Administration of fibroblasts and derivatives thereof for treatment of type 2 diabetes

Abstract

To provide methods of increasing insulin sensitivity in an individual in need thereof.SOLUTION: The increase in insulin sensitivity may derive from individuals that have diabetes, aging, low grade inflammation, obesity, pregnancy, metabolic syndrome X, congenital abnormality or a combination thereof. In specific embodiments, the methods encompass providing to an individual an effective amount of fibroblast cells of certain kinds.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 867,976, filed on June 28, 2019, which is hereby incorporated by reference in its entirety. (Technical Field) The disclosed embodiments include at least the fields of cell biology, molecular biology, cell therapy, and medicine .

Background Art

[0002] Diabetes is a disease of high blood sugar. There are mainly two types of diabetes: type 1 diabetes and type 2 diabetes In type 1 diabetes, also known as insulin - dependent diabetes mellitus (IDDM) or juvenile diabetes, the patient's pancreas produces little or no insulin, which is thought to be in part the result of autoimmunity attaching to the insulin produced by pancreatic beta - cells. It is one of the most costly chronic diseases in childhood, a non - growing one. It is believed that over one million Americans have IDDM. Severe IDDM patients must perform multiple daily insulin injections or continuously infuse insulin through a pump and prick their finger to test blood sugar more than six times a day. Dietary therapy and oral hypoglycemic drugs are not effective, and only insulin treatment is effective. Ketoacidosis and acidosis due to the loss of insulin - secreting ability, and diabetic coma may occur if untreated. Since a large number of factors such as stress, hormones, growth, physical activity, medication, illness / infection, fatigue, etc. affect insulin utilization, strict monitoring of insulin administration is required. ​Even the programs viewed do not mimic the endogenous functions of the pancreas, resulting in a number of complications occurring.

[0003] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, is associated with impaired peripheral tissue response to insulin. NIDDM is thought to affect approximately 18.2 million people in the United States, and as a result of the obesity epidemic, substantially younger patients are beginning to be diagnosed with this disease. The economic burden of NIDDM is witnessed in statistics showing that, on average, the medical costs of NIDDM patients are high.

[0004] Insulin resistance exists in almost all obese individuals [1]. However, compensatory insulin production by β-cells usually occurs, preventing hyperglycemia. With long-term insulin resistance and in response to other factors, β-cell insulin production ultimately loses its ability to cope with increasing insulin requirements, resulting in postprandial hyperglycemia and characterizing the transition between normal and abnormal glucose tolerance. Subsequently, in the liver, glucose secretion begins via gluconeogenesis (production of glucose from substrates other than glycogen and not from sugar), and hyperglycemia is observed even in the fasting state. In contrast to IDDM, NIDDM has only a very slight degree of ketosis and acidosis even though insulin action is reduced from normal, and insulin therapy is not necessarily required.

[0005] The greatest clinical challenge in this disease is the prevention of long-term complications, many of which involve the vascular, ocular and renal systems. Various drugs are utilized to increase glucose sensitivity or stimulate insulin secretion, but these approaches are for postprandial insulin secretion ​ Since it does not accurately mimic physiological control, it is not optimal. Thus, glucose fluctuations and downstream metabolic consequences ultimately lead to macrovascular pathologies such as coronary atherosclerotic heart disease, as well as an increased risk of stroke, and microvascular pathologies such as macular degeneration and renal insufficiency. Furthermore, neuropathy associated with hyperglycemia is often observed.

[0006] There are numerous treatment options available for NIDDM, and these depend on not only the severity of the disease but also the patient's specific characteristics. The treatment goal in diabetes management is to bring plasma glucose levels close to normal levels, for example, 80 - 120 milligrams per deciliter (mg / dl) before meals and 100 - 140 mg / dl at night. Many medical tests for monitoring glucose, as well as cholesterol and lipid levels, are known in the art. The goal of maintaining normal blood glucose levels is determined in several ways by the ability to prevent secondary complications such as retinopathy, neuropathy, vascular disease, and stroke.

[0007] In the early stages of NIDDM, patients are treated with various oral medications, and as the diabetes progresses, various forms of insulin may be administered. Tight blood glucose control is known to reduce the rate of diabetic complications, but achieving such control is very difficult and, even when achieved, a significant morbidity and mortality inevitably result. Some of the non - insulin treatments for NIDDM are listed below.

[0008] The main oral medications for diabetes, based on their mechanism of action, are sulfonylureas that induce insulin secretion ​​Hypoglycemic agents such as honil urea and meglitinide, and biguanides and α-glucosidase inhibitors that cause glucose uptake Can be divided into two groups of antihyperglycemic agents.

[0009] Sulfonylurea drugs are a type of drug that stimulates insulin release from β-cells. Essentially These drugs act by blocking the ATP-sensitive potassium channels in the pancreatic β-cell membrane. This action is mediated by the drug binding to the sulfonylurea receptor (SUR) subunit of the channel. When the potassium channel is inhibited, depolarization of the cell membrane And insulin secretion occur, which occurs in a way as if glucose was added to the cell. Glyburide is a second-generation sulfonylurea compound sold under the names Micronase, DiaBeta, or Glynas e. Glypidide, sold under the names Glucotrol and Glucotrol XL, is also a second-generation sulfonyl Urea drug. The third-generation sulfonylurea drug includes glimepiride (Amaryl). This Agent is considered to be safer in patients with ischemic heart disease compared to other sulfonylurea drugs. Glimepiride is the only sulfonylurea drug approved for combination use with insulin or metformin. Generally, sulfonylurea drugs Are troubled by the drawback that the induced insulin secretion amount depends on the timing and dose of drug administration, rather than on blood glucose levels. This causes not only various fluctuations in blood glucose levels, but also some patients who experience digestive disorders Such as loss of appetite. Point.

[0010] Meglitinide drugs (commonly known as glinide drugs) are short-acting insulin secretagogues It is a type of medicine that is administered after meals. Sulfonylurea drugs have a similar mechanism of inducing insulin secretion by blocking ATP-dependent potassium channels, but glinides are thought to have a shorter-term activity. Theoretically, these drugs induce less risk of hypoglycemia and cause an insulin release pattern similar to that of physiology. Repaglinide, sold under the name Prandin, and Nateglinide, sold under the name Starlix, are examples of the two glinides. Compared with sulfonylurea drugs, glinide drugs have been shown to have good control of postprandial hyperglycemia, do not induce hypoglycemia, and generally have a good safety profile, especially in patients with renal insufficiency [2].

[0011] Biguanide drugs are a type of medicine that reduces glucose production in the liver and increases insulin sensitivity. Metformin, sold under the names Glucophage, Glucophage XR, and Metformin XR, is an example of a biguanide drug. It is also the most widely prescribed oral diabetes treatment drug in the world and is usually the first choice for the initial treatment of obese patients with typical type 2 DM accompanied by mild to moderate hyperglycemia [3]. Metformin administration is associated with weight loss and improvement of lipid profile. Metformin is effective as monotherapy and may be able to reduce the need for insulin treatment when combined with both insulin secretagogues and thiazolidinedione (TZD) [4]. Metformin is known to induce an increase in glucose utilization and a decrease in leptin concentration [5]. Furthermore, metformin induces inhibition of dipeptidyl peptidase-IV activity and prolongs the half-life of GLP-1 ​​​​​​​​​​​​​​​​Extend [6]. Classical mechanisms of action include increased glucose utilization by anaerobic glycolysis, inhibition of gluconeogenesis in the liver, suppression of intestinal glucose absorption, etc. One adverse effect associated with various biguanide drugs is lactic acidosis.

[0012] Thiazolidinediones (glitazones) are a family of drugs that lower insulin resistance in both muscle and adipose tissue. They do not induce insulin secretion. Rosiglitazone, sold under the name Avandia, and Pioglitazone, sold under the name Actos, are two thiazolidinediones. These agents induce insulin sensitivity through activation of the insulin receptor kinase, thereby promoting glucose uptake by peripheral tissues and improving increased glucose production in the liver. Known side effects include gastrointestinal symptoms, edema, hematological changes, upregulation of plasma LDH, etc. Glitazones are interesting not only for their ability to increase insulin signaling but also for their anti-inflammatory effects. For example, rosiglitazone is known to suppress the interleukin-12 secreting ability of dendritic cells after stimulation via CD40 [7]. This is thought to occur through activation of the PPAR-γ pathway. Furthermore, treatment with rosiglitazone can suppress the development of colitis in animal models by preferentially inducing Th2 cytokine production [8].

[0013] α-Glucosidase inhibitors are used to slow the rate of sugar absorption. Acarbose, sold under the name Precose, and Miglitol, sold under the name Glyset, are two examples of drugs in this family.

[0014] Incretin mimetics reflect glucose-dependent insulin secretion and inhibit glucagon secretion. Exenatide is sold under the name Byetta. It is a glucagon-like peptide-1 (GLP-1) receptor agonist that activates β cells. Controlled clinical trials have shown that exenatide at 5-10 micrograms stimulates insulin secretion. When administered twice daily, glycemic control was comparable to that of conventional insulin therapy. Evidence was obtained showing this.

[0015] Currently available treatments for NIDDM involve inhibition of endogenous insulin secretion and insulin utilization responses. Therefore, the use of cell therapy to generate synthetic islets is currently being investigated. Various approaches have been pursued to address this issue. These approaches include GLP and Exam Ability of insulin-4 to induce differentiation of cells into insulin-producing or amylase-producing cells This patent includes U.S. Patent No. 7,056,734, which discloses a non-insulin GL to generate either insulin-producing cells or amylase-producing cells into insulin-producing cells. Use of P-1 or related molecules, as well as non-insulin-producing or amylase-producing cells The present invention includes the use of exendin-4 to make insulin-producing cells either:

[0016] U.S. Patent No. 6,903,073 describes a method for the administration of Hedgehog agonists to increase insulin production. This is because inhibiting Hedgehog signaling stimulates insulin expression. and transfection with hedgehog increased insulin production. This is based on the finding that [9]

[0017] U.S. Patent No. 6,967,019 conceptually discloses a method for expressing insulin in gastrointestinal organ cells and pancreatic cells in vitro for in vivo introduction. The patent essentially teaches that introducing the neuroendocrine class B basic helix-loop-helix (bHLH) transcription factor gene or the neurogenin3 (Ngn3) gene into gastrointestinal organ cells or pancreatic cells respectively results in the ability to produce insulin. Unfortunately, no evidence of glucose regulation was provided.

[0018] U.S. Patent No. 7,033,831 shows a method for generating insulin-producing cells from human embryonic stem cells through a process of first incubating the human embryonic stem cells with activin A and then incubating the cells with nicotinamide. Activin is a peptide involved in wound healing and morphogenesis, while nicotinamide is a form of vitamin B3 that improves the function of β cells. This patent includes culturing ES cells first in activin A and then in nicotinamide as a method for generating insulin-producing cells. Also included is a method for producing insulin-secreting cells that first grows embryoid bodies and then treats the embryoid bodies with one or more mitogens (to stimulate proliferation) using a TGF-β antagonist, followed by culturing the cells in nicotinamide. Further covered is the use of embryonic stem cells as a starting tissue for the generation of insulin-producing cells, rather than the use of embryoid bodies.

[0019] U.S. Patent No. 7,169,608 involves initial culturing in low-concentration glucose (at least ​​​​​​​​​​​​​​​a simple two-step culture approach that includes a first culture (for 3 days) and subsequent culture in high-concentration glucose (for at least 7 days) to induce the differentiation of bone marrow into pancreatic islets. A simple method for inducing the differentiation of bone marrow into pancreatic islets is described by this two-step culture approach. According to this patent, the obtained cells produce insulin in response to glucose and can prevent diabetes when administered in vivo to animals. This patent is interesting because the authors actually published some of the data from the patent

[10] . Notable about the published data is that when bone marrow-derived cells are administered in vivo, they appear to take on a structure similar to that observed in normal pancreatic islets. The transplanted cells produce insulin (I and II), glucagon, somatostatin, pancreatic polypeptide, and C-peptide. Furthermore, various animal models of diabetes were cured by the administration of bone marrow cells manipulated according to the present invention.

[0020] U.S. Patent No. 7,138,275 provides a program for dedifferentiation that confers stem cell-like capabilities to monocytes by culturing peripheral blood monocytes in the presence of IL-3 and M-CSF for about 6 days. The patent demonstrates that these monocytes can be converted into pancreatic islets and continues to show efficacy in a streptozotocin-induced diabetic mouse model of diabetes.

[0021] Regarding the above patents, although some generation of insulin-producing cells has been reported in vitro, it is clear that in some cases this therapeutic application is limited in vivo. In NIDDM, the high insulin requirement necessary to overcome insulin resistance places great stress on β cells. This "need" for high insulin production is related to hyperglycemia. Similar to other associated factors, Fas, ATP-sensitive K+ channels, insulin receptor substrate 2 , mechanisms such as oxidative stress, nuclear factor-κB, endoplasmic reticulum stress, mitochondrial dysfunction, etc. often accelerate β-cell apoptosis via

[11] . Therefore, even if an appropriate beta cell source can be generated as described in the above patent, due to the underlying causative factors that originally initiated diabetes onset, the possibility of bringing about long-term beneficial clinical outcomes is low. As described above, even if an appropriate beta cell source can be generated, due to the underlying causative factors that originally initiated diabetes onset, the possibility of bringing about long-term beneficial clinical outcomes is low.

[0022] This disclosure provides solutions to a long-felt need in the art for the treatment and prevention of type 2 diabetes.

Summary of the Invention

[0023] Embodiments of this disclosure relate to the field of metabolic diseases and their treatment or prevention. Certain embodiments provide methods by which this disclosure treats insulin resistance and provides an environment suitable for restoring insulin-producing cell function. In certain embodiments, this disclosure includes methods of treating and / or preventing insulin resistance using cell therapy, including, at least in certain cases, fibroblast therapy, and in some embodiments, combinations with one or more various pharmacological and medical interventions. Methods of treating type 2 diabetes include those described herein, including reducing its severity and / or delaying its onset. The methods also include those related to restoring insulin-producing cell function. Embodiments of this disclosure relate to methods of preventing, delaying, or reducing the severity of one or more complications from type 2 diabetes. The methods included herein also include methods of increasing insulin sensitivity, maintaining blood glucose at normal levels (50 ~110 mg / dL), increasing skeletal muscle perfusion, insulin responsiveness ~110 mg / dL), increasing skeletal muscle perfusion, insulin responsiveness Methods of conferring, methods of reducing inflammatory mediators, etc. are included. At least several In some embodiments, the methods and compositions utilized herein are not type 1 diabetes.

[0024] Embodiments of the present disclosure include methods of treating or preventing insulin resistance in an individual that include delivering a therapeutically effective amount of fibroblasts to the individual. The fibroblasts may be CD10 5+, CD34+, CD133+, or a mixture thereof. The fibroblasts may be CD90+, CD45 and / or CD14-. In certain embodiments, the fibroblasts have regenerative activity. In some cases, the fibroblasts are exposed to erythropoietin, prolactin , human chorionic gonadotropin, gastrin, EGF, FGF, and / or VEGF, and in some cases as a result, the fibroblasts have regenerative activity. In particular, insulin resistance is the result of diabetes, aging, low-grade inflammation, obesity, pregnancy, metabolic syndrome X , congenital abnormalities, or a combination thereof. The fibroblasts utilized herein are , umbilical cord blood, peripheral blood, menstrual blood, placental matrix, endometrium, umbilical cord blood, deciduous teeth, muscle tissue, placenta, skin , bone marrow, amniotic fluid, adipose, umbilical cord matrix, omentum, subintestinal mucosa, or a mixture thereof. The fibroblasts can be induced from, umbilical cord blood, peripheral blood, menstrual blood, placental matrix, endometrium, umbilical cord blood, deciduous teeth, muscle tissue, placenta, skin . The fibroblasts, when cultured in a 96-well plate in 10% fetal bovine serum in DMEM medium at a concentration of 20,000 cells per well, can have the ability to proliferate at a rate greater than 2-fold per 24 hours. The fibroblasts can be delivered systemically or locally to the individual. The fibroblasts can be delivered intramuscularly to the individual, and / or the fibroblasts can be delivered to the pancreas or near the pancreas to the individual. In some cases, the method is a therapeutically effective amount of 1 to the individual to the individual. In some cases, the method is a therapeutically effective amount of 1 further comprising the step of providing one or more anti-inflammatory agents, and / or the method treats an individual may further comprise the step of providing a therapeutically effective amount of any one or more types of diabetes treatment. The above will be better understood from the following detailed description of the features and technical advantages of the present disclosure has been outlined rather broadly. Additional features and advantages that form the subject matter of the claims in this specification will be described below. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments can be readily utilized as a basis for modifying or designing other structures to accomplish the same purposes of this design. Also, it should be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope set forth in the appended claims. The novel features that are considered to be the features of the design disclosed in this specification, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. However, it should be clearly understood that each drawing is provided for purposes of illustration and description only and is not intended as a definition of the limits of the present disclosure. It should be understood that the drawings are not to scale. The following description, when considered in conjunction with the accompanying drawings, will be better understood with respect to both the operation of the configuration and the method. However, it should be clearly understood that each drawing is provided for purposes of illustration and description only and is not intended as a definition of the limits of the present disclosure. It should be understood that the drawings are not to scale.

Brief Description of the Drawings

[0025]

Figure 1

Modes for Carrying Out the Invention

[0026] I. Definition Examples As used herein, "a" or "an" can mean one or more. As used in the claims as used herein, when used in conjunction with the term "comprising", the term "a" or "An" can mean one or more than one. As used herein, "another" can mean at least two or more. In certain embodiments, aspects of the present disclosure can "consist essentially of" or "consist of" one or more sequences of the present invention, for example. Some embodiments may comprise, or consist essentially of, one or more elements, method steps, and / or methods of the present invention. Any method or composition described herein is intended to be usable with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. As used herein, the terms "or" and "and / or" are utilized to describe combinations of multiple components or to describe them exclusively of one another. For example, "x, y, and / or z" can mean "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z", and specifically, it is contemplated that x, y, or z can be specifically excluded from an embodiment. Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" mean the inclusion of the stated step or element, or group of steps or elements, but do not mean the exclusion of any other step or element, or group of steps or elements. It is understood that any method or composition described herein is intended to be usable with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. As used herein, the terms "or" and "and / or" are utilized to describe combinations of multiple components or to describe them exclusively of one another. For example, "x, y, and / or z" can mean "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z", and specifically, it is contemplated that x, y, or z can be specifically excluded from an embodiment. Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" mean the inclusion of the stated step or element, or group of steps or elements, but do not mean the exclusion of any other step or element, or group of steps or elements. It is understood that any method or composition described herein is intended to be usable with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein.

[0027] As used herein, the terms "or" and "and / or" are utilized to describe combinations of multiple components or to describe them exclusively of one another. For example, "x, y, and / or z" can mean "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z", and specifically, it is contemplated that x, y, or z can be specifically excluded from an embodiment. For example, "x, y, and / or z" can mean "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z", and specifically, it is contemplated that x, y, or z can be specifically excluded from an embodiment. / or z" can mean "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y ) or z", "x or (y and z)" or "x or y or z", and specifically, it is contemplated that x, y, or z can be specifically excluded from an embodiment. It is contemplated that x, y, or z can be specifically excluded from an embodiment.

[0028] Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" mean the inclusion of the stated step or element, or group of steps or elements, but do not mean the exclusion of any other step or element, or group of steps or elements. e)", "comprises", and "comprising" mean the inclusion of the stated step or element, or group of steps or elements, but do not mean the exclusion of any other step or element, or group of steps or elements. It is understood that any method or composition described herein is intended to be usable with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. It is understood that any method or composition described herein is intended to be usable with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. It would be. "Consisting of" means including and not limited to what follows the phrase "consisting of". Therefore, the term "consisting of" indicates that the recited elements are essential or essential, and that no other elements are present. "Consisting essentially of" means including any element enumerated after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the enumerated elements. Therefore, the term "consisting essentially of" indicates that the recited elements are necessary or essential, but other elements are not optional and may or may not be present depending on whether they affect the activity or action of the recited elements. Throughout this specification, references to "one embodiment", "an embodiment", "a particular

[0029] embodiment", "related embodiments", "a particular embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the foregoing phrases at various places throughout this specification do not necessarily all refer to the same embodiment. Further, special features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0030] The terms "administered" or "administering", as used herein, refer to any method of providing a composition to an individual such that the composition has the intended effect on the For example, one method of administration is by an indirect mechanism using medical devices such as catheters, applicator guns, syringes, etc., but is not limited thereto. A second exemplary method of administration is by a direct mechanism, such as, for example, local tissue administration, oral ingestion, transdermal patch, topical, inhalation, suppository, etc.

[0031] As used herein, the term "allogeneic" refers to cells of the same species that are genetically different from the host's cells.

[0032] As used herein, the term "autologous" refers to cells derived from the same subject. As used herein, the term "engraftment" refers to the process of incorporating stem cells into a tissue of interest in vivo through contact with the existing cells of the tissue.

[0033] As used herein, the terms "about" or "approximately" refer to an amount, level, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, number, frequency, percentage, dimension, size, quantity, weight, or length. In certain embodiments, when the term "about" or "approximately" precedes a numerical value, it indicates a value that plus or minus in the range of 15%, 10%, 5%, or 1%. With respect to a biological system or process, this term can mean within an order of magnitude of the value, preferably within 5-fold, more preferably within 2-fold. Unless otherwise specified, the term "about" means within an acceptable error range of a particular numerical value.

[0034] As used herein, the term "activated fibroblast" refers to one or more changes in a cell: metabolism, immunology, growth factor secretion, surface marker expression, and / or production of microvesicles, induced ​​​​​​​​​​ Refers to fibroblasts treated with one or more agents and / or stimuli that can direct. Examples of agents include epidermal growth factor (EGF; (Peprotech)), transforming growth factor-α (TGF- α; Peprotech), basic fibroblast growth factor (bFGF; Peprotech ), brain-derived neurotrophic factor (BDNF; R&D Systems), and keratinocyte growth factor (KGF; Peprotech). EGF is a potent mitogenic factor for various cultured ectodermal and mesodermal cells, and has a significant impact on the differentiation of specific cells in vivo and in vitro, as well as on some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule and is cleaved by proteolysis to generate a 53-amino acid peptide hormone that stimulates cells . The preferred mitogenic growth factor is EGF. EGF is preferably added to the basal culture medium at a concentration of 5 - 500 ng / ml or at least 5 - 500 ng / ml . Preferred concentrations are at least 10, 20, 25, 30, 40, 45, or 50 n g / ml and 500, 450, 400, 350, 300, 250, 200, 150, or 100 ng / ml or less. More preferred concentrations are at least 50 ng / ml and 100 ng / ml or less. Even more preferred concentrations are about 50 ng / ml or 5 0 ng / ml. The same concentrations can be used for FGF, preferably FGF10 or FGF7. When two or more FGFs (e.g., FGF7 and FGF10) are used , the concentration of FGF is as defined above and refers to the total concentration of FGF used. During the culture of stem cells , the mitogenic growth factor is preferably added to the culture medium every 2 days, while the culture ​The culture medium is preferably refreshed every 4 days. Any member of the bFGF family can be used. In some cases, FGF7 and / or FGF10 are used. FGF 7 is also known as KGF (keratinocyte growth factor).

[0035] A "cell culture" is an artificial in vitro system containing viable cells, whether quiescent, senescent, or (actively) dividing. In cell culture, cells are grown and maintained at an appropriate temperature, typically 37°C, typically in an atmosphere containing oxygen and CO The culture 2 conditions can vary widely for each cell type, but variations in conditions for a particular cell type can result in different phenotypes being observed. The factor that most commonly varies in a culture system is the growth medium which can vary in the concentration of one or more of nutrients, growth factors, and other components. Growth factors used to supplement the medium often originate from animal blood such as bovine serum The most commonly varying factor in a culture system is the growth medium which can vary in the concentration of one or more of nutrients, growth factors, and other components. Growth factors used to supplement the medium often originate from animal blood such as bovine serum The growth factors used to supplement the medium often originate from animal blood such as bovine serum derived from animal blood.

[0036] As used herein, the term "conditioned medium of fibroblast regenerative cells" refers to the liquid medium in contact with cells, where the cells produce one or more factors that enter the medium and thus impart at least one therapeutic activity on the medium As used herein, the term "conditioned medium of fibroblast regenerative cells" refers to the liquid medium in contact with cells, where the cells produce one or more factors that enter the medium and thus impart at least one therapeutic activity on the medium and thus confer at least one therapeutic activity on the medium.

[0037] As used herein, the term "fibroblast derivative" refers to a dedifferentiated fibroblast, or an apoptotic body, or an exosome derived from a fibroblast As used herein, the term "fibroblast derivative" refers to a dedifferentiated fibroblast, or an apoptotic body, or an exosome derived from a fibroblast

[0038] As used herein, the term "individual" refers to a human or animal who may or may not be housed in a medical facility and may be treated as an outpatient in a medical facility As used herein, the term "individual" refers to a human or animal who may or may not be housed in a medical facility and may be treated as an outpatient in a medical facility receive, via the Internet and / or otherwise, one or more pharmaceutical compositions, or not. An individual can be of any age of a human or non-human animal, and thus includes both adults and infants (i.e., children) as well as neonates. The term "individual" is not intended to imply the necessity of medical treatment, and thus an individual may be part of an experiment either spontaneously or non-spontaneously, whether clinical or in support of basic science research. The terms "subject" or "individual" refer to any organism or animal subject to which the methods and / or materials are directed, including mammals such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), domestic animals (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The term "pharmaceutically" or "pharmacologically acceptable" as used herein refers to entities and compositions that do not produce harmful, allergic, or other untoward reactions when administered to an animal or a human.

[0039] The term "pharmaceutically acceptable carrier" as used herein includes, but is not limited to, water, ethanol, propylene glycol (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercially available detergents, etc., and any and all solvents or dispersion media. Supplementary bioactive ingredients can also be incorporated into such carriers.

[0040]

[0041] ​​​​​​The term "prevent" or "preventing" refers to a method of preventing the occurrence of the medical condition or onset of at least one of its symptoms.

[0042] As used herein, the terms "subject" or "individual" refer to a human or animal who may or may not be housed in a medical facility and may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the Internet. An individual may be of any age, human or non-human, and thus includes adults and infants (i.e., children) as well as newborns. The term "individual" is not intended to imply a need for medical treatment and thus an individual may be a subject for clinical or basic science research, either voluntarily or involuntarily. The terms "subject" or "individual" refer to any living or animal subject that is the subject of a method or material, including mammals such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0043] As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective amount," and / or "effective amount" and refers to the amount of a compound that elicits a biological, cosmetic, or clinical response required by a person of ordinary skill in the art in an individual in need thereof. By way of example, an effective amount is an amount sufficient to promote the formation of new blood vessels and associated vasculature (angiogenesis) and / or an amount sufficient to promote the repair or remodeling of existing blood vessels and associated vasculature. ​​​​​​is a quantity. The appropriate effective amount to be administered for a particular application of the disclosed method can be determined by one of ordinary skill in the art using the guidance provided in this specification. For example, the effective amount can be extrapolated from in vitro and in vivo assays as described herein. One of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of the compounds or compositions disclosed in this specification can be adjusted accordingly .

[0044] "Treatment", "treating", or "treat" means a procedure for reducing the effects of a disease or condition . Treatment can refer to a method of reducing not only the symptoms but also the disease or condition itself . Treatment can be any decrease from pre-treatment levels and cannot be limited to a complete ablation of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" includes a reduction in the severity of at least one symptom of a disease, a 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or disease progression. For example, a disclosed method for reducing the immunogenicity of cells is a treatment where there is a detectable reduction in the immunogenicity of the cells when compared to pre-treatment levels in the same subject or a control subject . Thus, the reduction can be a 10, 20, 3 0, 40, 50, 60, 70, 80, 90, 100%, or any amount in between reduction compared to natural or control levels . "Treatment" is understood and contemplated herein not necessarily to refer to a cure of a disease or condition but rather to an improvement in the outlook of a disease or condition. In certain embodiments, treatment ​​​​​​​​refers to a decrease in the severity or extent of at least one symptom, alternatively or additionally, It can also refer to a delayed onset of one symptom.

[0045] II. Method of manufacture and use Disclosed are methods for increasing insulin sensitivity and / or for treating a disease in need thereof. Methods, compositions, and cells useful for correcting deficiencies in insulin production in mice. One embodiment of the present disclosure is a method for producing fibroblasts and / or fibroblast-like cells and / or microvesicles thereof. Administration of the compound may stimulate angiogenesis and / or vascular responsiveness directly and / or indirectly. Another embodiment includes a method of increasing skeletal muscle perfusion through administration of host insulin. Insulin sensitivity can be improved by administration of a cell composition capable of being integrated into insulin-responsive tissue. Increases the activity of insulin, recruits host cells that can respond to insulin, and induces insulin resistance on other host cells. Recruitment of host cells that can confer insulin responsiveness, playing a role in insulin responsiveness The exogenously administered cells and / or the exogenously administered cells that endogenously induce insulin responsiveness on other host cells. In another embodiment, the present invention provides a means for upregulating responsiveness via either the To allow insulin sensitization and upregulated production of insulin during Included herein are methods for treating type 2 diabetes. The method also includes administering insulin to the patient, which may include reducing the severity and / or delaying the onset of the condition. The embodiments of the present disclosure relate to the recovery of cell function from type 2 diabetes. The present invention relates to a method for preventing, delaying, or reducing the severity of the above complications. The methods also include methods of increasing insulin sensitivity, maintaining blood glucose at normal levels, and Methods for increasing skeletal muscle perfusion, methods for conferring insulin responsiveness, methods for reducing inflammatory mediators, etc. are included. In at least some embodiments, the methods and compositions utilized herein are not type 1 diabetes. In at least some embodiments, the methods and compositions utilized herein are not type 1 diabetes. In at least some embodiments, the methods and compositions utilized herein are not type 1 diabetes.

[0046] Embodiments of the present disclosure include methods for increasing insulin sensitivity in a mammal by administering a therapeutically effective amount of a fibroblast population and / or fibroblast derivatives. In certain embodiments, the fibroblasts express the markers CD34 and / or CD133. Fibroblasts for any of the methods herein can be derived from any source, but in certain embodiments, the fibroblast population is derived from a group of tissues selected from the group consisting of cord blood, placenta, skin, bone marrow, amniotic fluid, adipose, umbilical cord matrix, omentum, and submucosa of the intestine; fibroblast derivatives can also be derived therefrom. Fibroblasts for any of the methods herein can be derived from any source, but in certain embodiments, the fibroblast population is derived from a group of tissues selected from the group consisting of cord blood, placenta, skin, bone marrow, amniotic fluid, adipose, umbilical cord matrix, omentum, and submucosa of the intestine; fibroblast derivatives can also be derived therefrom. In certain embodiments, the fibroblast population is derived from a group of tissues selected from the group consisting of cord blood, placenta, skin, bone marrow, amniotic fluid, adipose, umbilical cord matrix, omentum, and submucosa of the intestine; fibroblast derivatives can also be derived therefrom. In certain embodiments, the fibroblast population is derived from a group of tissues selected from the group consisting of cord blood, placenta, skin, bone marrow, amniotic fluid, adipose, umbilical cord matrix, omentum, and submucosa of the intestine; fibroblast derivatives can also be derived therefrom.

[0047] In certain cases, the fibroblast population has the ability to proliferate at a rate greater than two-fold per 24 hours when cultured at a concentration of 20,000 cells per well in a 96-well plate in DMEM medium containing 10% fetal bovine serum. In certain cases, the fibroblast population has the ability to proliferate at a rate greater than two-fold per 24 hours when cultured at a concentration of 20,000 cells per well in a 96-well plate in DMEM medium containing 10% fetal bovine serum. In certain cases, the fibroblast population has the ability to proliferate at a rate greater than two-fold per 24 hours when cultured at a concentration of 20,000 cells per well in a 96-well plate in DMEM medium containing 10% fetal bovine serum.

[0048] In certain embodiments, the fibroblast population capable of increasing insulin sensitivity has the ability to enhance skeletal muscle perfusion. In certain embodiments, the fibroblast population can increase skeletal muscle perfusion. In certain embodiments, the fibroblast population capable of increasing insulin sensitivity has the ability to enhance skeletal muscle perfusion. In certain embodiments, the fibroblast population can increase skeletal muscle perfusion. In certain embodiments, the fibroblast population capable of increasing insulin sensitivity has the ability to enhance skeletal muscle perfusion. In certain embodiments, the fibroblast population can increase skeletal muscle perfusion.

[0049] Cells capable of increasing insulin sensitivity are autologous or allogeneic fibroblasts that express the markers CD90, CD105, CD34, CD133, or a combination thereof. Cells capable of increasing insulin sensitivity are autologous or allogeneic fibroblasts that express the markers CD90, CD105, CD34, CD133, or a combination thereof. It may also be substantially lacking in CD45 and / or CD14 expression. In particular in certain cases, the cells have an adhesive phenotype and are derived from a source selected from the group consisting of a) bone marrow, b) peripheral blood, c) endometrium, d ) menstrual blood, e) cord blood, f) deciduous teeth, g) amnion, h) placental stroma, i) muscle tissue, and j) skin .

[0050] When providing fibroblasts to an individual, the fibroblasts can be administered, for example, intramuscularly . In certain embodiments, the fibroblast population is administered systemically or locally and, in certain cases, is administered in proximity to the pancreas.

[0051] In any method herein that requires fibroblasts and / or derivatives thereof, the individual may also receive one or more additional therapeutic agents (e.g., one or more anti-inflammatory agents).

[0052] Embodiments of the present disclosure include methods of increasing insulin sensitivity in a mammal, in at least some cases, through inhibition of one or more inflammatory processes, by administration of a cell population that includes anti-inflammatory activity.

[0053] With regard to methods of treating insulin resistance, insulin resistance can be caused by a number of factors including diabetes, aging, mild inflammation, obesity, pregnancy, metabolic syndrome X, and congenital abnormalities. In one aspect, the disclosure provides cells that can stimulate perfusion of skeletal muscle, which are administered systemically or locally to the skeletal muscle for the purpose of increasing blood flow and can be obtained.

[0054] In one aspect of the present disclosure, placental fibroblasts are utilized, which are commercially available be obtained or isolated from placental tissue and administered for the purpose of increasing skeletal muscle perfusion Placental fibroblasts can be identified based on the expression of one or more antigens selected from the group consisting of Oct-4, Rex-1, CD9, CD13, CD29, C D44, CD166, CD90, CD105, SH-3, SH-4, TRA-1-60, TRA-1-81, SSEA-4, Sox-2, and combinations thereof.

[0055] In another aspect of the disclosure, bone marrow fibroblasts are utilized and can be obtained commercially or isolated from bone marrow and administered for the purpose of increasing skeletal muscle perfusion. Bone marrow fibroblasts can be generated from bone marrow-derived mononuclear cells, including a population capable of differentiating into one or more cell types of endothelial cells, smooth muscle cells, and neuronal cells. In one embodiment, bone marrow fibroblasts can be selected based on the expression of one or more of the following antigens: CD34, c-kit, flk-1, Stro-1, CD1 05, CD73, CD31, CD56, CD146, vascular endothelial-cadherin, CD133 CXCR-4, and combinations thereof. Additionally, insulin sensitivity conferring activity can be enhanced by selecting cells that express the marker CD133. 05, CD73, CD31, CD56, CD146, vascular endothelial-cadherin, CD133 CXCR-4, and combinations thereof. Additionally, insulin sensitivity conferring activity can be enhanced by selecting cells that express the marker CD133. Furthermore, insulin sensitivity conferring activity can be enhanced by selecting cells that express the marker CD133. by selecting cells that express the marker CD133.

[0056] In another aspect of the disclosure, amniotic fluid fibroblasts can be utilized, for example, obtained commercially or isolated from amniotic fluid and used to stimulate skeletal muscle perfusion and / or enhance insulin sensitivity. Isolation can be achieved by purifying mononuclear cells and / or c-kit expressing cells from amniotic fluid, and the fluid can be extracted by means known to those skilled in the art, including the use of ultrasound guidance. Amniotic fluid fibroblasts are characterized by the expression of the following antigens: SSEA3, SSEA4, Tra-1 can be obtained commercially or isolated from amniotic fluid and used to stimulate skeletal muscle perfusion and / or enhance insulin sensitivity. Isolation can be achieved by purifying mononuclear cells and / or c-kit expressing cells from amniotic fluid, and the fluid can be extracted by means known to those skilled in the art, including the use of ultrasound guidance. Amniotic fluid fibroblasts are characterized by the expression of the following antigens: SSEA3, SSEA4, Tra-1 can be achieved by purifying mononuclear cells and / or c-kit expressing cells from amniotic fluid, and the fluid can be extracted by means known to those skilled in the art, including the use of ultrasound guidance. Amniotic fluid fibroblasts are characterized by the expression of the following antigens: SSEA3, SSEA4, Tra-1 can be achieved by purifying mononuclear cells and / or c-kit expressing cells from amniotic fluid, and the fluid can be extracted by means known to those skilled in the art, including the use of ultrasound guidance. Amniotic fluid fibroblasts are characterized by the expression of the following antigens: SSEA3, SSEA4, Tra-1 60, Tra-1-81, CD9, CD29, CD44, CD166, CD105, CD90, CD13, CD146, CD133, CXCR4, and combinations thereof. -60, Tra-1-81, Tra-2-54, HLA class I, CD13, CD44, one or more of CD49b, CD105, Oct-4, Rex-1, DAZL, Runx-1 expression, or combinations thereof, and / or the following antigens: CD34, CD45, HLA class II, or the absence of significant expression of one or more of their combinations.

[0057] In another aspect of the present disclosure, circulating peripheral blood fibroblasts are used for insulin sensitivity stimulation. Peripheral blood fibroblasts can be characterized by the ability to proliferate in vitro for a period of time, such as 1, 2, 3, 4, 5, 6, or more months, and / or characterized by the expression of CD34, CXCR4, CD117, CD113, c-met, or combinations thereof, and / or characterized by the absence of one or more differentiation-related markers. The markers can be selected from one or more of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD1 4, CD16, CD19, CD24, CD33, CD36, CD38, CD45, CD5 6, CD64, CD68, CD86, CD66b, HLA-DR, or combinations thereof.

[0058] In another aspect of the present disclosure, tissue fibroblasts are used for stimulating skeletal muscle perfusion. Tissue fibroblasts have the following markers: STRO-1, CD105, CD54, CD1 06, HLA-I marker, vimentin, ASMA, collagen-1, fibronectin LFA-3, ICAM-1, PECAM-1, P-selectin, L-selectin, CD 49b / CD29, CD49c / CD29, CD49d / CD29, CD61, CD18 , CD29, thrombomodulin, telomerase, CD10, CD13, STRO-2, express one or more of VCAM-1, CD146, THY-1, or combinations thereof and tissue fibroblasts may or may not express detectable levels of HLA-DR, CD117, CD45, or combinations thereof. Fibroblasts may be derived from bone marrow, adipose tissue, endometrium, menstrual blood, umbilical cord blood, placental tissue, peripheral blood mononuclear cells, differentiated embryonic stem cells, differentiated progenitor cells, or combinations thereof.

[0059] In another aspect of the disclosure, embryonic fibroblasts are utilized to stimulate the perfusion of skeletal muscle, and the cells express one or more markers selected from the group consisting of Oct4, Nanog, Dppa5 Rbm, cyclin A2, Tex18, Stra8 , Dazl, β1- and α6-integrin, Vasa, Fragilis, Nobox , c-Kit, Sca-1, Rex1, and combinations thereof.

[0060] In another aspect of the disclosure, adipose tissue-derived fibroblasts utilize, for example, the stimulation of skeletal muscle perfusion, where the adipose tissue-derived fibroblasts express one or more markers selected from one or more of CD13, CD29, CD44, CD63 , CD73, CD90, CD166, aldehyde dehydrogenase (ALDH), ABC G2, or combinations thereof. In certain embodiments, adipose tissue-derived fibroblasts can, for example, be derived from a population of purified mononuclear cells extracted from adipose tissue that can proliferate in culture for 1 month, 2 months, 3 months, or longer.

[0061] In another aspect of the present disclosure, the exfoliated tooth-derived fibroblasts are for the stimulation of skeletal muscle perfusion and are utilized herein, where the exfoliated tooth-derived fibroblasts express STRO-1, CD146 (MUC1 8), alkaline phosphatase, MEPE, bFGF, or a combination thereof and may express one or more markers selected therefrom.

[0062] In another aspect of the present disclosure, skin fibroblasts are utilized for the stimulation of skeletal muscle perfusion and herein, the cells express one or more markers selected from one or more of CD44, CD13, CD29, CD90, CD105, or a combination thereof and can proliferate in culture for at least 1, 2, 3, or more periods.

[0063] In another aspect of the present disclosure, side population fibroblasts (e.g., cells identified based on the expression of the multidrug resistance transport protein (ABCG2), and / or the ability to efflux intracellular dyes such as rhodamine-123 and / or Hoechst 33342, terms known in the art) are utilized for insulin sensitivity stimulation and skeletal muscle perfusion, and side population cells can be identified based on, for example, the expression of the multidrug resistance transport protein (ABCG2), and / or the ability to efflux intracellular dyes such as rhodamine-123 and / or Hoechst 3 3342. Side population cells include, by way of example, pancreatic tissue, liver tissue, smooth muscle tissue, skeletal muscle tissue, cardiac muscle tissue, bone marrow tissue, cartilage tissue, bone marrow tissue, cartilage tissue, liver tissue, pancreatic tissue, pancreatic duct tissue, spleen tissue, thymus tissue, Peyer's patch tissue, lymph node tissue, epidermal tissue, dermal tissue, subcutaneous tissue, cardiac tissue, lung tissue vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, gastrointestinal tissue, esophageal tissue, gastric tissue ​​​​​​It can be derived from tissues such as woven, small intestine tissue, large intestine tissue, adipose tissue, uterine tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, mesenteric tissue, etc.

[0064] In another aspect of the present disclosure, fibroblast progenitor cells are utilized for the stimulation of skeletal muscle perfusion be. In one aspect, fibroblast progenitor cells are harvested from mobilized peripheral blood. Mobilization can be achieved by administration of one or more mobilizing agents or therapies. The mobilizing agent is G-CSF , M-CSF, GM-CSF, 5-FU, IL-1, IL-3, hyaluronic acid fragment to, kit-L, VEGF, Flt-3 ligand, PDGF, EGF, FGF-1, FG F-2, TPO, IL-11, IGF-1, MGDF, NGF, HMG CoA) reduct tase inhibitor, a small molecule antagonist of SDF-1, and combinations thereof. Selected from. Mobilization therapies include exercise, hyperbaric oxygen, autologous blood therapy by ex vivo ozone treatment of peripheral blood, and / or induction of SDF-1 secretion in an anatomical region outside the bone marrow. One or more of the above may be selected. In some aspects of the disclosure, fibroblast progenitor cells express one or more markers such as CD3 1, CD34, AC133, CD146 and / or flk1. appear.

[0065] In one aspect of the present disclosure, the cells included herein as fibroblasts or fibroblast progenitor cells provide cellular and / or trophic support for the regeneration of insulin-producing cells and are administered systemically or in proximity to one or more specific tissues and / or organs. In certain embodiments, they are provided in proximity to the pancreas. They can also be administered intravenously, intramuscularly ly, intraperitoneally, or intralymphatically.

[0066] In one aspect of the present disclosure, one or more anti-inflammatory agents can be administered to an individual receiving fibroblasts that increase skeletal muscle perfusion, and / or for the regeneration of insulin-producing cells. Anti- inflammatory agents may inhibit molecular pathways such as the NF-kappa B pathway, the MyD88 pathway, the TNF signaling pathway, the Toll-like receptor signaling pathway associated with upregulation of MHC expression, upregulation of C-reactive protein production, and / or upregulation of TNFα production. Anti-inflammatory agents useful in the disclosed methods include at least , alclofenac; alclometasone dipropionate; algestone acetonide; α amylase; alpha lipoic acid; alpha tocopherol; amcinafal; amcinaprid; anfeclofenac sodium; amyloprox hydrochloride; anakinra; anileridine; anirolac; apazone; ascorbic acid; balsalazide disodium; bendazac; benoxaprofen; benzydamine hydrochloride; bromelain; broperamol; budesonide; carprofen; chlorogenic acid; cicloprofen; cintazone; cliprofen; clobetasol propionate; clobetasone butyrate; clopirac; cloticasone propionate; cormetasone acetate; cortodoxone; deflazacort; desonide; dexamethasone; dexamethasone dipropionate; diclofenac potassium; diclofenac sodium; diflorazone diacetate; diflumidone sodium; diflunisal; difluprednate; dithranol; dimethyl sulfoxide; drocinonide; ellagic acid; endrysone; enlimomab; enlimomab sodium; epirizole; etodolac; etofenamate; fen flufenamic acid; flumetasone pivalate; flunisolide; flunixin meglumine; fluprednisolone; fluticasone propionate; formocortal; gemfibrozole; glucocorticoid; halcinonide; hydrocortisone; hydrocortisone acetate; hydrocortisone butyrate; hydrocortisone valerate; ibuprofen; indomethacin; indoprofen; isoflupredone acetate; isoxepac; ketoprofen; ketorolac tromethamine; lofemizole hydrochloride; loteprednol etabonate; mefenamic acid; meprednisone; meprednisone acetate; mesalamine; methylprednisolone; methylprednisolone acetate; methylprednisolone succinate; mometasone furoate; nabumetone; naproxen; naproxen sodium; Rubinac; Phenamol; Fenbufen; Fenclofenac; Fenclorac; F endosal; Fenpiparon; Fentiazac; Fentiazac; Flazarone; Flua zacort; Flufenamic acid; Flumizole; Flunisolide acetate; Flunixin; F lunixin meglumine; Flucortin butyl; Fluorometholone acetate; Flucazone; Flu rubiprofen; Flurutazine; Fluretofen; Fluticasone propionate; Flap rofen; Frobufen; Glutathione; Halcinonide; Halobetasol propionate; Halopredone acetate; Hesperin; Ibufenac; Ibuprofen aluminum; Ibuprofen picnole; Ironidap; Indomethacin; Indomethacin sodium; Indopro fen; Indoxole; Intra zole; Isoflupredone acetate; Isosepac, Isochi cam, Ketoprofen, Lofemizole hydrochloride, Lornoxicam, Lote prednol etha bonate, Lycopene, Meclofenamic acid sodium, Meclofenamic acid, Meclorisone di butyrate, Mefenamic acid, Mesalamine, Meselazone, Methylprednisolone strepta nate, Moniflumate; Etabonate; Lycopene; Meclofenamic acid sodium; Meclo fenamic acid; Meclorisone dibutyrate; Mefenamic acid; Mesalamine; Mesecrazone; M ethylprednisolone streptate; Moniflumate; Nabumetone; Naproxen; Nap roxen sodium; Naproxyl; Nimazone; Oleropaine; Orsalazine sodium um; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Para ni rine hydrochloride; Pentosan polysulfate sodium; Phenylbutazone sodium glycerolate; Pirfenidone; Piroxicam; Piroxicam cinnamate; Piroxicam olamine; P Luprofen; Picnogenol; Polyphenol; Pranazate; Prefenon; Pro Doleic acid; Procazone; Proxazole; Proxazole citrate; Quercetin; Resve Ratrole; Remexolone; Romazalit; Rosmarinic acid; Rutin; Sarcorex; Sa Lunasedin; Salsalate; Sanguiinium chloride; Secrazone; Selmetacin; Sudoki Cam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosarate; Tebufenolone tenidap; Tenidap sodium; Tenoxicam; Tecicam; Tecimid ; Tetrahydrocurcumin; Tetradimine; Thiopyanac; Ticoxolol pivalate; Tolmetin; Tolmetin sodium; Triclonide; Triflumidate; Zidometacin; Zomepirac sodium, IL-4, IL-10, IL-13, IL-20, IL-1 receptor antagonists, TGF-beta and combinations thereof are included.

[0067] The present invention includes insulin sensitivity that can be increased by providing means for enhancing muscle perfusion, and / or reducing inflammation, and / or promoting pancreatic islet regeneration. The present disclosure relates to gestational diabetes; carbohydrate and lipid metabolism disorders; glucosuria; microvascular diseases; polyneuropathy and diabetic retinopathy; diabetic nephropathy; insulin resistance; glucose tolerance disorders (or glucose intolerance); obesity; hyperglycemia; hyperinsulinemia; hyperlipidemia; atherosclerosis; hypertension; congenital or acquired digestive absorption disorders including malabsorption syndrome; diseases caused by loss of intestinal mucosal barrier function; and / or various conditions related to insulin resistance other than IDDM and NIDDM including protein-losing gastroenteropathy, and provides means for treating the same. ); obesity; hyperglycemia; hyperinsulinemia; hyperlipidemia; hyperlipidemia; atherosclerosis ; hypertension; congenital or acquired digestive absorption disorders including malabsorption syndrome; diseases caused by loss of intestinal mucosal barrier function; and / or protein-losing gastroenteropathy, and provides means for treating various conditions related to insulin resistance other than IDDM and NIDDM.

[0068] ​​​​​ Other conditions associated with blood glucose concentrations above normal, in either acute or chronic form, are also encompassed by the present disclosure. Thus, embodiments of the present disclosure include methods of reducing blood glucose concentration in an individual having elevated blood glucose concentration by administering a therapeutically effective amount of fibroblasts and / or fibroblast derivatives. In one embodiment, the elevated glucose level is a fasting level above 100 mg / dL.

[0069] The present disclosure includes methods of using cells for the purpose of stimulating muscle perfusion, reducing the production of inflammatory mediators, and, in some circumstances, enabling pancreatic regeneration, and methods of using fibroblasts having the ability to differentiate into other cell types in certain aspects. Also provided are means for inducing pancreatic regeneration in the surrounding environment that helps maintain the survival and function of pancreatic islets or their components.

[0070] In one aspect of the present disclosure, an increase in the angiogenic ability of a subject is carried out for the purpose of increasing the vascularization of the pancreas and may include the delivery of fibroblasts and / or their derivatives. The increase in angiogenic ability can be achieved by administration of one or more angiogenic factors, cells having angiogenic ability, or combinations thereof. Angiogenesis can be stimulated in association with anti-inflammatory interventions, regardless of the presence or absence of administration of cells that can differentiate into insulin-producing cells.

[0071] In one aspect of the present disclosure, an individual is treated with one or more agents known to stimulate the generation of endogenous insulin-producing cells while simultaneously increasing anti-inflammatory activity and / or angiogenic activity. Methods for increasing the differentiation of endogenous insulin-producing cells are known in the art. is. An example of such a method is the combined administration of EGF and gastrin, which has been demonstrated to induce insulin secretion through the differentiation of endogenous stem cells into insulin-producing cells 12 - 14].

[0072] In one aspect of the present disclosure, one or more anti-inflammatory agents can be used in combination with fibroblasts and / or their derivatives that can increase angiogenesis and / or induce islet formation. / or can induce islet formation.

[0073] In one embodiment of the present disclosure, patients suffering from insulin resistance with a state of NIDDM are treated by intramuscular administration of fibroblasts and / or their derivatives. It is known that 7 0 - 80% of postprandial glucose is metabolized by skeletal muscle

[15] . In many NIDDM patients, severe atherosclerotic deposits are known to inhibit limb circulation. Without being bound by theory, the inhibition of circulation may occur in blood vessels such as the femoral artery, popliteal artery, and / or tibial artery and so on. Furthermore, the inhibition of circulation may occur at the level of the capillaries supplying various muscles. Circulatory disorders are known to occur not only due to atherosclerotic arteriosclerosis but also due to the inhibition of the vasodilatory mechanism

[16] . Due to the inhibition of circulation and the vasodilatory response, the insulin activation of GLUT4 membrane localization and the general insulin responsiveness are blunted. Therefore, in one embodiment of the present disclosure, the ability of muscle to respond to insulin can be restored by the administration of fibroblasts that can restore endothelial function and induce angiogenesis and is improved. Fibroblasts useful for this purpose can be of autologous, endogenous, or allogeneic origin. and is improved by the administration of fibroblasts that can restore endothelial function and induce angiogenesis. Fibroblasts useful for this purpose can be of autologous, endogenous, or allogeneic origin. origin. origin.

[0074] ​In one particular embodiment, an individual with NIDDM is treated with fibroblasts by administering them at a depth of approximately 1.5 cm into the gastric muscle. The injection can be performed to deliver a total number of fibroblasts in the range of 10 million to 10 billion mononuclear cells. In a preferred embodiment, an injection of approximately 1 to 3 billion mononuclear cells is administered. The injection can be performed with a total injection volume of 10 to 50 ml, and the injection is distributed over a grid placed on the gastric muscle. The number of injections can range from 1 to 10 0 injections, the optimal number ranges from about 10 to 50 injections, and more optimally is between 20 and 30 injections. The injection of fibroblast mononuclear cells can be specifically performed in occluded regions identified by methods known in the art such as digital subtractive tive angiography, Doppler imaging, positron emission tomography, and ultrasound. Alternatively, the administration of fibroblasts can be performed in regions where occlusion is suspected due to being unestablished. Further, means for evaluating tissue oxygenation such as transcutaneous pulse oximetry can be used to identify muscle regions lacking oxygenation. Global circulatory deficiencies can also be identified by measurements such as toe pulse or ankle - brachial blood pressure ratio. In one embodiment, the administration of bone marrow fibroblasts in the gastrocnemius muscle is performed when the ankle - brachial blood pressure ratio is below 0.9. In other embodiments, the administration of fibroblasts is performed in various muscles regardless of the perfusion state. For example, a patient with NIDDM can be injected with multiple aliquots of bone marrow in major skeletal muscles. Examples of major skeletal muscles suitable for injection include the deltoid muscle, pectoralis major muscle, biceps muscle, rectus abdominis muscle, external oblique abdominal muscle, gluteus medius muscle, gluteus maximus muscle, soleus muscle, tibialis anterior muscle, vastus medialis muscle, vastus intermedius muscle, vastus lateralis muscle, rectus femoris muscle, and sartorius muscle. In one embodiment, the administration of bone marrow fibroblasts in the gastrocnemius muscle is performed when the ankle - brachial blood pressure ratio is below 0.9. In other embodiments, the administration of fibroblasts is performed in various muscles regardless of the perfusion state. For example, a patient with NIDDM can be injected with multiple aliquots of bone marrow in major skeletal muscles. Examples of major skeletal muscles suitable for injection include the deltoid muscle, pectoralis major muscle, biceps muscle, rectus abdominis muscle, external oblique abdominal muscle, gluteus medius muscle, gluteus maximus muscle, soleus muscle, tibialis anterior muscle, vastus medialis muscle, vastus intermedius muscle, vastus lateralis muscle, rectus femoris muscle, and sartorius muscle. The deltoid muscle, pectoralis major muscle, biceps muscle, rectus abdominis muscle, external oblique abdominal muscle, gluteus medius muscle, gluteus maximus muscle, soleus muscle, tibialis anterior muscle, vastus medialis muscle, vastus intermedius muscle, vastus lateralis muscle, rectus femoris muscle, and sartorius muscle.

[0075] Fibroblasts are utilized in embodiments to increase muscle perfusion, including at least skeletal muscle perfusion In form, the effect may or may not be monitored. The effect of intramuscular fibroblast administration is observed, for example, not only by the ability to increase perfusion, but also by the ability to increase the blood flow-mediated dilation response (any vasodilation of arteries following an increase in luminal blood flow and an increase in wall shear stress). In specific embodiments, the effects of cell administration are evaluated by various means known in the art for the quantification of insulin sensitivity. For example, the hyperinsulinemic-euglycemic clamp technique is considered the gold standard for this purpose, but due to impracticalities such as time and cost, other techniques may also be used. Such techniques include the frequently sampled intravenous glucose tolerance test (FSIVGTT), insulin tolerance test ( ITT), insulin sensitivity test (IST), continuous infusion of glucose by model assessment (CIGMA), and oral glucose tolerance test (OGTT).

[0076] In some embodiments of the present invention, treatment with bone marrow mononuclear cells can be performed in combination with cytokines known to mobilize endogenous fibroblasts. It is known that intramuscular administration of bone marrow mononuclear cells mobilizes endogenous CD34 fibroblasts systemically from the bone marrow

[17] . Accordingly, the present disclosure encompasses a method wherein an increase in endogenous fibroblast mobilization following administration of bone marrow mononuclear cells to the muscle of a patient with NIDDM causes an enhanced therapeutic effect. Since intramuscularly administered fibroblasts have chemotactic activity, mobilization of bone marrow The agent will likely increase the therapeutic effect. Administration of G-CSF may be performed simultaneously with intramuscular injection of bone marrow cells or may be performed near the time point associated with maximal mobilization of CD34 cells. The time point may be determined experimentally or may be based on previously published data. For example, it has been reported that maximal CD34 mobilization following intramuscular administration of bone marrow cells occurs around 30 days prior. Thus, in one embodiment of the methods of the present disclosure, G-CSF is administered 30 days prior at a concentration sufficient to induce endogenous CD34 mobilization. In one embodiment, G-CSF is administered at a concentration of about 60 micrograms per day for 5 days by subcutaneous injection. Administration may be initiated, for example, on day 25 following intramuscular injection of bone marrow cells. In some embodiments, heparin may be co-administered to avoid the potential to cause embolism due to the high systemic white blood cell count caused by G-CSF injection. This may be useful in NIDDM patients who already have a high risk of embolism compared to the general

[0077] population. Anticoagulation methods are well known in the art and agents other than heparin may be utilized. However, when heparin anticoagulation is used, as an example, a dose of about 10,000 units per day may be useful. In another embodiment, one or more agents known to increase fibroblast . In one embodiment, administration of a TNFα neutralizing agent simultaneously administers with fibroblasts to relieve the inhibitory effect of this cytokine on circulating fibroblasts

[23] .

[0078] III. Fibroblasts and their modifications and preparations The fibroblasts utilized in the methods of the present disclosure can be prepared and provided to an individual in need thereof. Fibroblasts can be autologous, allogeneic, or xenogeneic with respect to the individual being treated.

[0079] In certain embodiments, fibroblasts having the ability to increase perfusion, increase insulin sensitivity, the ability to treat insulin resistance, the ability to provide an appropriate environment for the restoration of insulin-producing cell function, etc. are modified and / or prepared for use in the methods of the present disclosure. The fibroblasts of the present disclosure may or may not have a specific expression profile. In some embodiments, the fibroblasts of the present disclosure express one or more specific markers. In certain embodiments, the fibroblasts express CD34, CD133, or both. In certain embodiments, the fibroblasts express one or more markers selected from the group consisting of CD34, c-kit, flk-1, Stro-1, CD105, CD73, CD31, CD56, CD146, vascular endothelial cadherin, CD133, CXCR-4, and combinations thereof. In certain embodiments, the fibroblasts express CD34, CD133, as well as c-kit, flk-1, Stro-1, CD105, CD73, CD31, CD56, CD146, vascular endothelial cadherin, and In certain embodiments, the fibroblast expresses 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of the above. The cells are CD34+, CD133+, or both. The cells are CD90+, CD105+, and substantially lack CD45 and / or CD14 expression. .

[0080] In certain embodiments, the fibroblasts have regenerative activity.

[0081] In certain embodiments, fibroblasts are cultured, whether for storage and / or preparation. Various terms are used to describe cells in culture. Cell culture is Generally, cells taken from a living organism and grown under controlled conditions ("culture" or "culture"). Primary cell cultures refer to cells, tissues, or tissues that are taken directly from an organism prior to the first subculture. The cells are grown in a growth medium under conditions that facilitate cell growth and / or division. When placed in the ground, they are grown in culture to produce a larger population of cells. When grown at 4°C, cell proliferation rate is measured by the number of times the cells need to double in number. This is called the doubling time.

[0082] A cell line is a population of cells formed by one or more subcultures of a primary cell culture. Each round of subculture is called a passage. When cells are subcultured, they are passaged. A particular cell population, or cell line, is sometimes referred to by the number of times it has been passaged. For example, a cultured cell population that has been passaged 10 times is referred to as a P10 culture. The primary culture, i.e., the first culture after isolating cells from tissue, can be called P0. Following the first subculture, the cells are described as a subculture (P1 or passage 1). After the indicated passage, the cells are at passage three (P2 or passage 2), and so on. During the passage there can be many population doublings; thus, it is understood by those skilled in the art that the number of population doublings of the culture is greater than the passage number. The growth of the cells (i.e., the number of population doublings) during the period between passages depends on many factors including seeding density, glass substrate, medium, growth conditions, and the time between passages, but is not limited to these. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes.

[0083] In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes. In some embodiments, fibroblasts are included in, are in, or are exposed to a conditioning medium. A conditioned medium is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in a medium, they may provide nutritional support to other cells or secrete one or more cytokines having another function. Generally, a nutrient factor is defined as a substance that promotes or at least supports the survival, growth, proliferation, and / or maturation of cells or stimulates increased activity of the cells. Such nutrient factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A medium containing cytokines is a conditioning medium. Fibroblasts can be used alone or in combination with one or more other components to secrete one or more factors or entities (e.g., exosomes) for medical purposes.

[0084] As used herein, the term "growth medium" generally refers to a medium sufficient for the culture of any type of fibroblast. In particular, one medium for the culture of the cells of the invention herein includes Dulbecco's Modified Eagle Medium (also abbreviated herein as DMEM). Particularly preferred As used herein, the term "growth medium" generally refers to a medium sufficient for the culture of any type of fibroblast. In particular, one medium for the culture of the cells of the invention herein includes Dulbecco's Modified Eagle Medium (also abbreviated herein as DMEM). Particularly preferred As used herein, the term "growth medium" generally refers to a medium sufficient for the culture of any type of fibroblast. In particular, one medium for the culture of the cells of the invention herein includes Dulbecco's Modified Eagle Medium (also abbreviated herein as DMEM). Particularly preferred What is preferred is DMEM-low glucose (also DMEM-LG herein) (Invitr ogen, Carlsbad, CA). DMEM low glucose is preferably 15% (v / v) fetal bovine serum (e.g., defined fetal bovine serum, Hyclone, Logan U tah), antibiotics / antifungal agents (preferably penicillin (100 units / ml) , streptomycin (100 mg / ml), and amphotericin B (0 .25 μg / ml), (Invitrogen, Carlsbad, CA), and 0.001% (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 as supplements to the growth medium shown in the text.

[0085] Also, in connection with the present invention, the term "standard growth conditions" as used herein refers to culturing cells at 37°C in a standard atmosphere containing 5% CO 2 . Maintain the relative humidity at about 100% . The foregoing conditions are beneficial for culturing, but it should be understood that such conditions can be varied by those skilled in the art for culturing cells by varying options such as body temperature, CO , relative humidity, oxygen, growth medium, etc. 2 . Cells can be prepared for administration in a pharmaceutically acceptable carrier, such as a sterile physiological saline isotonic solution. In some embodiments, the pharmaceutically acceptable carrier is FASリ

[0086] gand, IL-2R, IL-2, IL-4, IL-8, IL-10, IL-20, IL- 41]] ​​35, HLA-G, PD-L1, I-309, IDO, iNOS, CD200, galectin 3, sCR1, arginase, PGE-2, aspirin, lovastatin, pravastatin simvastatin, pitavastatin, rapamycin, IVIG, naltrexone, TGF-β VEGF, PDGF-4, anti-CD45RB antibody, hydroxychloroquine, leflunomide dicyanoaurate, sulfasalazine, methotrexate, glucocorticoid, etanercept adalimumab, anakinra, cetuximab, golimumab, infliximab rituximab, lixizumab, cyclosporine, IFN-γ, everolimus, rapamycin VEGF-1, FGF-2, angiopoietin, HIF-1-α, or a combination thereof and may contain one or more additional agents such as combinations thereof.

[0087] In one embodiment of the present disclosure, the fibroblasts are administered to a subject by any suitable route including, but not limited to, injection (such as intramuscular injection) into a hypoxic region. Suitable routes include intravenous, subcutaneous, intrathecal, oral, rectal, intramedullary, intrasplenic, intraventricular, intrahepatic, and intrarenal. In certain embodiments, the fibroblasts may be derived from tissues including skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, adipose tissue, foreskin, placenta, and / or umbilical cord. In certain embodiments, the fibroblasts are placental, fetal, neonatal, or adult, or a mixture thereof. The number of cells administered to an individual depends at least in part on the factors described herein and can be optimized using methods conventional in the art. In certain embodiments, a single dose

[0088] In certain embodiments, the fibroblasts may be derived from tissues including skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, adipose tissue, foreskin, placenta, and / or umbilical cord. In certain embodiments, the fibroblasts are placental, fetal, neonatal, or adult, or a mixture thereof. In certain embodiments, the fibroblasts are placental, fetal, neonatal, or adult, or a mixture thereof.

[0089] The number of cells administered to an individual depends at least in part on the factors described herein and can be optimized using methods conventional in the art. In certain embodiments, a single dose Administration is necessary. In other embodiments, multiple administrations of the cells are required. Naturally however, this system is subject to variables such as the specific needs of the individual, which can change over time and circumstances, loss of cells, or the rate of loss of cell viability as a result of the viability of the individual cells. Thus each individual can be monitored for the appropriate dosage, and such an implementation of monitoring the individual is expected to be routine in the art.

[0090] In some embodiments, the cells are provided to one or more culture compositions comprising, consisting of, or consisting essentially of Roswell Park Memorial Institute (RPMI-16 40), Dulbecco's Modified Eagle Medium (DMEM), Eagle's Modified Eagle Medium (E MEM), Optimem, Iscove's Medium, or combinations thereof.

[0091] In one embodiment of the present disclosure, fibroblasts are obtained from amniotic fluid or amniotic membrane. Amniotic membrane-derived fibroblasts can be therapeutically utilized in an unpurified manner, optionally following matching. Amniotic fibroblasts can be administered locally, intramuscularly, or systemically to patients suffering from insulin resistance. In other embodiments, amniotic fibroblasts are substantially purified based on the expression of markers such as SSEA-3, SSE A4, Tra-1-60, Tra-1-81, and Tra-2-54 and subsequently administered. In other embodiments, the cells are cultured, expanded, for example, as described in U.S. Patent Application Publication No. 2005 / 0054093, and then injected into a patient. Amniotic fibroblasts are described in the following references [24-26]. One particular aspect of amniotic fibroblasts is mesenchymal progenitor cells and endothelial progenitor cells ​​​​Both phenotypic profiles as cells, thereby having anti-inflammatory functions, as well as angiogenesis functions are possible [25, 27]. This property is useful for the treatment of patients with insulin resistance and related diseases that would benefit from angiogenesis, but is also useful from the anti-inflammatory action of fibroblasts. The use of amniotic fibroblasts is particularly useful in situations such as ischemia-related pathologies and / or inflammatory conditions where hypoxia is known to sustain the degenerative process.

[0092] In one embodiment, allogeneic or autologous donors compatible with HLA or mixed lymphocyte reaction are mobilized by administration of G-CSF (filgrastim: Neupogen) at a concentration of about 10 μg / kg / day by subcutaneous injection for 2 - 7 days, for example 4 - 5 days. Peripheral blood mononuclear cells are collected using an apheresis device such as an AS104 cell separator (Fresenius Medical). 1 - 40 x 10 of mononuclear cells are collected, concentrated, and administered locally, systemically, or injected into an area close to the site pathology associated with a given degenerative disease. In situations where ischemia is localized, cell administration can be carried out within the context of the present invention. Methods for identifying such ischemic regions are routinely known in the art and include the use of techniques such as nuclear imaging or MRI imaging. Variations of this procedure can include subsequent culturing of the cells to enrich various populations known to have angiogenic and / or anti-inflammatory and / or anti-remodeling and / or regenerative properties. Furthermore, the cells can be purified for specific subtypes before and / or after culturing. The treatment can be during culturing or ex vivo to generate and / or expand specific subtypes and / or functional properties. 9 ​​​​​​​​​​​​​​​At certain times during the in vitro culture, but before injection, it can be performed on the cells. As described in this disclosure Various embodiments of the invention for other fibroblasts described herein can also be applied to circulating peripheral blood fibroblasts as well.

[0093] In one embodiment of the disclosure, allogeneic or autologous adipose tissue-derived fibroblasts are used as the cell supply source. Adipose tissue-derived fibroblasts express markers such as CD9; CD29 (integrin β1 ); CD44 (hyaluronic acid receptor); CD49d, e (integrin α4, 5); CD 55 (decay-accelerating factor); CD105 (endoglin); CD106 (VCAM-1); C D166 (ALCAM). These markers can be used not only for identification but also as a means of positive selection before and / or after culture to increase the purity of the desired cell population . For purification and isolation, devices for the rapid extraction and purification of cellular adipose tissue are known to those skilled in the art. U.S. Patent No. 6,316,247 describes an apparatus that allows for the purification of mononuclear adipose-derived fibroblasts in a closed environment without the need to establish a GMP / GTP cell processing laboratory, enabling the treatment of patients in a variety of settings. One embodiment of the disclosure achieves a starting lipospi rate of 10 - 200 ml, washes the lipospi rate in phosphate-buffered saline, digests the lipospi rate with 0.075% collagenase type I at 37 °C for 30 - 60 minutes, neutralizes the collagenase with gentle stirring, preferably neutralizes the collagenase with a medium containing DMEM or other autologous serum at a concentration of 10% v / v, centrifuges the treated lipospi rate at about 700 - 2000 g for 5 - 15 minutes, and then resuspends the cells in a suitable medium such as DM EM. Subsequently, a cell strainer is included. v / v of the collagenase with DMEM or other autologous serum-containing medium, centrifuges the treated lipospi rate at about 700 - 2000 g for 5 - 15 minutes, and then resuspends the cells in a suitable medium such as DM EM. Subsequently, a cell strainer is included. - First, filter the cells using, for example, a 100 μm nylon cell strainer to remove debris. . Subsequently, centrifuge the filtered cells again at approximately 700 - 2000 g for 5 - 15 minutes and resuspend them in a culture flask or similar container at about 1 × 10 6 / cm 2 . After culturing for 10 - 20 hours, remove non - adherent cells by washing with PBS and culture the remaining cells under the same conditions as described for culturing fibroblasts derived from umbilical cord blood. Once the desired concentration for clinical use is reached, harvest the cells, evaluate their purity, and administer them to patients in need as described above. Unique tissue - specific fibroblasts can be used in autologous or allogeneic settings for the practice of the methods of the present disclosure. These cells can be used, or induced to be used, fully for differentiation into endothelial or endothelial progenitor cells. Cells that exhibit the ability to efflux certain dyes (including, but not limited to, rhodamine - 123) are associated with stem - cell - like properties

[28] , and

[0094] these cells can be purified from tissues following cell dissociation based on their efflux properties. Thus, in one embodiment of the present disclosure, tissue - derived side population cells used in combination with fibroblasts can be newly isolated, classified into sub - populations, or utilized either after ex vivo culture for the treatment of altered states. For use in the disclosure, side population cells are obtained from pancreatic tissue, liver tissue, smooth muscle tissue, skeletal muscle tissue, bone tissue, bone marrow tissue, sponge tissue, bone tissue, cartilage tissue, liver tissue, pancreatic tissue, pancreatic duct tissue, spleen tissue, thymus tissue, Peyer's patch tissue, lymph node tissue, thyroid tissue, epidermal tissue, dermal tissue, subcutaneous tissue, heart tissue, lung tissue , vascular tissue, endothelial tissue, blood cell tissue, bladder tissue, kidney tissue, gastrointestinal tissue, esophageal tissue, gastric tissue In one embodiment of the present disclosure, side population cells derived from tissue used in combination with fibroblasts can be newly isolated, classified into sub - populations, or utilized either after ex vivo culture for the treatment of altered states. For use in the disclosure, side population cells are obtained from pancreatic tissue, liver tissue, smooth muscle tissue, skeletal muscle tissue, bone tissue, bone marrow tissue, sponge tissue, bone tissue, cartilage tissue, liver tissue, pancreatic tissue, pancreatic duct tissue, spleen tissue, thymus tissue, Peyer's patch tissue, lymph node tissue, thyroid tissue, epidermal tissue, dermal tissue, subcutaneous tissue, heart tissue, lung tissue , vascular tissue, endothelial tissue, blood cell tissue, bladder tissue, kidney tissue, gastrointestinal tissue, esophageal tissue, gastric tissue , vascular tissue, endothelial tissue, blood cell tissue, bladder tissue, kidney tissue, gastrointestinal tissue, esophageal tissue, gastric tissue , vascular tissue, endothelial tissue, blood cell tissue, bladder tissue, kidney tissue, gastrointestinal tissue, esophageal tissue, gastric tissue , small intestine tissue, large intestine tissue, adipose tissue, uterine tissue, ocular tissue, lung tissue, testicular tissue, ovarian tissue, pro duced from tissues such as static glandular tissue, connective tissue, endocrine tissue, and mesenteric tissue. In one embodiment form, purification of side population cells dissociates cardiac valve cells into 10 6 cells / ml and resuspends them in HBSS+ without calcium and magnesium (supplemented with 2% FCS, 10 mM Hepes, and 1% penicillin / streptomycin) medium containing 6.0 μg / ml of Hoech hst 33342 and stain at 37 °C for 90 minutes. Then , the cells are run on a flow cytometer and evaluated for the efflux of Hoechst 33342. The purified cells can be evaluated for their ability to form cardiac spheres, which is in lin (25 μg / ml), transferrin (100 μg / ml), progesterone (2 0 nM), sodium selenite (30 nM), putrescine (60 nM), recombinant mouse E GF (20 ng / ml), and recombinant human FGF2 in DME / M199 (1:1 ) serum-free growth medium at a density of 1 - 2 × 106 cells / ml in a non-coated 10 cm dish by suspending the side population cells. Half of the medium is changed every 3 days . Subculture can be performed using 0.05% trypsin and 0.53 mM EDT A-4Na every 7 - 14 days. Then, the cardiac spheres are dissociated into single cell suspensions and then , for therapeutic purposes, or for evaluating the therapeutic ability in vitro or in animal models before clinical use and used. Cardiac spheres can be induced to differentiate into endothelial cells by culturing them in angiogenic factors prior to administration. These methods are the methods of the present disclosure . More can be induced to differentiate into endothelial cells. These methods are the methods of the present disclosure In publications referred to by the implementers, other tissue side population cells are described. 29-31]. Various embodiments of the invention for other fibroblasts described in this disclosure may also be applicable to side population fibroblasts.

[0095] In one embodiment of the present disclosure, "young" fibroblasts are used to compensate for the deterioration of the function of aging tissues. The term "young" is used to indicate cells derived from donors of a younger age than the recipient. In some embodiments, the young cells can be cells of the same recipient harvested earlier in the day until cell injection. The use of young cells for implementing the methods of the present disclosure has certain advantages. For example, it is known that old animals have impaired physiological responses compared to younger animals. Aging is known to be associated with impaired insulin responsiveness [32, 33]. In some cases, aging is associated with an increase in the production of inflammatory cytokines such as TNF-α that cause insulin resistance. For example antibodies against TNF-α have been demonstrated to be able to inhibit age-related insulin resistance in the muscles of Sprague-Dawley rats

[34] . For example, in an experiment by the group of Edelberg, when 3-month-old ROSAβ-galactosidase transgenic bone marrow cells were transplanted into 18-month-old recipients, it was demonstrated that they could enter the bone marrow and cause chimeric hematopoiesis without pretreatment of the recipient

[35] . Furthermore interestingly, endothelial progenitor cells derived from young 3-month-old bone marrow donors have the ability to "rejuvenate" the ability of 18-month-old recipient mice to sustain angiogenesis in the ectopically transplanted neonatal heart. Specifically, 18-month-old recipients have neonatal hearts ​​​​​​​​​​​​​ When transplanted together, the donor heart lost viability due to lack of angiogenesis. 18 When 3-month-old bone marrow cells were administered to 18-month-old recipients, the angiogenesis ability of neonatal hearts was still impaired. However, 3-month-old bone marrow injection was able to establish angiogenesis in a dose-dependent and PDGF -B-dependent manner.

[0096] In one embodiment of the present disclosure, fibroblasts that are substantially younger than the recipient are administered to the recipient for the production of cells that directly or indirectly increase responsiveness to insulin. As previously described, fibroblasts derived from umbilical cord blood, bone marrow, and adipose tissue have the ability to differentiate into skeletal muscle cells. Considering the observation that younger cells can integrate with older tissues and re-establish the function of older tissues the present invention teaches the use of younger fibroblasts to increase responsiveness to insulin. In one embodiment, umbilical cord blood fibroblasts are utilized as a source of "young" fibroblasts for generating cells that are similar to skeletal muscle cells in vivo to reduce insulin resistance . This should not be construed as being bound by theory, as differentiation into muscle-like cells is one of several mechanisms by which the present invention discloses the ability of umbilical cord blood fibroblasts to reverse insulin resistance . In one embodiment, umbilical cord blood fibroblasts are obtained from umbilical cord blood samples obtained from healthy pregnancies. Umbilical cord blood is purified according to routine methods

[36] . In one embodiment, a standard Baxter 450 ml blood donor containing CPD A anticoagulant (citrate / phosphate / dextrose / adenine) (Baxter Health Ca re, Deerfield, IL) is used. / phosphate / dextrose / adenine) (Baxter Health Ca re, Deerfield, IL) containing standard Baxter 450 ml blood donor Insert a 16-gauge needle from the set and use it to puncture the umbilical vein of the placenta obtained from the mother, which was tested for viral and bacterial infections according to international donor standards. Umbilical cord blood is drained by gravity so as to drip into the blood bag. The placenta is placed in an absorbent cotton pad lined with plastic suspended from a specially constructed support frame to allow collection and reduce contamination by maternal blood and other secretions. 63 ml of CPD A used in a standard transfusion bag calculated for 450 ml of blood is reduced to 23 ml by draining 40 ml into a graduated cylinder immediately before collection. An aliquot of umbilical cord blood is removed for safety testing according to the guidelines of the National Marrow Donor Program (NMDP). Safety testing includes detection by routine clinical tests for human immunodeficiency virus 1 and 2, human T-cell lymphotropic virus I and II, hepatitis B virus, hepatitis C virus, cytomegalovirus, and syphilis. Subsequently, 6% (wt / vol) hydroxyethyl starch is added to the anticoagulated umbilical cord blood to a final concentration of 1.2%. Then, the umbilical cord blood hydroxyethyl starch mixture in the original blood collection bag is centrifuged (50×g, 5 minutes at 10°C) to separate the supernatant rich in white blood cells. The supernatant rich in white blood cells is transferred from the bag to a 150-ml plasma transfer bag (Baxter Health Care) and centrifuged (400×g, 10 minutes) to sediment the cells. The excess supernatant plasma is transferred to a second plasma transfer bag without cutting the connecting tube. Finally, the sedimented white blood cells are resuspended in the supernatant plasma to a total volume of 20 ml. For each umbilical cord, approximately 5x10 -7x10 ... (the rest of the text continues as in the original but with English translation inserted where applicable). ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 8 ... 9Nucleated cells are obtained. The cells are cryopreserved according to the method described by Rubi nstein et al.

[36] .

[0097] In some embodiments, donor cells are matched to the recipient, and in other situations, matching is not performed. As an example, a plurality of purified and cryopreserved cord blood fibroblast sources as described above can be utilized for the treatment of patients in need of fibroblast therapy. Aliquots of mononuclear cells are taken from each of the plurality of cord blood fibroblast sources, and the aliquots contain approximately 10 cells. The cells are plated in a Nunc 96-well plate at a concentration of 10 cells per well in 9 wells at a volume of 100 uL per well . Prior to plating, the cells are washed and reconstituted in DMEM-LG medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum. The cord blood cells are considered "stimulator" cells for the purpose of the matching procedure. To generate "responding" cells, 20 ml of peripheral blood is drawn from a patient in need of fibroblast therapy via venipuncture 5 . The 20 ml of peripheral blood is collected in a heparinized Vacutainer®, heparinized, layered on a Ficoll® density gradient, and centrifuged at 500 g for approximately 60 minutes . The mononuclear layer is collected and washed with phosphate-buffered saline supplemented with 3% fetal bovine serum 4 . For every 9 wells of stimulator cells, 3 wells are added with a concentration of 10 responding cells, 3 wells are added with a concentration of 10 responding cells, and for 3 wells, medium without cells is added to have a control for the natural activity of the stimulator cells. Prior to adding the responding cells to the stimulator cells the responding cells are washed and reconstituted in DMEM-LG medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum . The 20 ml of peripheral blood is collected in a heparinized Vacutainer®, heparinized, layered on a Ficoll® density gradient, and centrifuged at 500 g for approximately 60 minutes . The mononuclear layer is collected and washed with phosphate-buffered saline supplemented with 3% fetal bovine serum . For every 9 wells of stimulator cells, 3 wells are added with a concentration of 10 4 responding cells, 3 wells are added with a concentration of 10 responding cells, and for 3 wells, medium without cells is added to have a control for the natural activity of the stimulator cells. Prior to adding the responding cells to the stimulator cells 5 the responding cells are washed and reconstituted in DMEM-LG medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum . The responding cells are added to the stimulator cells Reconstitute in DMEM-LG medium supplemented with 10% heat-inactivated fetal bovine serum. The responder cells and the medium contain a volume of 100 μL before being added to the stimulator cells. Additionally, to have a control for the spontaneous activity of the responder cells, 10 in a volume of 100 μL 4 and 10 5 responder cells are added in triplicate to 100 μL of medium without stimulator cells. To obtain a control for background or other contamination, 3 wells are plated with 200 μL of medium alone Thus, the total culture consists of 25 fibroblast sources × 9 wells = 225 wells, i.e., a total of 3 96-well plates are used. Additionally, 9 wells without addition of stimulator cells or without any cells added are used as responder controls. A 72-hour mixed lymphocyte reaction is performed, and the cells are pulsed with 1 μCi [3H] thymidine for the last 18 hours. The cultures are harvested onto glass fiber filters (Wallac, Turku, Finland) . Radioactivity is counted using a Wallac 1450 Microbeta liquid scintillation counter and the data are analyzed with UltraTerm 3 software (Micros oft, Seattle, WA). If lymphocyte proliferation is more than 2-fold higher compared to lymphocytes cultured without stimulator cells, after subtracting the background proliferation of the stimulator alone if the case, the cord blood batch is not used for treatment. According to this criterion, two or more of the multiple batches of fibroblast sources can be selected for administration to the patient. The purified cells can be utilized for delivery. In one embodiment, cord blood is used to generate fibroblasts regardless of matching with the recipient

[0098] Obtain fibroblasts, and deplete the umbilical cord blood of specific immunogenic components that can cause host-versus-graft It may be taken steps to cause, and / or alternatively, the graft is manipulated to neutralize immunocytes that may have the ability to cause graft-versus-host. Specifically, umbilical cord blood fibroblasts are concentrated in GMP (Good Manufacturing Practices) grade Hanks' balanced salt solution (containing Ca 2+ ). The cells are washed prior to preconcentration so that the cells are substantially free of plasma and erythrocytes and granulocytes are depleted. The volume of the mononuclear cell suspension is adjusted so that the cell density is about 3×10 7 / mL, and CAMPATH-1M or C AMPATH-1H is added to a final concentration of 0.1 mg / mL. The mixture is incubated at room temperature for 15 minutes, then recipient serum is added to achieve a final concentration of 25% (vol / vo l). Thereafter, the mixture is incubated at 37 °C for an additional 30 minutes . The treated umbilical cord blood cells are washed once, evaluated for viability, and injected into patients in need of treatment.

[0099] The ability of fibroblasts to differentiate into various tissues is well known, but a less well-known ability of various fibroblasts is their anti-inflammatory function. NIDDM has been demonstrated to be associated with elevated inflammatory mediators. This was elegantly summarized in the review by Pickup et al. that described "low-grade inflammation" as part of the process associated with the development of insulin resistance and subsequent NID DM. This is because elevated circulating inflammatory markers such as C-reactive protein and interleukin-6 predict the onset of type 2 diabetes, and several The drug is based on the observation that it reduces both acute phase reactants and blood glucose (aspirin and thiazolidinedione), and probably reduces the risk of developing type 2 diabetes (statins). Furthermore, Pickup hypothesizes that features of type 2 diabetes such as fatigue, sleep disturbance, and depression may be the result of fibroblastic "hypercytokinemia"

[37] . TNF -α and IL-6 are known to be secreted at basal levels by the adipose compartment, and a correlation has been made between the levels of these cytokines and insulin resistance. For example, Kern et al. measured TNF and IL-6 levels in non-diabetic liposuctioned and obese patients. Comparing subjects with liposuction [body mass index (BMI) < 25 kg / m(2)] and obesity (BMI 30 - 40 kg / m(2)), a 7.5-fold increase in TNF secretion was observed, and TNF secretion was inversely correlated with insulin sensitivity measured by intravenous glucose tolerance test

[38] . In many other studies, it has been demonstrated that plasma TNF concentrations are high in insulin-resistant patients [39, 40]. Furthermore, a decrease in TNF -α is associated with responses to various insulin sensitizers

[41] . The insulin resistance-inducing ability of TNF-α is thought to be based on the induction of serine phosphorylation of insulin receptor substrate-1 (IRS-1). When IRS-1 serine is phosphorylated, IRS protein dissociates from the insulin receptor, blocking insulin signaling

[42] . Despite the important role of TNF-α in insulin resistance, it is not the only causal factor. Treatment with TNF-α blockers does not increase insulin sensitivity [43, 44]. However, this is secreted from adipose tissue and, in addition to TNF-α, inst ulin​​​​​​​​​ This is most likely because of the large amounts of inflammatory substances such as leptin, IL-6, resistin, visfatin, and IL-1 that are associated with insulin resistance. In rheumatoid arthritis, TNF-α is one of the major cytokines produced, resulting in the development of insulin resistance. Interestingly, blocking TNF-α with infliximab in RA patients increases insulin sensitivity

[47] . This finding may be explained by the fact that RA is associated with one major inflammatory mediator, whereas obesity is associated with several mediators. Thus, in one embodiment of the present disclosure, fibroblasts are used to induce an anti-inflammatory state or to reduce inflammation in patients with NIDDM. Inflammatory states can be diagnosed by a number of means available to those skilled in the art, including the assessment of C-reactive protein concentration, IL-1, IL-6, TNF, leptin, and IL-18. A variety of fibroblast sources can be used in the practice of the present invention. Furthermore, combinations of fibroblasts for angiogenesis generation and fibroblasts for induction of an anti-inflammatory state are disclosed in the present disclosure. Useful cells can, in some embodiments, include fibroblasts. These cells have been shown to have immunosuppressive and anti-inflammatory functions. In some embodiments of the present disclosure, fibroblast populations are used in combination with one or more agents known to stimulate insulin production or protect the islets from damage. For example, such agents may be amylin analogs. These compounds delay gastric emptying, reduce postprandial glucagon release,

[0100] and regulate appetite, thereby mimicking the actions of amylin. ​ reproduce the effect. Pramlintide acetate is sold under the name "Symlin" and is indicated as an adjunct to mealtime insulin for the treatment of type 1 and type 2 diabetes. In a number of clinical trials, adjunctive pramlintide treatment resulted in improved postprandial blood glucose management and significant decreases in A1C and body weight compared to insulin alone. A variety of agents that can stimulate insulin secretion and / or sensitize peripheral tissues to insulin activity have been the subject of numerous patents. These include, for example, U.S. Patent Nos. 6,121,282 , 6,057,343, 6,048,842, 6,037,359, 6,030 ,990, 5,990,139, 5,981,510, 5,980,902, 5 ,955,481, 5,929,055, 5,925,656, 5,925,64 7, 5,916,555, 5,900,240, 5,885,980, 5,84 9,989, 5,837,255, 5,830,873, 5,830,434, 5,817,634, 5,783,556, 5,756,513, 5,753,7 90, 5,747,527, 5,731,292, 5,728,720, 5,7 08,012, 5,691,386, 5,681,958, 5,677,342 , 5,674,900, 5,545,672, 5,532,256, 5,531, 991, 5,510,360, 5,480,896, 5,468,762, 5, 444,086, 5,424,406, 5,420,146, RE34,878 , 5,294,708, 5,268,373, 5,258,382, 5,019, , 5,268,373, 5,258,382, 5,019, 580, 4,968,707, 4,845,231, 4,845,094, 4, 816,484, 4,812,471, 4,740,521, 4,716,163 No., 4,695,634, 4,681,898, 4,622,406, 4,499 ,279, 4,467,681, 4,448,971, 4,430,337, 4 ,421,752, 4,419,353, 4,405,625, 4,374,14 8, 4,336,391, 4,336,379, 4,305,955, 4,26 2,018, 4,220,650, 4,207,330, 4,195,094, 4,172,835, 4,164,573, 4,163,745, 4,141,8 98, 4,129,567, 4,093,616, 4,073,910, 4,0 52,507, 4,044,015, 4,042,583, 4,008,245 , 3,992,388, 3,987,172, 3,961,065, 3,954, 784, 3,950,518, 3,933,830 are included, and these are incorporated herein by reference in their entirety.

[0101] IV. Kits of the Disclosure Certain aspects of the present disclosure also relate to kits comprising the compositions of the present disclosure or compositions for practicing the methods of the present disclosure. In some embodiments, the kit can be used to provide fibroblasts, including fibroblast regenerative cells, populations thereof, progeny thereof, or conditioned media thereof. In some cases, the kit includes one or more reagents for producing and / or identifying fibroblasts containing regenerative cells.

[0102] The kit may include components that can be individually packaged or placed in a container, such as tubes, bottles, vials, syringes, or other suitable container means. It may include components that can be individually packaged or placed in a container.

[0103] The individual components may also be provided in the kit in concentrated amounts; in some embodiments the components are provided individually at the same concentration as they are in solution with other components. The concentration of the components can be 1x, 2x, 5x, 10x, or 20x or more.

[0104] In certain embodiments, negative and / or positive control agents are included in some kit embodiments. The control molecules can be used to verify enhanced regenerative activity of fibroblasts. The control molecules can be used to verify enhanced regenerative activity of fibroblasts.

[0105] The kit may include a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container can be made of various materials such as glass or plastic. The container can hold a composition containing a probe useful for prognostic or non-prognostic applications as described above. The label on the container can indicate that the composition is for use in a specific prognostic or non-prognostic application and can also indicate directions for any of the in vivo or in vitro uses as described above. The kit may include the above-described container and one or more other containers containing materials desirable from a commercial and user perspective, including buffer solutions, diluents, filters, needles, syringes, and an accompanying document with instructions for use. The container can be made of various materials such as glass or plastic. The container can hold a composition containing a probe useful for prognostic or non-prognostic applications as described above. The label on the container can indicate that the composition is for use in a specific prognostic or non-prognostic application and can also indicate directions for any of the in vivo or in vitro uses as described above. The kit may include the above-described container and one or more other containers containing materials desirable from a commercial and user perspective, including buffer solutions, diluents, filters, needles, syringes, and an accompanying document with instructions for use. including buffer solutions, diluents, filters, needles, syringes, and an accompanying document with instructions for use.

[0106] The kit may include metformin, one or more sulfonylureas, one or more meglitinides, one or more thiazolidinediones, one or more DPP-4 inhibitors, one or more GLP-1 receptor An agonist, one or more SGLT2 inhibitors, and / or one or more additional treatments for a medical condition including diabetes, such as insulin, may or may not include. The kit may or may not include one or more devices and / or reagents for the diagnosis of diabetes and / or the monitoring of blood glucose levels.

Examples

[0107] The following examples are included to demonstrate specific embodiments of the present disclosure. In the following examples The disclosed technology represents the technology discovered by the inventors to function well in the implementation of the disclosed materials, and thus is considered to constitute a specific form for its implementation It should be understood by those skilled in the art that it is possible. However, those skilled in the art can make many changes in the specific embodiments disclosed in light of the present disclosure, and still without departing from the spirit and scope of the present invention, it should be understood that similar or analogous results can be obtained by making many changes in the specific embodiments disclosed, and still without departing from the spirit and scope of the present invention, it should be understood that similar or analogous results can be obtained by making many changes in the specific embodiments disclosed, and still

[0108] 〔Example 1〕 Enhancement of insulin responsiveness in type 2 diabetes

[0109] 100 patients with type 2 diabetes who receive insulin injections daily are recruited. 50 patients are treated with a placebo control, and 50 patients are administered allogeneic umbilical cord blood-derived fibroblasts . As described in the literature (Durdu et al. J Vasc Surg. 2006 Oct;44(4):73 2-9), the cells are intramuscularly injected into the gastrocnemius muscle at a concentration of 40 million cells per limb. The extraction and expansion of umbilical cord blood CD34 will be described later. Umbilical cord blood is obtained by a conventional method The extraction and expansion of umbilical cord blood CD34 will be described later. Umbilical cord blood is obtained by a conventional method method (Rubinstein et al., Processing and cryopreserv Placental / Umbilical cord blood collection for unrelated bone marrow reconstitution .(Proc Natl Acad Sci USA 92:10119-10122) Therefore, it is purified. Briefly, CPD A anticoagulant (citrate / phosphate / dextrose / adenine) (Baxter Health Care, Deerfield, IL) from a standard Baxter 450 ml blood donor set is used to insert a 16-gauge needle and puncture the umbilical vein of the placenta obtained from a healthy delivery from a mother who has been tested for viral and bacterial infections according to international donor standards. Cord blood is drained by gravity so as to drip into the blood bag. The placenta is suspended from a specially constructed support frame to allow collection and reduce contamination by maternal blood and other secretions, and is placed in an absorbent cotton pad lined with plastic. The 63 ml of CPD A used in a standard transfusion bag calculated for 450 ml of blood is reduced to 23 ml by discharging 40 ml into a graduated cylinder immediately before collection. The volume of this anticoagulant better matches the volume of cord blood normally collected (<170 ml).

[0110] An aliquot of the blood is taken for safety testing according to the guidelines of the National Marrow Donor Program (NMDP). Safety testing includes routine clinical tests for human immunodeficiency virus 1 and 2, human T-cell lymphotropic virus I and II, hepatitis B virus, hepatitis C virus, cytomegalovirus, and syphilis. Detection by [method] is included. Subsequently, 6% (wt / vol) hydroxyethyl starch is added to cord blood up to a final concentration of 1.2%. Next, the hydroxyethyl starch mixture in the original blood collection bag is centrifuged (50×g, 5 minutes at 10°C) to separate the supernatant rich in white blood cells. The supernatant rich in white blood cells is extruded from the bag into a 150 ml plasma transfer bag (Baxter Health Care) and centrifuged (400×g, 10 minutes) to sediment the cells. The excess supernatant plasma is transferred to a second plasma transfer bag without cutting the connecting tube. Finally, the sedimented white blood cells are resuspended in the supernatant plasma to a total volume of 20 ml. Approximately 5×10 -7×10 nucleated cells are obtained per umbilical cord. (Baxter Health Care) and centrifuged (400×g, 10 minutes) to sediment the cells. The excess supernatant plasma is transferred to a second plasma transfer bag without cutting the connecting tube. Finally, the sedimented white blood cells are resuspended in the supernatant plasma to a total volume of 20 ml. Approximately 5×10 -7×10 nucleated cells are obtained per umbilical cord. The cells are cryopreserved for subsequent cell therapy according to the method described by Rubinstein et al. (Rubinstein et al., Processing and cryopreservation of placental / umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 92: 10119 - 10122). CD105 cells can be expanded by culture and utilized for the methods of the present disclosure. CD105+ cells are purified from the mononuclear cell fraction by immunomagnetic separation using the Magnetic Activated Cell Sorting (MACS) CD34+ progenitor cell separation kit (Miltenyi - Biotec, Auburn, CA) according to the manufacturer's recommendations. The purity of the obtained CD34+ cells is 8 -7×10 9 nucleated cells are obtained per umbilical cord. The cells are cryopreserved for subsequent cell therapy according to the method described by Rubinstein et al. (Rubinstein et al., Processing and cryopreservation of placental / umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 92: 10119 - 10122). CD105 cells can be expanded by culture and utilized for the methods of the present disclosure. CD105+ cells are purified from the mononuclear cell fraction by immunomagnetic separation using the Magnetic Activated Cell Sorting (MACS) CD34+ progenitor cell separation kit (Miltenyi - Biotec, Auburn, CA) according to the manufacturer's recommendations. The purity of the obtained CD34+ cells is d cryopreservation of placental / umbilica l cord blood for unrelated bone marrow r econstitution.Proc Natl Acad Sci USA 92: 10119 - 10122). CD105 cells can be expanded by culture and utilized for the methods of the present disclosure. CD105+ cells are purified from the mononuclear cell fraction by immunomagnetic separation using the Magnetic Activated Cell Sorting (MACS) CD34+ progenitor cell separation kit (Miltenyi - Biotec, Auburn, CA) according to the manufacturer's recommendations. The purity of the obtained CD34+ cells is determined by flow cytometry. CD105+ cells are purified from the mononuclear cell fraction by immunomagnetic separation using the Magnetic Activated Cell Sorting (MACS) CD34+ progenitor cell separation kit (Miltenyi - Biotec, Auburn, CA) according to the manufacturer's recommendations. The purity of the obtained CD34+ cells is determined by flow cytometry. - Site metric evaluation (FACScan flow cytometer, Becton-Dick inson, Immunofluorometry systems, Mountain View, CA) is in the range of 95% to 98%. Cells are cultured in a 24-well culture plate (Falcon; Becton D ickinson Biosciences) with a final volume of 0.5 ml in DMEM supplemented with 20 ng / ml IL-3, 250 ng / ml IL-6, 10 ng / ml SCF , 250 ng / ml TPO, 100 ng / ml flt-3L, and 50% of LPCM at a concentration of 10.sup. 4 cells / ml. LPCM is generated by obtaining fresh human placenta from vaginal delivery and placing it in a sterile plastic container. The placenta is rinsed with an anticoagulant solution containing 1: 1000 concentration of heparin (1% w / w) (American Pharmaceutic al Partners, Schaumburg, IL) in phosphate-buffered saline water (Gibco-Invitrogen, Grand Island, NY). Then, the placenta is covered with DMEM medium (Gibco) in a sterile container, immersing the entire placenta in the medium, and incubating at 37°C for 2 4 hours in a humidified 5% CO incubator. At the end of 24 hours, the fresh placenta conditioned medium (LPCM) is isolated from the container and sterile filtered using a commercially available sterile 0.2 micron filter (VWR). Cells are grown, checked for purity using CD34-specific flow cytometry, and immunologically matched to the recipient using a mixed lymphocyte reaction. Against recipient lymphocytes The placenta is immersed in the medium, and the whole placenta is immersed in the medium, and incubated at 37°C for 2 2 4 hours in a humidified 5% CO incubator. At the end of 24 hours, the fresh placenta conditioned medium (LPCM) is isolated from the container and sterile filtered using a commercially available sterile 0.2 micron filter (VWR). Cells are grown, checked for purity using CD34-specific flow cytometry, and immunologically matched to the recipient using a mixed lymphocyte reaction. Against recipient lymphocytes The placenta is immersed in the medium, and the whole placenta is immersed in the medium, and incubated at 37°C for 2 4 hours in a humidified 5% CO incubator. At the end of 24 hours, the fresh placenta conditioned medium (LPCM) is isolated from the container and sterile filtered using a commercially available sterile 0.2 micron filter (VWR). Cells are grown, checked for purity using CD34-specific flow cytometry, and immunologically matched to the recipient using a mixed lymphocyte reaction. Against recipient lymphocytes ​Cells that induce low levels of allogeneic stimulatory activity are selected for transplantation. The cells are administered as described above. Patients in the treatment group show an increase in responsiveness to insulin beginning two weeks after the injection of the cells.

[0111] [[Example 2]] Increase in insulin responsiveness after allogeneic fibroblast regenerative cells Embodiments of the present disclosure include methods of increasing insulin responsiveness and / or insulin sensitivity upon administration of fibroblasts comprising specific fibroblasts. In certain embodiments, the fibroblasts are CD34+ and / or CD133+.

[0112] In one study, a group of 100 patients with type 2 diabetes who were receiving daily insulin injections was utilized. Fifty patients were treated with a placebo control and 50 were administered allogeneic skin-derived fibroblasts as an example of fibroblasts. Patients in the treatment group show an increased responsiveness to insulin beginning, for example, 1, 2, 3, 4 weeks or more after the injection of the cells.

[0113] [[Example 3]] Intravenous administration of fibroblasts increases glucose tolerance Five-week-old C57BL / 6 mice were fed a high-fat diet (HFD) in which 60% of the calories were obtained from fat for a total of 24 weeks. After 23 weeks of HFD feeding, the mice were injected daily for 3 consecutive days with 40 mg / kg of streptozocin (Sigma-Aldrich, St. Louis, MO, USA). Streptozocin kills pancreatic cells, creating a model of diabetes including type 2 diabetes (T2D) in mice.

[0114] Human dermal fibroblasts, bone marrow mesenchymal stem cells (BM-MSC) or adipose MSC, passage 3​​​​​​​​​​​ cultured in Opti-MEM medium containing 10% fetal bovine serum. The cells were washed in phosphate-buffered saline (PBS) and injected intravenously (5×10 / mouse, 0.2 ml PBS) via the tail vein into T2D recipients generated by HFD and STZ injection after 24 weeks of HFD. 5 A group of 10 mice per treatment was used. Non-fasting blood glucose levels were measured daily using a Freestyle Lite blood glucose meter

[0115] (Abbott). An intraperitoneal glucose tolerance test was performed 2 weeks after cell injection with blood samples collected at baseline ( 0 minutes), 30 minutes, and 120 minutes after administration of (2 g / kg glucose). In animals administered fibroblasts, better control of blood glucose levels was shown compared to the other two types of MSCs.

[0116] References All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the present invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated as being incorporated by reference.

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Pharmacol Toxicol, 2002. 91(6): p. 414-20. 13. Suarez-Pinzon, W.L.,ら, Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet {beta}-cells from pancreatic duct cells and an increase in functional {beta}-cell mass. J Clin Endocrinol Metab, 2 005. 90(6): p. 3401-9. 14. Suarez-Pinzon, W.L.,ら, Combination therapy with epidermal growth factor and gastrin increases beta-cell mass and reverses hyperglycemia in diabetic NOD mice. Diabetes, 2005. 54(9): p. 2596-601. 15. DeFronzo, R.A., Lilly lecture 1987. The triumvirate: beta-cell, muscle, l iver. A collusion responsible for NIDDM. Diabetes, 1988. 37(6): p. 667-87. 16. Cheetham, C.,ら, Losartan, an angiotensin type I receptor antagonist, imp roves conduit vessel endothelial function in Type II diabetes. Clin Sci (Lond), 2001. 100(1): p. 13-7. 17. Kajiguchi, M.,ら, Safety and efficacy of autologous progenitor cell trans plantation for therapeutic angiogenesis in patients with critical limb ischemia. Circ J, 2007. 71(2): p. 196-201. 18. Tsai, P.T., et al., A critical role of erythropoietin receptor in neurogenesis and post-stroke recovery. J Neurosci, 2006. 26(4): p. 1269-74. 19. Ogueta, S., et al., Prolactin is a component of the human synovial liquid and modulates the growth and chondrogenic differentiation of bone marrow-derived mes enchymal stem cells.Mol Cell Endocrinol, 2002. 190(1-2): p. 51-63. 20. Banerjee, M., M. Kanitkar, and R.R. Bhonde, Approaches towards endogenous pancreatic regeneration. Rev Diabet Stud, 2005. 2(3): p. 165-76. 21. Wang, C., et al., Mechanical, cellular, and molecular factors interact to modu late circulating endothelial cell progenitors. Am J Physiol Heart Circ Physiol, 2004. 286(5): p. H1985-93. 22. Yildirim, S., et al., Expansion of cord blood CD34+ hematopoietic progenitor c ells in coculture with autologous umbilical vein endothelial cells (HUVEC) is su prior to cytokine-supplemented liquid culture. 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[0119] 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 The present invention is limited to certain embodiments of the process, machine, manufacture, composition of matter, means, methods and steps. As will be readily understood from this disclosure, those skilled in the art will appreciate that the present disclosure is not intended to 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, method 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] 2. A method for treating insulin resistance in an individual, comprising delivering a therapeutically effective amount of fibroblasts to the individual. A method for treating or preventing

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