DMEM) is particularly preferred. Fluorescence (also DMEM-LG here) (Invitrogen, Carlsbad) DMEM low glucose is considered to be 15% (v / v) fetal bovine serum (e.g., (e.g., defined fetal bovine serum, Hyclone, Logan Utah), antibiotic / antimycotic (e.g., penicillin (100 units / ml), streptomycin (100 ml) grams / milliliter), and amphotericin B (0.25 micrograms / milliliter (Invitrogen, Carlsbad, CA), and 0.001% (v (v / v) 2-mercaptoethanol (Sigma, St. Louis, MO) In some cases, different growth media are used or different supplements are provided. and these are usually referred to in the text as supplements to growth media.

[0029] Fibroblasts can be cultured under standard growth conditions. As used herein, the term "standard growth conditions" refers to conditions in a standard atmosphere containing 5% CO2 at 37°C. The relative humidity is maintained at approximately 100%. While beneficial, such conditions may vary depending on the options available to the skilled artisan for culturing cells, e.g. For example, by changing body temperature, CO2, relative humidity, oxygen, growth medium, etc. I want you to understand that it is possible.

[0030] In one embodiment of the present disclosure, RNA-treated fibroblasts are In such cases, the term "inflammatory condition" is used to treat conditions such as, for example, ) Acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, pressure injury, multiple organ failure, hypovolemia Tissue damage caused by ischemia-reperfusion, such as shock, intestinal ischemia, spinal cord injury, and traumatic brain injury; (2) Inflammatory diseases (burns, endotoxemia and septic shock, adult respiratory distress syndrome, Cardiopulmonary bypass, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's disease , sickle cell anemia, poststreptococcal glomerulonephritis, membranous nephritis, pancreatitis; (3) transplant rejection, e.g. (4) pregnancy-related disorders such as recurrent fetal loss and preeclampsia; (5) Drug allergies, IL-2-induced vascular leak syndrome, and X-ray contrast media allergies Adverse reactions: Myasthenia gravis, Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, systemic erythema Erythematosus, insulin-dependent diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, antiphospholipids Complement-mediated inflammation associated with autoimmune diseases such as antibody syndrome, autoimmune hepatitis, and Crohn's disease. Phospholipid syndrome, autoimmune hepatitis, Crohn's disease, Goodpasture's syndrome, Graves' disease Disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, pemphigus, Sjogren's Syndrome, Takayasu's arteritis, and the like can also be treated with the methods described herein.

[0031] In some embodiments of the present disclosure, the RNA-treated fibroblasts comprise one or more Used to treat neurodegenerative conditions. A "neurodegenerative condition" (or disorder) is a condition that affects the central nervous system or It includes acute and chronic conditions, disorders or diseases of the peripheral nervous system. Neurodegenerative conditions are age-related may be caused by injury or trauma, or may be related to a particular disease or disorder. Acute neurodegenerative conditions may be associated with, for example, stroke, focal or diffuse traumatic brain injury, diffuse brain injury, spinal cord injury or peripheral nerve injury, e.g., physical or chemical burns , conditions related to neuronal cell death or cerebrovascular insufficiency resulting from deep amputation or limb amputation Examples of acute neurodegenerative diseases include, but are not limited to: thromboembolism; Cerebral ischemia or infarction, including thrombotic occlusion and thrombotic occlusion, reperfusion after acute ischemia, perinatal hypoxic ischemia Blood injuries, cardiac arrest, and any type of intracranial bleeding (epidural, subdural, subarachnoid and cerebral) intracranial and intravertebral lesions (such as contusion, perforation, shear, compression, and laceration), and Chronic neurodegenerative conditions include Alzheimer's disease, Pico- Page 11 11 Page ... Lewy body disease, progressive supranuclear palsy (Shy-Drager syndrome), neurology Chronic status epilepticus with degeneration, motor neuron diseases including amyotrophic lateral sclerosis, corticobasal Degeneration, ALS-Parkinsonism Dementia Complex in Guam, Huntington's Disease, Synucleinopathy (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Jo Seph disease / Spinocerebellar ataxia type 3 and olivopontocerebellar degeneration, Gilles de la Tourette Kennedy's disease, bulbar palsy, spinal and spinal bulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, Dongig-Hoffmann disease, Kugelberg-Welander disease, Sandhoff disease, familial spastic disorder Disease, spastic paraparesis, progressive multifocal leukoencephalopathy, familial autonomic neuropathy (Lee-Day syndrome) , prion diseases (including but not limited to Creutzfeldt-Jakob disease, Gerstmann- (including Straussler-Scheiker disease, rickets, and fatal familial insomnia), demyelinating diseases, and These include disorders including glaucoma and multiple sclerosis, as well as genetic disorders such as leukodystrophies, Not limited to these.

[0032] Fibroblasts for use in any of the methods of the present disclosure may be of any mammalian origin, including, but not limited to, In one embodiment of the present disclosure, the fibroblasts may be derived from a mammal (e.g., a human, a rat, a primate, a pig, etc.). The cells are derived from the human umbilicus. Umbilicus-derived cells are capable of self-renewal and proliferation in culture, and are comparable to other Umbilical cord tissue fibroblasts can be derived from human umbilical cord tissue. The cells are capable of self-renewal and proliferation in culture and are capable of expressing other proteins. The method comprises the steps of (a) obtaining human umbilical tissue; (b) obtaining human umbilical tissue; and (c) obtaining human umbilical tissue. (c) removing substantially all of the blood to obtain substantially blood-free umbilical tissue; (d) dissociating the tissue by chemical or enzymatic treatment, or both; and (e) resuspending the cells in a medium capable of self-renewal and proliferation in culture and other Providing growth conditions that allow the expansion of human umbilical cord-derived cells with the ability to differentiate into cells of this phenotype The tissue may be extracted from the vagina or other routes (e.g., surgically). Any completed pregnancy, birth, or delivery, whether delivered via elective Caesarean section or Obtaining tissue from a tissue bank is also possible in the methods of the present invention. It is considered to be within the range.

[0033] The tissue is rendered substantially free of blood flow by any means known in the art. For example, blood may be washed, rinsed, or removed before or after bulk blood removal by aspiration or drainage. The blood cells can be removed physically by dilution or the like. Other means may include enzymatic or chemical treatment. Dissociation of umbilical tissue involves mechanical disruption. This can be accomplished by any of a variety of techniques known in the art, for example, by removing tissue. can be cut aseptically with scissors, or can be cut using a scalpel, or Otherwise, such cells may be collected in any manner compatible with the recovery of intact or viable cells from human tissue. Such tissues can be minced, blended, ground, or homogenized.

[0034] In one embodiment, the isolation procedure also utilizes an enzymatic digestion process. Isolating individual cells from complex tissue matrices to facilitate their growth in culture As described above, it is known in the art that the method is useful for isolating cells from tissues. A wide range of digestive enzymes are available to those skilled in the art for use in isolation. , deoxyribonuclease and neutral protease, dispase) to strong digestive enzymes (e.g. Such enzymes range from phospholipids (e.g., papain and trypsin) to phospholipids (e.g., phospholipids), and are commercially available. A non-exhaustive list of enzymes suitable for this application includes mucolytic enzyme activity, metalloproteinase activity, and proteases, neutral proteases, serine proteases (trypsin, chymotrypsin, or enzymes) These include enzymes such as ribosomal enzymes (e.g., ribosomal enzymes), and deoxyribonucleases. an enzymatic activity selected from metalloprotease, neutral protease and mucolytic activity. For example, collagenase is known to be useful for isolating various cells from tissues. Deoxyribonuclease can digest single-stranded DNA and is used during isolation. It can minimize cell clumping. Enzymes can be used alone or in combination. Since serine proteases can degrade other enzymes, it is preferable to use other enzymes. Following the use of the enzyme, the temperature and time of contact with the serine protease are determined. Serine proteases are present in serum α2-microglobulin. The medium used for digestion can be serum-free. Nase is commonly used to improve yield or efficiency. Specific methods include , collagenase and dispase, or collagenase, dispase, and hyaluronidase Such methods are provided, and in certain preferred embodiments, include enzymatic treatment with hydroxylase. A mixture of collagenase and the neutral protease dispase is used in the dissociation step. The specific method is to identify a small number of strains of Clostridium histolyticum At least one collagenase, as well as protease activity, dispase and thermolysin Even more preferred are methods using digestion in the presence of collagenase. This method uses digestion with both collagenase and dispase enzyme activity. Also preferred are methods that include digestion with hyaluronidase activity in addition to dispase activity. Those skilled in the art will recognize that many such enzymatic treatments for isolating cells from various tissue sources are well known. For example, LIBERASE BLENDZYME (Roc The enzyme combinations of collagenase and neutral protease from the he series are very useful. Other sources of enzymes are known and may be used in the present method. Such enzymes can be obtained directly from their natural sources. Those skilled in the art can also isolate the cells of the present invention. Evaluate new or additional enzymes or enzyme combinations for their usefulness in The specific enzyme treatment may be performed for 0.5, 1, 1.5, or 2 hours or more. In another specific embodiment, the tissue is maintained at 37°C during the enzymatic treatment of the dissociation step. Diluting the digest also allows cells to become trapped within the viscous digest. This may improve cell yield.

[0035] The use of enzymatic activity is utilized in some embodiments, but is not limited to the methods provided herein. Such isolation methods are not required. Methods based solely on mechanical separation are, as described above, The cells can be successfully isolated from

[0036] The cells are resuspended after the tissue is dissociated in any culture medium as described herein above. The cells may be resuspended after a centrifugation step to separate the cells from tissue or other debris. Resuspension can involve mechanical methods of resuspension or simply the addition of culture medium to the cells.

[0037] Providing growth conditions includes culture medium, supplements, atmospheric conditions, and relative humidity of the cells. The specific temperature is 37°C, but the temperature can be adjusted to other culture conditions. The temperature may range from about 35°C to 39°C, depending on the conditions and the desired use of the cells or cultures.

[0038] Presently contemplated in some embodiments are supplemented blood samples provided with the growth medium. Provides cells that do not require exogenous growth factors except for those available in the supernatant. Also disclosed are methods for inducing umbilical cells that are capable of proliferation in the absence of specific growth factors. Also provided herein are methods similar to those described above, except that the cells The cells are finally resuspended and grown in a culture medium containing specific growth factors (for this reason, In this sense, the method requires the absence of a specific Selective for cells that can divide in the absence of growth factors. In some embodiments, cells are grown and cultured in a chemically defined growth medium without added serum. In such cases, the cells are able to grow and proliferate in a medium to support and maintain the cells. In certain embodiments, the cells may require specific growth factors that can be added to the serum-free medium. The factors added for proliferation in the culture medium are one of FGF, EGF, IGF, and PDGF. In some embodiments, two, three, or all four factors are present in a serum-free medium. In another embodiment, LIF is added to a chemically defined medium. It is added to serum-free media to support or improve growth.

[0039] Also, a method in which cells can expand in the presence of about 5% to about 20% oxygen in their atmosphere. A method for obtaining cells that require L-valine is also provided, comprising incubating the cells in the presence of L-valine. After the cells are obtained, they are tested for their L-valine requirement and then cultured in the presence of L This can be confirmed by growing the strain on a D-valine-containing medium lacking the -isomer.

[0040] Before reaching the senescent state, cells undergo at least 25, 30, 35, or 40 doublings. A method is provided that can 14 can double to reach more than 100 cells Preferably, at least about 10 14 , 10 15 , 10 16 , or 10 17 More than 10 cells in culture 3 from about 10 6 cell / cm 2 This is a method to derive cells that can double sufficiently to generate when seeded up to Preferably, these cell numbers are produced within 80, 70, or 60 days. isolated and expanded umbilical cord tissue fibroblasts and expressed CD10, CD13, CD44, and CD73 , CD90, CD141, PDGFr-α, or HLA-A, B, C. Additionally, the cells have one or more markers that are associated with CD31, CD34, CD45, C They do not produce one or more of D117, CD141, or HLA-DR, DP, DQ.

[0041] In some embodiments, fibroblasts are collected from donors and information about each donation is recorded. In certain embodiments, the recorded information includes the type of cells, their origin, at least one of the following: the number of tissues, the date of their collection, and the identity of the donor; In other particular embodiments, the recorded information includes various characterization algorithms. Examples include results obtained from HLA typing, the presence of specific markers, determination of specific SNP alleles and / or performing nucleated cell counts on stem cell units In some embodiments, the collected cells are subjected to at least one criterion. In some particular embodiments, they are selected by their type, their They are classified according to tissue of origin, their date of collection, and donor identity. Based on this, specific fibroblast populations can be utilized.

[0042] In some embodiments, the harvested fibroblasts are purified by filtration to render the cells viable and functional. In some particular embodiments, the fibroblasts are stored under conditions appropriate to maintain the integrity of the fibroblasts. The cells are stored under cryopreservation conditions. In another embodiment, the fibroblasts are for allogeneic use. In some embodiments, the stored stem cells are used in allogeneic transplants. In other embodiments, the stored fibroblasts are used in various applications, such as research and pharmaceutical applications. It is used for the establishment of cell lines with good viability and other desired characteristics for the purpose of

[0043] In some embodiments, fibroblasts stored in a repository such as a bank are arranged in units. According to these embodiments, each donation (each deposition of fibroblasts) to the bank is In some exemplary embodiments, the units are divided into units of In some cases, the fibroblasts contain a population of the same type collected from a single donor. In an exemplary embodiment, the units are fibroblasts expressing one or more specific markers. In some embodiments, the units are further determined by the number of nucleated cells present in the sample. Upon request, one or more units may be In some embodiments, a portion of the units may be allocated to a recipient in need. In some exemplary embodiments, the number of units to be allocated The number of nucleated cells in each unit depends on the medical condition being treated. In embodiments, the amount of fibroblasts available for allocation to an individual, or the number of units, may be determined by the number of donations made. Depends on the amount of

[0044] In some embodiments, the fibroblasts are subjected to further processing after their collection. In certain embodiments, the collected fibroblasts are cultured, expanded and / or purified. In a further specific embodiment, the collected fibroblasts may be cultured or expanded. In some embodiments, the therapeutic agent is a compound that is used to treat certain pathological conditions. To achieve this, an optimal combination of fibroblasts can be selected from a reservoir of cells.

[0045] According to another aspect, the present disclosure provides a method of fibroblast banking, the method comprising: It involves collecting multiple donations periodically from an individual over the life of the body. In embodiments, the method includes collecting fibroblasts from two or more sources. In some embodiments, the method includes collecting more than one type of fibroblast. Includes.

[0046] In some embodiments of the present disclosure, the donor cells are adapted to have enhanced therapeutic properties. It is adjusted accordingly.

[0047] In some embodiments of the present disclosure, the fibroblasts have enhanced neuromodulatory properties and The transfection is carried out by a virus that has protective properties. This can be achieved by the use of any type of vector, including viral or non-viral vectors. Examples of viral vectors include lentiviruses, adenoviruses, and retroviruses. or adeno-associated viral vectors. In one embodiment, a lentiviral vector The methods for performing lentiviral-mediated transfection are well known in the art. These are discussed in the following references [5-11]. A specific example of gene transfection is stem cell homing, particularly hematopoietic stem cells. Transfection of SDF-1 to promote homing

[12] in animal models GDNF to treat Parkinson's disease

[13] and promote remyelination in brain injury models HGF, which promotes cardiomyocyte remodeling, is one of the key factors in the pathological remodeling of the heart. Protection from cell death

[15] , induction of tumor cell apoptosis by TRAIL [16-19] , cardioprotection by PGE-1 synthase

[20] , migration promotion by NUR77

[21] , B DNF reduces ocular nerve damage associated with hypertension

[22] , and HIF-1α promotes bone formation.

[23] , and attenuation of pulmonary fibrosis by dominant-negative CCL2

[24] . Interferon-β suppresses immune responses

[25] , HLA-G enhances immune suppression

[26] , and hepatocytes hTERT

[27] , which induces differentiation along the cellular pathway, cytosine deaminase

[28] , and senescence OCT-4 [29,30] suppresses TGF expression and parasite activity, while BAMBI

[31] suppresses TGF-β expression and parasite activity. , HO-1 for radioprotection

[32] , and LIGHT for inducing antitumor activity

[33] . iR-126 promotes angiogenesis [34,35], and bcl-2 induces nucleus pulposus cell generation.

[36] , induction of neurogenesis by telomerase

[37] , and hematopoietic recovery by CXCR4.

[38] and unnecessary immune suppression

[39] , and the production of regenerative cytokines by wnt11. These include the promotion of growth

[40] and the suppression of cancer by the HGF antagonist NK4

[41] . do. [Example]

[0048] The following examples are included to demonstrate preferred embodiments of the invention. The techniques described are techniques discovered by the inventors to work well in the practice of the present invention. and therefore can be considered to constitute a preferred mode for practicing the invention. However, it should be understood by those skilled in the art that, having regard to the present disclosure, Therefore, the spirit and scope of the invention are not to be construed in any way as limiting the scope of the invention, as defined by the specific embodiments disclosed and in which similar results can be obtained. It will be understood that many variations are possible without departing from the scope of the invention.

[0049] Example 1 Stromal cell-derived factor 1 :sdf-1) Cells were incubated under normoxia for 2 hours to induce chemokine-dependent responses to the indicated chemokine (SDF-1). Chemotaxis was assessed (Figure 1). Migrated cells were collected from the lower transition chamber compartment and counted. . Transwell system (3mm hole diameter, Corning Costar, 3415 Cells were seeded at 2.5 × 106 / mL in the upper chamber of a 10% FBS RPMI 1640 medium alone or recombinant human CXCL12 (100 ng / mL) (Pepro tech,300-28A), or CCL19 (0.3 μg / mL), or CCL2 Medium supplemented with 1 (0.6 μg / mL) was added to the lower compartment. The cells were allowed to migrate under the conditions of 0.01% CO₂ at 37°C for 2 hours. The cells that migrated to the lower compartment were then circulated. Collected and counted. Example 2 Enhancement of fibroblast hepatocyte growth factor (HGF) production from fibroblasts Poly(I:C)

[0050] Fibroblasts were cultured as in Example 1 and treated with InvivoGen® (San D Cells were treated with control, low molecular weight, or high molecular weight poly(I:C) from Iego, CA. After 48 hours of culture, HGF levels were assessed using ELISA. The effect of HGF on stem cell proliferation was observed in both high-molecular-weight and low-molecular-weight poly(I:C) (Fig. 2). This is an example of a cytokine that mediates the therapeutic effect of cytotoxic T cells.

[0051] Thus, in some embodiments, fibroblasts are exposed to cytokines such as HGF. HGF was significantly increased after exposure to effective amounts of poly(I:C) compared to untreated fibroblasts. The therapeutic properties of HGF include the enhancement of liver regeneration. Promotes proliferation of renal tubular epithelial cells, promotes proliferation of keratinocytes, promotes angiogenesis, and promotes proliferation of cancer cells Growth inhibition, hematopoiesis promotion, accelerated recovery of kidney function after injury, promotion of keratinocyte proliferation, and promotion of angiogenesis Promotes angiogenesis, inhibits cancer cell proliferation, promotes hematopoiesis, activates B cells, and stimulates bronchial epithelium Promoting cell growth, activating type 2 pneumocytes, inhibiting epithelial cell apoptosis, and promoting lung healing Promotes pulmonary fibrosis, reduces lung fibrosis, promotes pancreatic regeneration, promotes nerve cell survival, promotes axon growth, and improves muscle health. Activation of stellate cells, promotion of intestinal epithelial cell reconstitution, promotion of recovery after myocardial infarction, suppression of cardiomyopathy, Suppression of autoimmune myocarditis, reduction of endothelial cell injury, reduction of graft-versus-host disease, and reduction of stroke Promotes recovery, inhibits neuronal cell death, increases cerebral hypoperfusion, and inhibits the progression of neurodegenerative diseases. Generate more oligodendrocytes, improve the effectiveness of pancreatic islet transplants, and reverse hearing loss , promotion of neuronal migration

[0116] , suppression of inflammatory bowel disease, attenuation of learning impairment caused by ischemia , enhancing synaptic plasticity, preventing blindness, promoting the production of interleukin-1 receptor antagonists, etc. Contains

[0052] (References) All publications mentioned in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications are specifically and entirely incorporated by reference as if each individual publication were incorporated by reference. are incorporated by reference herein to the same extent as if individually indicated.

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[0094] Although the present disclosure and its advantages have been described in detail, the scope of the present invention is defined by the appended claims. Various changes, substitutions, and alterations may be made herein without departing from the spirit and scope of the present design. It is understood that the scope of the present application is limited to the Any disclosures expressly incorporated herein by reference are limited to particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps. As those skilled in the art will readily understand from this disclosure, perform substantially the same function or achieve substantially the same result as the corresponding embodiment described in Any now existing or later developed process, machine, manufacture, composition of matter, or technique that accomplishes the Any stage, method, or step may be utilized in accordance with the present disclosure. The appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, or methods. or steps.

Claims

[Claim 1] 1. A method for enhancing one or more therapeutic properties of a fibroblast population, comprising treating the fibroblast population with an effective amount of exogenous RNA sufficient to enhance one or more therapeutic properties of the fibroblast population.

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