Treatment of neurological damage associated with diabetes and its complications with fibroblasts and fibroblast-immune cell combinations

Abstract

To provide compositions for effective treatment of chronic traumatic encephalopathy (CTE) and other types of subconcussive-associated and concussive-associated neurological damage.SOLUTION: Embodiments of the disclosure include treatments of subconcussive and / or concussive brain damage by administering fibroblasts and / or fibroblasts cultured with one or more types of immunocytes. In one specific embodiment, fibroblasts are cultured with monocytes in the presence of patient-specific T cells, and subsequently the T cells are re-administered into the patient. In one particular embodiment, products derived from fibroblast-immunocyte mixtures are comprised of cellular lysate, apoptotic bodies, exosomes, and / or other microvesicles. In one embodiment, the fibroblast cells and / or products derived from the fibroblast cells are administered subsequent to one or multiple head injuries. In other embodiments, products are administered in combination with neurorestorative and / or neuroprotective interventions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (Reference to Related Application) This application was filed on September 9, 2019, and is incorporated herein by reference in its entirety. This application claims priority to U.S. Provisional Patent Application No. 62 / 897,428.

[0002] (Technical field) The disclosed embodiments are directed to at least one of the following fields: cell biology, molecular biology, neurology, physiology, biochemistry, It encompasses the fields of immunology and medicine. [Background technology]

[0003] Chronic traumatic encephalopathy (CTE) was originally identified in the sport of boxing as a condition known as "punch-drunk." A professional boxer with multiple seizures and repeated head injuries has been described as a syndrome. It is known that repeated head injuries can lead to chronic traumatic encephalopathy (CTE). CTE can result in rotational acceleration of the brain, diffuse axonal injury, and other neuropathological features. Motor changes such as tremor, dysarthria, and parkinsonism; cognitive changes such as mental retardation and memory impairment These include psychiatric changes such as explosive behavior, pathological fantasies, pathological addictions, and [1-6] can be.

[0004] In the UK, at least 17% of boxers suffer from CTE, which causes problems with walking and coordination, and In addition to inarticulate speech and tremors, they have brain dysfunction that causes cognitive and neurobehavioral disorders. [7] One study found that conventional MR imaging did not reveal Diffusion tensor imaging (DTI), which is sensitive to microscopic white matter changes [8,9], was used to assess executive function. Neuropsychological testing of memory along with tract-based spatial statistics In conjunction with TBSS (Training Statistics), male amateur boxers A cohort of 31 people and 31 age-matched controls and an additional IQ A matched subgroup of 19 participants was studied. Participants underwent neurological and conventional MRI showed normal findings. The fractional anisotropy was significantly reduced, but the diffusion These changes were due in part to the effects of the competition. This study showed that TBSS was related to the number of fights in amateur boxers. These findings demonstrated widespread white matter damage associated with incident T The findings are very similar to those in BI patients, and amateur boxers also show similar histological changes.

[10]

[0005] In addition to boxing, jockeys have also been reported to suffer from CTE. In a 1976 publication, Foster et al. National Hunt jockeys (PJJs) presented with post-traumatic brain injury with epilepsy and significant reported that two patients had significant intellectual and psychological deterioration

[11] . Other reports of jockeys with similar conditions have been described

[12] . It is also used in whiplash,

[13] shaken baby syndrome,

[14] wrestling,

[15] and military combat. Fighting [16, 17], soccer [18-22], rugby

[23] , football [24, 25 ], prison head injuries

[26] , shotgun injuries

[27] , and mixed martial arts

[28] . And many other things are mentioned.

[0006] One study in JAMA looked at 202 football players whose brains were donated for research. A case series was investigated. Neuropathological evaluation by informants and retrospective clinical observations by telephone were performed. A floor assessment (including head trauma history) was conducted in a blinded manner. Exercise history was assessed by an online questionnaire. and military history. Neurological disorders, including CTE, based on defined diagnostic criteria. Physical diagnosis; neuropathological severity of CTE (stage I to stage IV, or mild [stage I and II]) and severe [stages III and IV]); informant-reported exercise history; and For players who died after 2014, clinical features such as behavior, mood, and cognitive symptoms, as well as dementia, were examined. 202 former soccer players died (median age at death: 66 years [interquartile range: 47-76 years]) ), 177 athletes (87%; median age at death) were diagnosed with CTE neuropathologically. Age 67 years [interquartile range 52–77 years]; mean years of soccer participation 15.1 [SD, 5.2] ) 0 out of 2 former high school students, 3 out of 14 high school students (21%), and 48 out of 53 university students (91%). , 9 out of 14 semi-professionals (64%), 7 out of 8 Canadian Soccer League players (88%), none The neuropathological significance of CTE was 110 of 111 players (99%) from the National Soccer League. The severity of the disease was distributed across the highest levels of play, with all three former high school players having mild pathology. The following were pre-university (27 people [56%]), semi-professional (5 people [56%]), and professional (101 people [86%]) players: The majority of athletes had severe pathology. Of the 27 participants with mild CTE pathology, Of these, 26 (96%) had behavioral or mood symptoms or both, and 23 (85%) ) had cognitive symptoms, and 9 (33%) had signs of dementia. Of the 84 participants with a history of psychiatric disorders, 75 (89%) had behavioral or mood symptoms or both. 80 patients (95%) had cognitive symptoms, and 71 patients (85%) had signs of dementia. In a convenience sample of cadaveric football players who donated their brains for research, a high percentage Neuropathological evidence of CTE was found in 2014, and CTE was associated with pre-participation in football. It has been suggested that these may be related.

[29]

[0007] In another study, the authors compared tackle football players with neuropathologically confirmed CTE. Pathological severity and neurobehavioral manifestations of chronic traumatic encephalopathy (CTE) in soccer players The effect of age on age at first exposure to tackle football was examined. The study included 246 soccer players who provided brains for neuropathological examination. 211 cases were diagnosed with CTE (126 of the 211 cases did not have a concomitant neurodegenerative disease), and 35 None of the cases had CTE. Informed interviews revealed that the age of first exposure Age at onset of cognitive and behavioral / mood symptoms was ascertained. Analyses were performed over a 10-year and recurrence period. Age at exposure was associated with pathological severity of CTE, Alzheimer's disease, or Lewy body pathology. In the 211 participants with CTE, each year of life increased the risk of developing a 2.44-year-old younger participant. Cognitive symptom onset reported by age (p<0.0001) and behavioral / mood symptoms by age 2.5 years The earlier they started playing tackle football, the better they predicted their condition (p<0.0001). Age at exposure 2 years or earlier was associated with earlier cognitive decline (p<0.01) by 13.39 and 13.28 years, respectively. <0.0001) and behavioral / mood (p<0.0001) symptoms predicted dementia participation. In patients with CTE, a younger age at exposure corresponded to an earlier onset of functional impairment. A similar effect was observed in 126 participants with CTE. In participants without CTE, the effect size was In this sample of deceased Thakur footballers, the incidence was similar at a younger age. Tackle football exposure was not associated with CTE pathological severity, but earlier Exposure in young people playing soccer predicted the development of neurobehavioral symptoms. This may reduce resilience to learning.

[30]

[0008] The present disclosure provides information on the effects of CTE and other types of sub-aggregation-related and concussion-related neurological damage. provides effective treatment or prevention. Summary of the Invention

[0009] The present disclosure relates to the field of head trauma, and more particularly to methods for the treatment of CTE. It concerns the field of chronic traumatic encephalopathy (CTE), including the field of regenerative medicine.

[0010] Disclosed are altered states and conditions such as chronic traumatic encephalopathy (CTE). fibroblasts and / or cells cultured with any kind of immune cells and / or stem cells Novel and useful methods for the treatment of concussive and / or post-concussive brain injury using fibroblasts In one embodiment of the present disclosure, the cell composition The combination is intended to a) reduce oxidative stress; b) inhibit inflammation; and c) promote neuroprotection. and / or d) to stimulate axonal regrowth. In embodiments, fibroblasts (e.g., derived from umbilical cord and / or other perinatal tissues, for example) The resulting cells are cultured with immune cells (e.g., monocytes) in the presence of patient-specific T cells, followed by In another embodiment, the fibroblasts are derived from bone marrow. In yet another embodiment, the fibroblasts are adipose-derived. -Products derived from immune cell mixtures include cell lysates, apoptotic bodies, exosomes, and In one embodiment, the microvesicles are composed of fibroblasts and / or fibroblast-derived microvesicles. In another embodiment, the product is administered following one or more head injuries of any kind. The product may be administered concurrently with or without administration of any cells to provide neurorestorative and / or neuroprotective effects. It is administered in conjunction with protective interventions.

[0011] The foregoing describes the features and technical advantages of the present disclosure in order that the following detailed description may be better understood. have been outlined quite broadly. Additional features and advantages form the subject of the claims herein. The disclosed concepts and specific embodiments accomplish the same objectives of the present design. Those skilled in the art will recognize that the present invention may be readily utilized as a basis for modifying or designing other structures for Such equivalent constructions are also within the scope of the appended claims. It should be understood by those skilled in the art that the present invention will not depart from the spirit and scope of the invention as described herein. The novel features believed characteristic of the designs disclosed herein provide further objects and advantages. together with the points, both as to construction and method of operation, when considered in conjunction with the accompanying drawings. However, the figures are for purposes of illustration and explanation only and are not intended to be limiting unless otherwise specified. It should be expressly understood that these figures are provided for purposes of illustration only and are not intended as a definition of the limits of the present disclosure. is. [Brief explanation of the drawings]

[0012] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0013] [Figure 1] Figure 1 shows that fibroblasts synergize with monocytes to produce brain-derived neurotrophic factor (BDNF). Bars from left to right are control, fibroblasts alone, monocytes alone, and a mixture of fibroblasts and monocytes.

[0014] [Figure 2] Figure 2 shows that fibroblasts synergize with CD34 in producing BDNF. Bars from left to right are control, fibroblasts alone, CD34 alone, and a mixture of fibroblasts and CD34.

[0015] [Figure 3] Figure 3 demonstrates that fibroblasts synergize with MSCs in producing BDNF. Bars from left to right are control, fibroblasts alone, MSCs alone, and a mixture of fibroblasts and MSCs.

[0016] [Figure 4] Figure 4 shows that fibroblasts synergize with monocytes to inhibit neuronal death. Bars from left to right are control, fibroblasts alone, monocytes alone, and a mixture of fibroblasts and monocytes.

[0017] [Figure 5] Figure 5 shows that fibroblasts synergize with CD34 to inhibit neuronal death. Bars from left to right are control, fibroblasts alone, CD34 alone, and a mixture of fibroblasts and CD34.

[0018] [Figure 6] Figure 6 shows that fibroblasts synergize with MSCs to inhibit neuronal death. Bars from left to right are control, fibroblasts alone, MSCs alone, and a mixture of fibroblasts and MSCs.

[0019] [Figure 7]Figure 7 shows that fibroblasts synergistically stimulate neurogenesis with monocytes. Bars from left to right are control, fibroblasts alone, monocytes alone, and a mixture of fibroblasts and monocytes.

[0020] [Figure 8] Figure 8 shows that fibroblasts synergize with CD34 to stimulate neurogenesis. Bars from left to right are control, fibroblasts alone, CD34 alone, and a mixture of fibroblasts and CD34.

[0021] [Figure 9] Figure 9 demonstrates that fibroblasts synergize with MSCs to stimulate neurogenesis. Bars from left to right are control, fibroblasts alone, MSCs alone, and a mixture of fibroblasts and MSCs.

[0022] [Figure 10] FIG. 10 shows that fibroblast conditioned medium protects neurons from injury. DETAILED DESCRIPTION OF THE INVENTION

[0023] I. Definition example In accordance with long-standing patent law convention, the terms "a" and "an" include the claims, As used herein, "one or more" refers to some embodiments of the disclosure. consists of or essentially consists of one or more elements, method steps, and / or methods of the disclosure Any of the methods or compositions described herein may be It is contemplated that it may be used with respect to any other method or composition described.

[0024] As used herein, the terms "about" or "approximately" refer to a reference amount, level, number, frequency, or , percentage, dimension, amount, weight, or length 30, 25, 20, 25, 10, Amount, level, number, or percentage that varies by 9, 8, 7, 6, 5, 4, 3, 2, or 1% , size, percentage, dimension, amount, weight or length. When "about" or "approximately" precedes a numerical value, it indicates a range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, this term refers to the value It can mean within an order of magnitude, such as within 5 times or within 2 times. Unless otherwise stated, the term "about" refers to an acceptable error range for a particular numerical value.

[0025] The terms "administered" or "administering" as used herein refer to the administration of a composition to an individual. It refers to any method of providing a composition to an individual so that it has its intended effect on the individual. For example, one method of administration is via a medical device such as a catheter, applicator gun, or syringe. A second exemplary administration includes, but is not limited to, an indirect mechanism using a Methods include direct mechanisms, e.g., local tissue administration, oral ingestion, transdermal patch, topical, inhalation, suppository, etc. by.

[0026] As used herein, "allogeneic" refers to a gene derived from one or more individuals of the same species, but Another antibody that is or may be immunologically incompatible in its natural setting It refers to tissues or cells or other materials derived from the body.

[0027] As used herein, the term "allograft" refers to the transplantation of an organ from a donor to a recipient. Transplantation of tissue and / or cells, where the donor and recipient are different individuals. Tissue transplanted by such a procedure is called an allograft or This is called an allograft.

[0028] As used herein, the terms "allo-stimulatory" and "allo-responsive" refer to the alloantigen or Stimulation of the immune system to respond to "alloantigens" or cells expressing foreign HLA haplotypes and reactants.

[0029] As used herein, "autologous" refers to any organism that originates from or is derived from the same individual's body. refers to tissues or cells or other materials transferred from a donor (i.e., autologous blood donation; autologous bone marrow transplant). .

[0030] As used herein, the term "autologous transplant" refers to the transfer of a tissue from a part of the body of an individual to the same tissue. Transplantation of organs, tissues, and / or cells into another part of the same individual (i.e., donor (The transplanter and recipient are the same individual.) The tissue is called an autograft or autotransplant.

[0031] The term "biologically active" refers to any molecule that has a structural, regulatory, or biochemical function. For example, biological activity refers to the production of a protein that is a wild-type increase in a cell lacking the protein activity. Cells lacking protein activity can be identified by a number of methods (i.e., e.g., recovery of growth). complementation can be achieved by a number of mutations, including point mutations and frameshift mutations; Transfecting cells lacking the gene with an expression vector that expresses the protein, its derivative, or a portion thereof. In other cases, this is achieved by transfecting a fragment of the gene product. A construct (e.g., a protein) is biologically active if it retains the activity of the full-length gene product. Although it may be considered functionally active (or referred to as functionally active), it is not the full-length gene. The activity of the product may be reduced but still detectable.

[0032] "Cell culture" refers to any cell culture, whether quiescent, senescent, or (actively) dividing. An artificial in vitro system containing living cells. In cell culture, cells are grown at a temperature of, for example, 37°C. They are grown and maintained at suitable temperatures and typically in an atmosphere containing oxygen and CO2. In other cases, these are altered. Culture conditions can vary widely for each cell type, but Variation in conditions for a particular cell type can result in different phenotypes being expressed. The factor most commonly varied is the growth medium. The growth medium contains nutrients, growth factor concentrations, The growth factors used to supplement the medium may vary in the presence of ATP and other components. Often derived from animal blood, such as bovine serum.

[0033] "Comprising" refers to the inclusion of any listed steps or elements or groups of steps or elements. means one step or element, or the exclusion of another step or element, or group of steps or elements. It will be understood that "consisting of" is not This means that "consist" means "to include, but not be limited to, anything that follows." The phrase "ing of" indicates that the listed elements are required or essential and that other elements may be present. "Consisting essentially of" means that the material does not contain any of the elements listed after the phrase. and does not interfere with the activity or function specified in the disclosure for the recited elements. Therefore, the term "essentially different from" is used to refer to the product or service that is not essential to the product or service. "Required" indicates that the listed elements are required or mandatory, but other elements are not optional, and may or may not be present depending on whether it affects the activity or operation of the listed elements. good.

[0034] As used herein, the term "drug," "agent," or "compound" refers to any substance that achieves a desired effect. Drugs or compounds refer to any pharmacologically active substance that can be administered to a patient. or naturally occurring, non-peptide, protein or peptide, oligonucleotide, or It can be a nucleotide (DNA and / or RNA), a polysaccharide or a sugar.

[0035] As used herein, the term "individual" refers to a person who may or may not be housed in a medical facility. An individual is a human or animal that may be treated as an outpatient in a medical facility. The individual may receive one or more medical compositions via any of the following methods: and therefore includes both adults and young children (i.e., children) and infants. The term "individual" is not intended to imply a need for medical treatment and therefore does not refer to an individual. People may volunteer or contribute to research, whether clinical or in support of basic science. The term "subject" or "individual" refers to a mammal, e.g., a human , laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep) , goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), and transgenic non-human animals, including rodents, horses, and transgenic non-human animals. refers to any living organism or animal subject.

[0036] Throughout this specification, the terms "one embodiment," "one embodiment," "particular embodiment," "related embodiment," "related" and "related" are used interchangeably. "related embodiments," "particular embodiments," "additional embodiments," or "further embodiments" Reference to any particular feature, structure, or combination thereof described in connection with an embodiment is not intended to be limiting. It is intended that the composition or feature be included in at least one embodiment of the present invention. Therefore, appearances of the foregoing phrases in various places throughout this specification do not necessarily all refer to the same implementation. Moreover, any particular feature, structure, or characteristic may be incorporated into one or more embodiments. The components may be combined in any suitable manner.

[0037] As used herein, the term "pharmaceutically" or "pharmacologically acceptable" refers to an animal or will not produce adverse, allergic, or other untoward reactions when administered to humans. It refers to the fruiting body and composition.

[0038] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier such as water, ethanol, polyisoprene, or the like. ols (e.g., glycerol, propylene glycol, and liquid polyethylene, glycosyl and suitable mixtures thereof, as well as vegetable oils, coatings, isotonic and absorption-delaying agents. Any suitable anti-inflammatory agent, including but not limited to anti-inflammatory agents, liposomes, commercially available detergents, etc. Supplementary biologically active ingredients may also be used in such compositions. The signal processing unit 100 may be incorporated into a carrier.

[0039] Refers to the occurrence of any symptoms in untreated subjects compared to treated subjects. When referring to (including "lower," "smaller," etc.) refers to the amount and severity of symptoms in treated subjects. and / or magnitude are not recognized as clinically relevant by any medically trained personnel. Any amount of the compound administered will result in a reduction in the amount and / or magnitude of symptoms in the treated subject. In one embodiment, the amount and / or magnitude of symptoms in a treated subject is compared to an untreated subject. At least 10% lower and at least 25% lower than the amount and / or size of symptoms in elephants at least 50% lower, at least 75% lower, and / or at least 90% lower.

[0040] II. Embodiment The present disclosure encompasses the treatment and prevention of brain-related medical conditions, including injury and / or disease. Certain methods of the present disclosure treat or prevent neurological damage. It may be a disability. It is not possible to treat or prevent any type of brain injury, including traumatic brain injury. Injuries may include hematoma, bleeding, concussion, edema, a mixture of these, etc. Types of traumatic brain injury include contusion, second impact syndrome, and Coup-Contrec oupThere is brain injury, shaken baby syndrome, and / or penetrating injury.

[0041] A medical condition may be the result of a single injury or repeated injuries. The wound may be due to physical contact, including as a result of work and / or sports. The damage may occur over the course of an individual's lifetime, years, months, or years before any one or more symptoms appear. It may have occurred over a period of time, including days or weeks. In certain embodiments, the medical condition The condition is called chronic traumatic encephalopathy (CTE), which includes pugilistic a) Dementia is included. Individuals may be involved in sports such as football, boxing, wrestling, soccer, and hockey. recreational sports such as basketball, baseball, softball, and rugby. Athletes may be athletes, including those involved in sports or professional activities. Individuals may be at risk for injury due to sports and require prevention. construction workers, first responders, warehouse workers, and others who have suffered head injuries related to their jobs. Any type of worker may have a medical condition. This may put you at risk of injury.

[0042] In certain embodiments, the present disclosure provides methods for the treatment of various types of stem cells, such as mesenchymal stem cells, monocytes, and / or hematopoietic stem cells. fibroblasts and administration of fibroblasts with other cells enhances production of one or more regenerative factors triggering a series of biochemical and cellular reactions that allow for the development of CTE associated with regeneration / protection / prevention in individuals suffering from concussive and / or subcontact injuries More specifically, the term encompasses novel, useful, and unexpected discoveries that are useful in preventing or preventing The present invention relates to fibroblasts, and / or fibroblasts in combination with one or more other compositions (including cells). The combination of In one embodiment, the present invention provides a method for inhibiting mitochondrial activation in an individual having a CTE. As a means of enhancing neuronal function and / or protecting neurons in the body, The therapeutically effective amount of fibroblasts is administered.

[0043] In some embodiments, the fibroblasts are utilized in an autologous manner for the individual. In embodiments, allogeneic fibroblasts are utilized for the practice of the methods of the present disclosure. Various sources of fibroblasts can be used to practice the disclosed methods, including: a) follicle fibroblasts; skin; b) adipose tissue; c) skin biopsy; d) bone marrow; e) placenta; f) umbilical cord; g) amniotic fluid; h) umbilical cord blood; i) earlobe skin; j) embryonic fibroblasts; k) plastic surgery-related byproducts; and / or l) nails. Contains a matrix.

[0044] In one embodiment, the present disclosure provides methods for the activation of fibroblasts prior to therapeutic use, and and / or administration of one or more agents that act as fibroblast "regenerative adjuvants" In certain embodiments, when the cells in the formulation are grown in a culture monolayer, they exhibit a typical fibroblastic Cell morphology is shown, specifically cells have an elongated spindle or spindle-shaped appearance with elongated extensions The cells may appear as larger, flattened astrocytes that may show a cytoplasmic leading edge. A mixture of these morphologies can also be observed. Cells can be, for example, fibroblasts. specific marker, CD90 (Thy-1), a 35 kDa cell surface glycoprotein, and Extracellular matrix proteins, including collagen, characteristic of normal fibroblasts Fibroblast dosage formulations can be cultured in individual cells using standard tissue culture procedures. Autologous cell therapy generation consisting of a suspension of autologous fibroblasts grown from a skin biopsy. In one embodiment, the fibroblasts of the present disclosure are also used in tissue repair and / or regeneration. It can be used to generate other cell types for growth.

[0045] Embodiments of the present disclosure provide an individual in need thereof with a medicament containing a medicament, cultured with one or more types of immune cells. Concussive and / or concussive fibroblasts administered by administering In one particular embodiment, the fibroblasts are T Cultured with monocytes in the presence of cells (autologous, allogeneic, or xenogeneic to the recipient individual). In certain embodiments, the fibroblast-immunocyte T cells are administered to the individual. The products derived from the cell mixture include cell lysates, apoptotic bodies, exosomes, and / or In one embodiment, the microvesicles are composed of fibroblasts and / or fibroblast-derived microvesicles. In another embodiment, the product is administered following one or more head injuries. , administered in combination with neurorestorative and / or neuroprotective interventions.

[0046] Embodiments of the present disclosure include a therapeutically effective amount of regenerative cells and / or one or more regenerative cell-derived administering the product; and, if necessary, administering the regenerative cells and / or the product derived from the cells. and treating a subconcussive or concussive brain injury in an individual, comprising repeating the administration of a product comprising: The regenerative cells include one or more types of immune cells and / or one or more In certain cases, the fibroblasts may be cultured with stem cells of the same type. The cells have been cultured with monocytes, mesenchymal stem cells, and / or hematopoietic stem cells, and in some cases In this case, the product from the fibroblasts at any step of this method can be used by an individual in need thereof. In some cases, an individual may experience one or more inflammations following a head and / or neck injury. are identified as having an elevation of the marker.

[0047] Embodiments in which fibroblasts and / or products derived therefrom are delivered to an individual in need thereof In this embodiment, fibroblasts can be naturally regenerative or engineered to be regenerative. In such cases, the fibroblasts may express one or more specific markers and / or For example, fibroblasts lack Oct-4, Nan og, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, C Selected from the group consisting of D105, CD117, CD344 Stella, and combinations thereof Fibroblasts may express markers such as CD10, CD13, CD44, and CD73. , CD90, CD141, PDGFr-α, HLA-A, HLA-B, HLA-C, and and the expression of a marker selected from the group consisting of these combinations. Fibroblasts express MHC class I, MHC class II, CD45, CD13, and CD 49c, CD66b, CD73, CD105, CD90, and combinations thereof The fibroblasts may additionally or alternatively lack expression of a marker selected from: CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP , HLA-DQ, and combinations thereof. Or alternatively can be absent.

[0048] In certain embodiments, any cell used in any of the methods encompassed herein is regenerated. cells, such cells being fibroblasts and hematopoietic stem cells (and / or mesenchymal stem cells), optionally Optionally, any type of immune cell can be included in combination. Hematopoietic stem cells can be used to treat immunodeficient host cells. Hematopoietic stem cells express the c-kit protein, Sc a-1 protein, CD34, and / or CD133, which They may lack expression of one or more lineage markers; they may lack expression of CD38. They may be positive for the expression of c-kit and Sca-1 and may contain one or more may substantially lack expression of multiple lineage markers.

[0049] When mesenchymal stem cells are utilized, they may express one or more markers, and may lack expression of one or more markers. In certain cases, they include: a) CD7 b) CD90; c) CD105; and d) combinations thereof. and / or they express markers that are a) CD14; b) CD45; c) CD34 and d) lacking expression of a marker selected from the group consisting of combinations thereof. Cut.

[0050] In certain embodiments, the mesenchymal stem cells are derived from a particular tissue, and as a result, can be derived from one or more myeloid cells. markers, and they may lack expression of one or more markers. In some cases, they are a) bone marrow; b) peripheral blood; c) adipose tissue; d) mobilized peripheral blood; e) Umbilical cord blood; f) Wharton's jelly; g) umbilical cord tissue; h) skeletal muscle tissue; i) subepithelial umbilical cord; j) endometrial tissue; k) menstrual blood; l) fallopian tube tissue; and m) combinations thereof. may be derived from a selected tissue

[0051] In one embodiment, the fibroblasts utilized in the present disclosure are derived from the recipient's own tissue. (e.g., skin) (in the case of autologous preparations), or tissue (e.g., skin) from a healthy donor (in the case of In some embodiments, the cells are produced by propagation from a biopsy (in the case of a seed preparation). The fibroblasts are used from a young donor. In another embodiment, the fibroblasts are augmented. Transformation with one or more genes to allow controlled growth and overcoming the Hayflick limit Following cell derivation, the cultures can be expanded using standard cell culture techniques. Skin tissue (dermal and epidermal layers) can be biopsied, for example, from the post-auricular region of a subject. In one embodiment, the starting material is collected using standard sterile techniques. Consists of a 3 mm punch skin biopsy. The biopsy was collected by the treating physician. Place the biopsy in a vial containing sterile phosphate-buffered saline (PBS). Store the biopsy in a refrigerator at 2-8°C. In one embodiment, after arriving at the manufacturing facility, the biopsy is inspected. Once accepted, they are transported directly to the manufacturing area. The biopsy tissue is washed prior to enzymatic digestion. After washing, Liberase Digestiv e Add Enzyme Solution without cutting the biopsy tissue into small pieces and heat to 37.0°C. Incubate for 1 hour at 2°C. The duration of biopsy tissue digestion determines the viability and survival of cells in culture. Liberase is a key process parameter that can affect growth rate. Formulated by Walkersville, Inc. (Walkersville, Md.) Roche Diagnostics Corp. (Indianapolis, I It is a collagenase / neutral protease enzyme cocktail not prescribed by N. Alternatively, other commercially available collagenases (e.g., Serva Collagenase NB6) may be used. (Helidelburg, Germany) can be used. After digestion, the starting growth medium (IMDM, GA, 10% fetal bovine serum (FBS)) was added to neutralize the enzyme, and the cells were centrifuged. Pellet by centrifugation and resuspend in 5.0 mL of starting growth medium. No isolation is performed and complete inactivation of the enzyme occurs by addition of the initial growth medium only. Before seeding the cell suspension into T-175 cell culture flasks for the initiation of growth and proliferation, Add growth initiation medium. T-75, T-150, T-18 flasks instead of T-75 flasks. Incubate the cells at 37±2.0°C for 5.0±1 min in a T-225 flask. Incubate with 0.0% CO2 and feed with fresh complete growth medium every 3-5 days. Every feed in this process involves removing half of the complete growth medium and replacing it with the same volume of fresh Alternatively, a complete feeding can be performed. Cells should not be left in the T-175 flask for more than 30 days before culturing. Monitored throughout the process to ensure proper seeding density during fertilization When cell confluence is 40% or greater in a T-175 flask, the spent medium is Remove the cells, wash them, and treat with trypsin-EDTA to remove the adherent cells in the flask. The cells are then passaged by releasing them into a T-5 Alternatively, seed one or two T-300 flasks to continue cell expansion. SC, 1 layer cell stack (1CS), 1 layer cell factory (1CF) or 2 A layered cell stack (2CS) can be used in place of a T-500 flask. Morphology was assessed prior to passaging and harvest to ensure culture purity throughout the process. Morphology is monitored by transferring the observed samples to a laboratory for morphological examination of cell cultures. The cells are assessed by comparison with a visual standard. In this case, the cells show a typical fibroblast morphology. The cells are elongated, spindle-shaped or fusiform with long, thin extensions. They appear as larger, flattened astrocytes that may have a cone-shaped appearance or have a leading edge of cytoplasm. A mixture of these morphologies can also be observed. Fibroblasts may be similarly shaped but randomly oriented. Keratinocytes in cell culture The presence of keratinocytes is also assessed. Keratinocytes appear rounded and irregularly shaped, and the higher the confluency, the better. At lower confluence they appear organized into cobblestone formations. In this case, keratinocytes were observed in small colonies. Incubate at 4°C with 5.0±1.0% CO2 and culture in T-500 flasks every 3–5 days. The cells were then passaged every 5-7 days in 10-layer cell stacks (10CS). The bulk drug substance should not remain in the T-500 flask for more than 1 hour. Quality Control (QC) release testing includes sterility testing and endotoxin testing. When the cell confluence in the culture medium is approximately 95%, the cells are passaged into 10CS culture vessels. Alternatively, two 5-layer cell stacks (5CS) or 10-layer cell factories (10CF) can be used instead of 10CS. Passage through 10CS removes spent medium. The cells were then washed and treated with trypsin-EDTA to bring the adherent cells in the flask into solution. The cells are then transferred to 10CS. Additional complete growth medium is added. In addition, trypsin was neutralized and the cells from the T-500 flask were transferred to a 1000 ml plate containing fresh complete growth medium. Transfer the contents of the 2L bottle to a 10CS tube and seed all layers. The cells were then incubated at 37±2.0°C with 5.0±1.0% CO2. Feed fresh complete growth medium every 5-7 days. Cells should be kept in 10CS for at least 20 days before passaging. In one embodiment, the passaged dermal fibroblasts are expanded fibroblasts. Incubation of the cells in a protein-free medium for a period of time allows the cells to be isolated from the culture medium. The cells in the primary harvest 10CS are substantially free of immunogenic proteins present in the When the cell confluence is 95% or more, the cells are harvested. The cells were washed and treated with trypsin-EDTA to release adherent cells into solution, followed by additional complete This is done by neutralizing the trypsin with the addition of growth medium. The cells are then harvested by centrifugation. The cells were then resuspended and in-process QC tests were performed to determine total viable cell count and cell viability. Determine.

[0052] In certain embodiments, about 50 million to 500 million fibroblasts are administered to a subject. , approximately 50 million to 100 million fibroblasts, approximately 50 million to 200 million fibroblasts, approximately 5000 Approximately 100 million to 300 million fibroblasts, approximately 50 million to 400 million fibroblasts, approximately 100 million to 200 million fibroblasts Cells, approximately 100 million to 300 million fibroblasts, approximately 100 million to 400 million fibroblasts, approximately 100 million to 500 million glands Fibroblasts, approximately 200 million to 300 million fibroblasts, approximately 200 million to 400 million fibroblasts, approximately 200 million fibroblasts Cells: Approximately 200 million to 500 million fibroblasts, approximately 300 million to 400 million fibroblasts, approximately 300 million to 500 million fibers Fibroblasts, approximately 400 million to 500 million fibroblasts, approximately 50 million fibroblasts, approximately 100 million fibroblasts Approximately 150 million fibroblasts, approximately 200 million fibroblasts, approximately 250 million fibroblasts Fibroblasts, approximately 300 million fibroblasts, approximately 350 million fibroblasts, approximately 400 million fibroblasts cells, about 450 million fibroblasts, or about 500 million fibroblasts are administered to the subject. It is possible.

[0053] In some embodiments, fibroblast exosomes can be derived from any type of cell. The exosomes for use in the present invention are used to reduce L-17 production. The fibroblasts can be purified as follows: In one embodiment, the fibroblasts are purified by filtration of the viable fibroblasts. To preserve viability and proliferation potential, the cells are cultured using means known in the art. Disclosed are methods for preparing and establishing individualized autologous exosomes for experimental or biological use. The present invention can be applied to both exosome preparations obtained from selected cell lines. The present disclosure relates more specifically to a method for preparing membrane vesicles, in particular to a method for preventing potential biological contamination. Based on the use of chromatographic separation methods to separate membrane vesicles from substances, wherein The microvesicles are exosomes and are utilized to generate the exosomes. The cells are fibroblasts.

[0054] For the practice of the present disclosure, certain embodiments involve the use of fibroblasts alone or mesenchymal stem cells (MS C) in combination with a compound selected from the group consisting of benzodiazepines (B1, B2, B3, B4), benzodiazepines (B1, B2, B3 ... It is administered intravenously.

[0055] In some embodiments, "mesenchymal stem cells" or "MSCs" are (1) plastic-bound adherent, (2) expressing CD73, CD90, and CD105 antigens, while CD14, CD 34, CD45, and HLA-DR negative, and (3) osteogenic, chondrogenic, and adipogenic systems. Other cells with mesenchymal-like properties are called "mesenchymal stem cells." Included within the definition are cells that: a) have regenerative activity; b) produce growth factors; c) directly; or have at least one of the following abilities: to induce a healing response through the induction of endogenous host repair mechanisms; As used herein, "mesenchymal stromal cells" or mesenchymal stem cells are , may be used interchangeably.

[0056] MSCs can be derived from, but are not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, and trophoblast. tissue, umbilical cord blood, Wharton's jelly, placenta, amniotic tissue, derived from pluripotent stem cells, and MSCs can be derived from any tissue, including the brain and / or teeth. In this study, cells were CD34 positive upon initial isolation from tissue, but were not phenotypically or functionally distinct. As used herein, "MSC" includes cells similar to those described above. NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56 , CD63, CD73, CD105, CD106, CD140b, CD146, CD27 1, MSCA-1, SSEA4, STRO-1 and STRO-3, and any of them The cells isolated from the tissue using cell surface markers selected from a list consisting of a combination of and may include cells that meet ISCT criteria either before or after expansion. As used herein, "MSC" refers to bone marrow stromal stem cells (BMSSC), bone marrow isolated multipotent stem cells (BMSCs). Adult progenitor cells (MAPCs), mesenchymal adult stem cells (MASCs), MultiStem ( (Registered Trademark), Prochymal (Registered Trademark), Mesenchymal Progenitor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX-PAD, AlloStem®, Ast rostem®, Ixmyelocel-T, MSC-NTF, NurOwn (registered trademark), Stempeucel (registered trademark), Stempeucel CLI, St empeucelOA, HiQCell, Hearticellgram-AMI, Re vascor (registered trademark), Cardiorel (registered trademark), Cartistem (registered trademark) Registered trademark), Pneumostem (registered trademark), Promostem (registered trademark), Ho meo-GH, AC607, PDA001, SB623, AC607, endometrial regeneration cells (ERCs), and cells described in the literature as adipose-derived stem and regenerative cells (ADRCs). Contains cells.

[0057] The MSCs may be expanded and utilized by administration itself, or may be grown to obtain conditioned medium. The term "growth medium" generally refers to a medium sufficient for the culture of umbilicus-derived cells. In particular, one particular medium for culturing the cells of the present invention herein is Darbe Particularly preferred are: DMEM-low glucose (also DMEM-LG herein) (Invitrogen, DMEM low glucose is preferably 15% (v / v) fetal bovine serum (e.g., defined fetal bovine serum, Hyclone, Logan Utah); Antibiotic / antifungal medication (preferably penicillin (100 units / milliliter), streptococcus aureus, or flu-like substance) tomycin (100 mg / ml), and amphotericin B (0.25 mg / ml) (Invitrogen, Carlsbad, CA), and 0.001% (v / v) 2-mercaptoethanol (Sigma, St. Louis, MO). In some cases, a different growth medium is used or Different supplements are provided and these are usually referred to herein as supplements to the growth medium. is shown.

[0058] Also, in the context of the present invention, the term "standard growth conditions" as used herein refers to conditions such as 5% CO This refers to culturing cells at 37°C in a standard atmosphere containing 2.5% CO₂ and a relative humidity of approximately 1. While the above conditions are beneficial for cell culture, these conditions may be harmful to the cells. The options available to one skilled in the art for controlling the temperature, CO2, relative humidity, oxygen, growth medium, etc. It should be understood that this can be varied by changing the

[0059] Mesenchymal stem cells ("MSCs") are originally derived from the embryonic mesoderm and subsequently from adult bone marrow and It has been isolated from other adult tissues, including muscle, bone, cartilage, fat, bone marrow stroma, and / or tendon. Mesoderm can also differentiate to form cardiac muscle, smooth muscle, and They differentiate into muscle or visceral mesoderm, which can give rise to blood islands consisting of endothelium and hematopoietic progenitor cells. The differentiation potential of described mesenchymal stem cells is well known for their mesenchymal origin, including the most well-characterized mesenchymal stem cells. Cell-restricted (Pittenger et al., Science (1999) 284:143 -147 and U.S. Patent No. 5,827,740 (SH2 + SH4 + CD29 + CD 44 + CD71 + CD90 + CD106 + CD120a + CD124 + CD 14 - CD34 - CD45 - ). The present disclosure is incorporated herein by reference. It encompasses the use of a variety of mesenchymal stem cells.

[0060] In one embodiment, MSC donor lots are generated from umbilical cord tissue. The procedures for constructing the umbilical cord have been previously published and are incorporated by reference [31-37]. "Tissue-derived cells (UTC)" are described, for example, in U.S. Patent No. 7,510,873. No. 413,734. U.S. Pat. Nos. 7,524,489 and 7,560,276 UTC refers to cells of any mammalian origin (e.g., human, rat, In one embodiment of the present disclosure, the UTC is derived from the human umbilicus. Umbilical cord-derived cells versus human cells that are fibroblasts, mesenchymal stem cells, or iliac crest bone marrow cells. The cells have reduced expression of one or more of the following genes: chemokine (cXc motif) ligand 2 (stromal cell-derived factor 1); elastin (supravalvular aortic stenosis, Williams-Beulen syndrome); cDNA DKFZp586M 2022 (derived from clone DKFZp586M2022); mesenchymal homeobox 2 (growth stop-specific homeobox; sine oculis homeobox 1 (Drosophila fly); ZFKP586B2420 protein; morphogenesis-associated activator; neurogenesis Similar to larin 1; tetranectin (plasminogen-binding protein); src homology 3 (SH3) and cysteine-rich domain; cholesterol 25-hydroxylase; Interleukin-11 receptor, α; procollagen c-endo peptide cytidase enhancer; frizzled homolog 7 (Drosophila); hypothetical gene BC0 08967;Collagen, type VIII, alpha 1;Tenascin(c) Hexabrachion, Iroko Protein 5, integrin, integrin, β8, neuroblastoma, tumorigenicity suppression, 36 kDa, insulin-like growth factor binding protein 2, clone MAMMA1001744, Kali Intermediate / small conductance calcium-activated channel, subfamily N, β7, I Integrin, transcriptional coactivator with PDZ-binding motif (TAZ), sine oc ulis homeobox homolog 2 (Drosophila), KIA1034 protein, Vesicle-associated membrane protein 5 (myobrevin), an EGF-containing fibrin-like extracellular matrix Protein 1, Early Growth Response 3, Distal Non-Homeobox 5, Hypothetical Protein FLJ2037 3. Aldo-keto reductase family 1 member C3 (3-alpha hydroxylase) asteroid dehydrogenase, type II); PDZ-binding motif (TAZ); proenkepha Integrin, beta-like 1 (with EGF-like repeat domain); Homo sapiens mRNA full-length insert cDNA clone EUROIMAGE 1968422;EphA3; KIAA367 protein; natriuretic peptide receptor c / guanylate cyclase c (A Triatriuretic peptide receptor c); Hypothetical protein FLJ14054; Homosapien mRNA; cDNA DKFZp564B222 (clone DKFZp564B222 BCL2 / adenovirus E1B 19kDa interacting protein 3-like; AE-binding protein White matter 1; and cytochrome C oxidase subunit VIIa polypeptide 1 (muscle) Furthermore, these isolated human umbilical cord-derived cells express interleukin-8; reticulon-1; chemokine (CXC motif) ligand 1 (melonoma growth stimulatory activity, alpha); Chemokine (CXC motif) ligand 6 (granulocyte chemotactic protein 2); ChemoKai (CXC motif) ligand 3; and tumor necrosis factor alpha-induced protein 3 The gene for each is expressed, where expression is observed in fibroblasts, mesenchymal stem cells, iliac crest bone marrow, The cells are cultured in a manner similar to that of human placenta-derived cells. They are capable of self-renewal and proliferation and have the potential to differentiate into cells of other phenotypes.

[0061] A method for deriving umbilical cord tissue mesenchymal stem cells from human umbilical cord tissue is provided. They are capable of self-renewal and proliferation in the cytoplasm and have the potential to differentiate into cells of other phenotypes. The method includes (a) obtaining human umbilical tissue; (b) removing substantially all blood to substantially (c) obtaining blood-free umbilical tissue; and (c) dissociating the tissue by mechanical or enzymatic treatment. (d) resuspending the tissue in culture medium; and (e) culturing the tissue. Human umbilical cord-derived cells capable of self-renewal and proliferation and the ability to differentiate into cells of other phenotypes. This includes providing growth conditions that allow the cells to grow.

[0062] Whether the tissue is delivered vaginally or via other routes (e.g., surgical Caesarean section), The pregnancy may be obtained from any completed pregnancy, birth, or sub-birth period, regardless of whether the pregnancy is a gestational age or a gestational age. Obtaining tissue from a tissue bank is also considered within the scope of this disclosure.

[0063] The tissue is rendered substantially free of blood flow by any means known in the art. For example, blood may be washed, removed, or re-used before or after bulk blood removal, e.g., by aspiration or drainage. It can be physically removed by rinsing, dilution, etc. It is substantially free of blood cells. Other means of obtaining tissue may include enzymatic or chemical treatment.

[0064] Dissociation of umbilical tissue can be performed by any of a variety of techniques known in the art, including mechanical disruption. This can be achieved, for example, by cutting the tissue aseptically with scissors or by using surgical instruments. Intact or viable cells from human tissue that can be cut or otherwise removed using a scalpel Such tissue may be comminuted, blended, ground, or homogenized in any manner compatible with the recovery of the tissue. It can be sized.

[0065] In this embodiment, the isolation procedure also utilizes an enzymatic digestion process. Isolating individual cells from complex tissue matrices to facilitate growth in culture As described above, it is known in the art that the method is useful for isolating cells from tissues. A wide range of digestive enzymes are available to those skilled in the art for use. Oxyribonuclease and neutral protease, dispase) to strong digestive enzymes (e.g. Such enzymes range from ribosomal enzymes (e.g., papain and trypsin) and are commercially available. A non-exhaustive list of enzymes compatible with this specification includes enzymes with mucolytic enzyme activity, metalloproteases, and proteases, neutral proteases, serine proteases (trypsin, chymotrypsin, or elas Certain embodiments include metalloplasts, The enzymatic activity is selected from protease, neutral protease and / or mucolytic activity. For example, collagenase is known to be useful for isolating various cells from tissues. Deoxyribonuclease can digest single-stranded DNA and is used to isolate cells during isolation. Aggregation can be minimized. Enzymes can be used alone or in combination. Since serine proteases can degrade other enzymes, it is preferable to use other enzymes. The temperature and time of contact with the serine protease are monitored. Serine proteases are inhibited by α2 microglobulin in serum. Therefore, the medium used for digestion is preferably serum-free. A and DNase are commonly used and can improve yield or efficiency. The method may involve, for example, collagenase and dispase, or collagenase, dispase, and hyaluronan. Such methods are provided, and certain preferred embodiments involve enzymatic treatment with arginidase. In this embodiment, a mixture of collagenase and the neutral protease dispase is used for the dissociation step. More preferably, Clostridium histolytii is used in at least one collagenase from icum, as well as protease activity, dispersant activity, A more preferred method is to use digestion in the presence of either enzyme or thermolysin. A more suitable method is to use digestion with both collagenase and dispase enzymatic activity. Methods involving digestion with hyaluronidase activity in addition to collagenase and dispase activity Those skilled in the art will appreciate the availability of many such enzymes for isolating cells from various tissue sources. It is understood that such blending processes are known in the art. For example, LIBERASE BLEND ZYME (Roche) series collagenase and neutral protease enzyme combinations are very useful and may be used in the present method. Other sources of enzymes are known and are known in the art. One skilled in the art can also obtain such enzymes directly from their natural sources. Novel or additional enzymes or enzyme combinations are being investigated for their utility in isolating soluble cells. The preferred enzyme treatments are 0.5, 1, 1 In another preferred embodiment, the tissue is treated with an enzyme for the dissociation step. The digest is then incubated at 37°C. Diluting the digest also prevents the cells from becoming viscous within the digest. The use of enzymes is currently preferred, but This is not necessary for the isolation methods provided herein. Methods based solely on mechanical separation As described above, the present cells can be successfully isolated from the umbilicus. The cells may be dissociated and resuspended in any culture medium, such as those described above. To separate the cells from the debris, a centrifugation step may be followed by resuspension. Providing growth conditions may involve mechanical methods such as culturing, or simply adding culture medium to the cells. A wide range of options are available for culture media, supplements, atmospheric conditions, and relative humidity of the cells. The specific temperature is 37°C, but the temperature may vary depending on other culture conditions and the desired properties of the cells or cultures. Depending on the use, the temperature may range from about 35°C to 39°C.

[0066] Exogenous growth medium, except as available in supplemented serum provided with the growth medium. Specific methods for providing cells that do not require a growth factor are encompassed herein. Also provided herein are methods for deriving umbilical cells that are capable of proliferation in the absence of offspring. This method is similar to the above method, except that the cells are finally resuspended and then The cells are grown in a culture medium containing specific growth factors (for which the cells are not required). In this sense, this method allows differentiation in the absence of specific growth factors. The specific cells are selective for cells that can be cleaved. and can grow and proliferate in a chemically defined growth medium without added serum. In such cases, the cells may be cultured in a medium containing specific growth factors that may be added to support and maintain the cells. Specific factors added for growth in serum-free medium include FGFs. In some embodiments, the 2 One, three, or all four factors are added to serum-free or chemically defined media. In another embodiment, LIF is added to serum-free media to support or improve cell growth. will be added.

[0067] Also, a method in which cells can expand in the presence of about 5% to about 20% oxygen in their atmosphere. A method for obtaining cells that require L-valine is also provided, comprising incubating the cells in the presence of L-valine. After the cells are obtained, they are tested for their L-valine requirement and then cultured in the presence of L This can be confirmed by growing the strain on a D-valine-containing medium lacking the -isomer.

[0068] Before reaching the senescent state, cells undergo at least 25, 30, 35, or 40 doublings. A method is provided that can 14 can double to reach more than 100 cells Preferably, at least about 10 14 , 10 15 , 10 16 , or 10 17 More than 10 cells in culture 3 from about 10 6 cell / cm 2 This is a method to derive cells that can double sufficiently to generate when seeded up to In certain cases, these cell numbers are produced within 80, 70, or 60 days. In embodiments, umbilical cord tissue mesenchymal stem cells are isolated, expanded, and labeled with CD10, CD13, CD44 , CD73, CD90, CD141, PDGFr-α, or HLA-A, B, C Furthermore, the cells have one or more markers selected from CD31, CD34, C D45, CD117, CD141, or one or more of HLA-DR, DP, DQ do not.

[0069] Flow cytometry was performed to determine the quality of MSC cultures, SH-2, SH-3 , surface expression of SH-4 MSC markers, and contaminating CD14-positive and CD-45-positive cells All cultures were tested for cell deficiency. Cells were incubated with 0.05% trypsin- Separated with EDTA, washed with DPBS + 2% bovine albumin, and then resuspended in 1% paraformaldehyde. The cells were fixed in 0.5% PBS, blocked in 10% serum, and then incubated with primary SH-2, SH-3, and SH-4 antibodies. The cells were incubated separately with PE-conjugated anti-mouse IgG (H+L) antibody. 2 Confluent MSCs in flasks were washed with Tyrode's salt solution and then resuspended in Medium 199 (M199 ) for 60 minutes and then lysed with 0.05% trypsin-EDTA (Gibco). Dissociate cells from 10 flasks and add MSCs to 40 ml of M199 + 1% human Serum albumin (HSA; American Red Cross, Washington, DC) MSCs collected from each set of 10 flasks were incubated at 4°C for 4 h. The time was kept until the end of collection. 6 / kg MSCs M19 The cells were resuspended in 9 + 1% HSA and centrifuged at 460g for 10 minutes at 20°C. Resuspend in fresh M199 + 1% HSA medium and repeat at 460g for 10 min at 20°C for three more times. The total harvest time is 2-4 days based on the MSC yield and target dose per flask. The time was 4 hours. The collected MSCs were then diluted with 10% DMSO (Research Industry ries, Salt Lake City, UT, USA) and a final concentration of 5% HSA. Cryocyte (Baxter, Deerfield, MA) was frozen using a controlled rate freezer. The units were frozen and stored in a freezer bag (Hall, IL, USA). On the day of injection, the frozen units were Thaw at bedside in a 37°C water bath, transfer to a 60 ml syringe within 5 min, and infuse for 10-15 min. The patient received an intravenous infusion over 15 minutes. Premedication is given orally with 12.5-25 mg of diphenhydramine. Heart rate, respiratory rate, temperature, and oxygen saturation were monitored at the time of infusion and every 15 minutes thereafter for three consecutive days. Monitor every two times for six sessions.

[0070] In one embodiment, the MSCs are derived from bone marrow-derived MSCs, a process previously used to treat patients. Specifically, bone marrow is extracted under local anesthesia (with or without sedation). Aspirate (10-30 ml) from the posterior iliac crest and collect in a tube containing sodium heparin. GMP (Good Manufacturing Practices) cleanroom The bone marrow cells are then transferred to a wash chamber. ), RPMI, or PBS supplemented with autologous plasma), and then rinse with 25 ml of Percoll. (1.073g / ml) above, approximately 1-2 x 10 7 The cells were then layered at a concentration of 1000 cells / ml. Cells were cultured at 900 g for approximately 30 minutes or until separation of mononuclear cells from debris and red blood cells was achieved. The cells were then washed with PBS and resuspended in DME containing 10% FCS. 175cm in M 2 Approximately 1 x 10 cells per mL in a tissue culture flask 6 Plate at a concentration of cells The flasks are then loaded with a minimum of 30 million bone marrow mononuclear cells. MSCs are engrafted for 72 hours. After the cells had adhered, the medium was changed every 3-4 days. Removed at 175cm 2 Hit 1'10 6 Bone marrow MSCs are replated at a concentration of Intravenously, or in certain embodiments intrathecally, in patients suffering from radiation-associated neurodegenerative conditions Dosage may be determined by one skilled in the art and may take into account various patient characteristics. Intravenous administration can be performed at concentrations ranging from 1 to 10 million MSCs per kg, depending on the patient. , the preferred dosage is about 2 to 5 million cells per kg.

[0071] In one embodiment, the hematopoietic stem cells are C isolated from peripheral blood, bone marrow, or umbilical cord blood. Specifically, hematopoietic stem cells are derived from, but not limited to, human, mouse, and rat. These hematopoietic stem cells may be derived from the blood system of any mammal, and may be derived from the blood or Hematopoietic stem cells may be collected by isolating them from tissues and organs. The antibody can be collected from a donor by any method described in, for example, U.S. Patent Publication No. 2013 / 014 9286 details procedures for obtaining and purifying stem cells from mammalian cadavers. Stem cells may be obtained from a human by bone marrow harvest or peripheral blood stem cell harvest, These techniques are all well known in the art. Once obtained from the source, they may be cultured using stem cell expansion techniques. No. 6,338,942 to Kraus et al., entitled "S selective expansion of target cell population tions,” Issue Date: January 15, 2002; and U.S. Patent No. 5,0002, entitled "Selective expansion of target cell popu lations”, Publication date: January 15, 2002. 6,3 to Rodgers et al. No. 35,195, titled “Method for promoting hematopoiesis ietic and cell proliferation and differe "Integration," published January 1, 2002, which are incorporated herein by reference in their entireties. In some embodiments, the stem cells obtained from the donor are incorporated into a population of stem cells. In another preferred embodiment, stem cells harvested from a donor source are cultured to expand. The cells are not expanded using such techniques. Stem cells are stored in a cytoprotective medium using standard methods. It is possible.

[0072] Some of the methods of the present disclosure have risks associated with certain types of stem cells (e.g., pluripotent stem cells). In some embodiments, the stem cells are encapsulated by a membrane and a capsule prior to transplantation. It is contemplated that any of the many methods of cell encapsulation available may be used. In some embodiments, the cells are individually encapsulated. In an embodiment, many cells are encapsulated within the same membrane. After implantation, the cells are removed. In some embodiments, a relatively large structure encapsulating many cells, such as within a single membrane, is used for collection. Various experiments for the microencapsulation of stem cells have been carried out. In embodiments, a wide variety of materials can be used. For example, polymer capsules, alginate-poly-L-lysine-alginate microcapsules Poly-L-lysine alginate barium capsule, alginate barium capsule, poly Acrylonitrile / Polyvinyl Chloride (PAN / PVC) Hollow Fiber and Polyether Sulfone Hollow fibers (PES) are examples of cell microtubules that can be used for the administration of stem cells. Techniques for encapsulation are known to those skilled in the art and are described, for example, in Chang, P. et al., 1999 9;Matthew, HW et al., 1991;Yanagi, K. et al., 1989;Cai ZH et al., 1988; Chang, TM, 1992 and U.S. Pat. No. 5,639,277 5. (This is, for example, for the long-term propagation of cells stably expressing biologically active molecules. Further methods of encapsulation are described in the European Patent Application No. Publication No. 301,777 and U.S. Patent Nos. 4,353,888; 4,744,933; ,749,620;4,814,274;5,084,350;5,089,272;5 , 578,442; 5,639,275; and 5,676,943. All of the above are incorporated herein by reference in their entirety with respect to stem cell encapsulation. Certain embodiments involve the use of stem cells in a polymer (e.g., a biopolymer or a synthetic polymer). Examples of biopolymers include fibronectin, fibrin, and fibrinogen. , thrombin, collagen, and proteoglycans. Other factors, such as the cytokines mentioned above, can also be incorporated into the polymer. In another embodiment of the present invention, stem cells may be incorporated into the interstices of the three-dimensional gel. The polymer or gel is typically surgically implanted. Polymers or gels that can be polymerized can be delivered by other common, more convenient, non-surgical routes. It can be administered as follows. [Example]

[0073] The following examples are included to demonstrate preferred embodiments of the invention. The techniques described below have been discovered by the inventors to work well in the practice of the present invention. and can therefore be considered to constitute a preferred mode for carrying out the invention. However, those skilled in the art will understand that, given the present disclosure, It is understood that the spirit and scope of the invention may be exceeded in specific embodiments disclosed and similar results may be obtained. It will be understood that many variations are possible without departing from the spirit and scope of the present invention.

[0074] Example 1 Neuroregenerative cytokine BDNF in response to inflammation by fibroblasts and their combination Stimulation Fibroblasts were co-cultured with either monocytes, mesenchymal stem cells, or hematopoietic stem cells at a 1:1 ratio. Cells were cultured for 48 hours in the presence of increasing concentrations of TNF-α to stimulate inflammatory signaling. The presence of BDNF was determined by ELISA. As can be seen in Figures 1-3, All cell combinations showed additive or synergistic increases in the production of this neuroprotective cytokine. brought about.

[0075] Example 2 Neuronal apoptosis in response to oxidative stress in fibroblasts and fibroblast combinations Decrease in Fibroblasts were co-cultured with either monocytes, mesenchymal stem cells, or hematopoietic stem cells at a 1:1 ratio. Cells were incubated for 4 h in the presence of increasing concentrations of hydrogen peroxide to stimulate oxidative stress. The conditioned medium from the cells was then cultured for 8 hours. Apoptosis was assessed using Annexin V staining to determine the percentage of apoptotic cells. Fibroblasts were measured by flow cytometry, as shown in Figure 4. It synergizes with CD34 (Fig. 5) or MSCs (Fig. 6) to inhibit neuronal death.

[0076] Example 3 Stimulation of neural progenitor cell proliferation by a combination of fibroblasts and fibroblasts Fibroblasts were co-cultured with either monocytes, mesenchymal stem cells, or hematopoietic stem cells at a 1:1 ratio. Cells were cultured for 48 hours in the presence of increasing concentrations of interleukin-1 beta, and myeloid leukemia was observed. The conditioned medium from the cells stimulated the production of inflammatory factors. Fibroblasts were added for 48 hours and proliferation was measured by thymidine incorporation. , synergizing with monocytes (Figure 7), CD34 (Figure 8), or MSCs (Figure 9) to stimulate neurogenesis. Become excited.

[0077] Example 4 Fibroblast-conditioned medium protects neurons from injury Cells are triple-cloned sublines of the SH-SY5Y bone marrow biopsy-derived line SK-N-SH (Sigma) has dopamine-β-hydroxylase activity and mediates the neurotransmission of glutamate. These cells are known to be able to convert about 70% of apoptosis into the neurotransmitter GABA. 24-hour treatment with 70 mM glucose, which induces cytotoxicity, served as a model of neurotoxicity. To assess whether fibroblasts have neuroprotective properties, fibroblast-conditioned medium Increasing concentrations of the medium were added to SH-SY5Y cells.

[0078] To obtain fibroblast conditioned medium, skin fibroblasts (ATCC) were cultured in 10% fetal bovine serum. The cells were cultured for 24 hours in OPTI-MEM medium containing serum and non-essential amino acids. The cells were cultured in a T-75 flask at confluency in 1 ml of medium. After 24 hours, the cells were cultured in liquid medium. Fibroblast conditioned medium was generated by extracting

[0079] Administration of conditioned medium increased glucose uptake as assessed by Annexin-V staining. A dose-dependent reduction in thrombus-induced neuronal death was observed (Figure 10).

[0080] References All patents and publications are incorporated by reference as if each individual publication were incorporated by reference. Each of the foregoing is incorporated by reference herein to the same extent as if specifically and individually indicated to be incorporated by reference. U.S. Patent No. 4,353,888 U.S. Patent No. 4,744,933 U.S. Patent No. 4,749,620 U.S. Patent No. 4,814,274 U.S. Patent No. 5,084,350 U.S. Patent No. 5,089,272 U.S. Patent No. 5,578,442 U.S. Patent No. 5,639,275 U.S. Patent No. 5,676,943 U.S. Patent No. 5,827,740 U.S. Patent No. 7,413,734 U.S. Patent No. 7,510,873 U.S. Patent No. 7,524,489 U.S. Patent No. 7,560,276 European Patent Publication No. 301,777 1. Zhuo, Y.ら, White matter impairment in type 2 diabetes mellitus with and w ithout microvascular disease. Neuroimage Clin, 2019. 24: p. 101945. 2. Mendez, M.F., The neuropsychiatric aspects of boxing.Int J Psychiatry Med, 1995. 25(3): p. 249-62. 3. Jordan, B.D., Neurologic aspects of boxing. 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[0081] Although the present disclosure and its advantages have been described in detail, the scope of the present invention is defined by the appended claims. Various changes, substitutions, and alterations may be made herein without departing from the spirit and scope of the present design. It is understood that the scope of the present application is limited to the Any disclosures expressly incorporated herein by reference are limited to particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps. As those skilled in the art will readily understand from this disclosure, perform substantially the same function or achieve substantially the same result as the corresponding embodiment described in Any now existing or later developed process, machine, manufacture, composition of matter, or technique that accomplishes the Any stage, method, or step may be utilized in accordance with the present disclosure. The appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, or methods. or steps.

Claims

1. 1. A pharmaceutical composition for treating a subconcussive or concussive brain injury in an individual, comprising: a) optionally identifying the individual with one or more elevated inflammatory markers following a head and / or neck injury; b) administering a therapeutically sufficient amount of regenerative cells and / or one or more products derived from said regenerative cells; and c) optionally repeat administration of said regenerative cells and / or products derived from said regenerative cells, The regenerative cells comprise fibroblasts and hematopoietic stem cells (HSCs), fibroblasts and mesenchymal stem cells (MSCs), and / or fibroblasts and monocytes.

2. 2. The composition of claim 1, wherein the inflammatory markers assessed after the head injury are selected from the group consisting of a) C-reactive protein, b) interleukin-1, c) interleukin-6, d) interleukin-8, e) interleukin-33, f) erythrocyte sedimentation ratio, g) TNF-α, h) interferon-γ, and i) combinations thereof.

3. 3. The composition of claim 1 or 2, wherein the increase comprises a concentration that is at least 20% higher compared to a standard laboratory value or a value in the general population.

4. (a) the fibroblasts express a marker selected from the group consisting of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344 Stella, and combinations thereof; (b) the fibroblasts express markers selected from the group consisting of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-α, HLA-A, HLA-B, HLA-C, and combinations thereof; (c) the fibroblasts do not express a marker selected from the group consisting of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, CD90, and combinations thereof; and / or (d) the fibroblasts do not express a marker selected from the group consisting of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, HLA-DQ, and combinations thereof; The composition of claim 1.

5. (a) the hematopoietic stem cells are capable of multilineage reconstitution in an immunocompromised host; (b) the hematopoietic stem cells express c-kit protein; (c) the hematopoietic stem cells express Sca-1 protein; (d) the hematopoietic stem cells express CD34; (e) the hematopoietic stem cells express CD133; (f) the hematopoietic stem cells lack expression of lineage markers; (g) the hematopoietic stem cells lack expression of CD38; (h) the hematopoietic stem cells are positive for expression of c-kit and Sca-1 and substantially lack expression of lineage markers; and / or (i) the hematopoietic stem cells are derived from: a) peripheral blood; b) mobilized peripheral blood; c) bone marrow; d) umbilical cord blood; e) adipose stromal vascular fraction; f) progenitor cells; or g) a combination thereof. The composition of claim 1.

6. (a) the mesenchymal stem cells are plastic-adherent; (b) the mesenchymal stem cells express a marker selected from the group consisting of: a) CD73, b) CD90, c) CD105, and d) combinations thereof; (c) the mesenchymal stem cells lack expression of a marker selected from the group consisting of: a) CD14, b) CD45, c) CD34, and d) combinations thereof; and / or (d) the mesenchymal stem cells are derived from a tissue selected from the group consisting of: a) bone marrow, b) peripheral blood, c) adipose tissue, d) mobilized peripheral blood, e) umbilical cord blood, f) Wharton's jelly, g) umbilical cord tissue, h) skeletal muscle tissue, i) subepithelial umbilical cord, j) endometrial tissue, k) menstrual blood, l) fallopian tube tissue, and m) combinations thereof. The composition of claim 1.

7. (a) the mesenchymal stem cells derived from the umbilical cord tissue express a marker selected from the group consisting of: a) oxidized low density lipoprotein receptor 1, b) chemokine receptor ligand 3, c) granulocyte chemotactic protein, and d) combinations thereof; (b) the mesenchymal stem cells derived from the umbilical cord tissue do not express a marker selected from the group consisting of: a) CD117, b) CD31, c) CD34, d) CD45, and e) a combination thereof; (c) the mesenchymal stem cells derived from the umbilical cord tissue express increased levels of interleukin 8 and / or reticulon 1 compared to human fibroblasts; (d) the umbilical cord tissue-derived mesenchymal stem cells have the potential to differentiate into cells of at least skeletal muscle, vascular smooth muscle, pericyte, or vascular endothelial phenotype; and / or (e) the mesenchymal stem cells derived from the umbilical cord tissue express a marker selected from the group consisting of: a) CD10, b) CD13, c) CD44, d) CD73, e) CD90, and f) a combination thereof; The composition of claim 6.

8. (a) the umbilical cord tissue mesenchymal stem cells are umbilical cord tissue cells isolated from substantially blood-free umbilical cord tissue that are capable of self-renewal and proliferation in culture; (b) the umbilical cord tissue mesenchymal stem cells have the ability to differentiate into cells of other phenotypes; (c) the cord tissue-derived mesenchymal stem cells are capable of, or have undergone, at least 20 doublings in culture; (d) the cord tissue-derived mesenchymal stem cells maintain a normal karyotype during passage; (e) the mesenchymal stem cells derived from the cord tissue express a marker selected from the group consisting of A) CD10, b) CD13, c) CD44, d) CD73, e) CD90, f) PDGFr-α, g) PD-L2, h) HLA-A, B, C, and i) combinations thereof; (f) the umbilical cord tissue mesenchymal stem cells do not express one or more markers selected from the group consisting of a) CD31; b) CD34; c) CD45; d) CD80; e) CD86; f) CD117; g) CD141; h) CD178; i) B7-H2; j) HLA-G; k) HLA-DR, DP, DQ; and l) combinations thereof. (g) the umbilical cord tissue-derived cells secrete a factor selected from the group consisting of a) MCP-1, b) MIP1 beta, c) IL-6, d) IL-8, e) GCP-2, f) HGF, g) KGF, h) FGF, i) HB-EGF, j) BDNF, and k) TPO, l) Rantes, m) TIMP1, and n) combinations thereof; (h) the umbilical cord tissue-derived cells express a marker selected from the group consisting of a) TRA1-60, b) TRA1-81, c) SSEA3, d) SSEA4, e) nanog, and f) combinations thereof. (i) the umbilical cord tissue-derived cells stain positive for alkaline phosphatase, and / or (j) the umbilical cord tissue-derived cells are capable of differentiating into one or more lineages selected from the group consisting of: a) ectoderm; b) mesoderm; c) endoderm; and d) combinations thereof. The composition according to claim 6 or 7.

9. 9. The composition of claim 8, wherein the other phenotype comprises a) osteocytic, b) adipogenic, c) chondrogenic differentiation, or d) a combination thereof.

10. (a) the bone marrow-derived mesenchymal stem cells have a marker selected from the group consisting of: a) CD73, b) CD90, c) CD105, and d) a combination thereof; (b) the bone marrow-derived mesenchymal stem cells have a marker selected from the group consisting of: a) LFA-3, b) ICAM-1, c) PECAM-1, d) P-selectin, e) L-selectin, f) CD49b / CD29, g) CD49c / CD29, h) CD49d / CD29, i) CD29, j) CD18, k) CD61, l) 6-19, m) thrombomodulin, n) telomerase, o) CD10, p) CD13, q) integrin β, and r) combinations thereof; and / or (c) the bone marrow-derived mesenchymal stem cells are mesenchymal stem cell precursor cells; The composition of claim 6.

11. The mesenchymal progenitor cells are a population of bone marrow mesenchymal stem cells enriched for cells containing STRO-1, or the mesenchymal progenitor cells express both STRO-1 and VCAM-1. The composition of claim 10.

12. 11. The composition of claim 10, wherein the STRO-1 expressing cells are negative for at least one marker selected from the group consisting of A) CBFA-1, b) type II collagen, c) PPAR.gamma.2, d) osteopontin, e) osteocalcin, f) parathyroid hormone receptor, g) leptin, h) H-ALBP, i) aggrecan, j) Ki67, k) glycophorin A, and l) combinations thereof.

13. (a) the bone marrow mesenchymal stem cells lack expression of CD14, CD34, and / or CD45; (b) the bone marrow mesenchymal stem cells express a marker selected from the group consisting of: a) CD13, b) CD34, c) CD56, d) CD117, and e) a combination thereof; (c) the bone marrow mesenchymal stem cells do not express CD10; (d) the bone marrow mesenchymal stem cells do not express one or more of CD2, CD5, CD14, CD19, CD33, CD45, and DRII; (e) the bone marrow mesenchymal stem cells express one or more of CD13, CD34, CD56, CD90, CD117, and nestin, and do not express one or more of CD2, CD3, CD10, CD14, CD16, CD31, CD33, CD45, and CD64; and / or (e) the bone marrow mesenchymal stem cells express one or more of CD13, CD34, CD56, CD90, CD117, and nestin, and do not express one or more of CD2, CD3, CD10, CD14, CD16, CD31, CD33, CD45, and CD64; The composition of claim 6.

14. 14. The composition of claim 13, wherein the STRO-1 expressing cells are positive for a marker selected from the group consisting of a) VACM-1, b) TKY-1, c) CD146, d) STRO-2, and d) combinations thereof.

15. (a) the skeletal muscle mesenchymal stem cells express a marker selected from the group consisting of: a) CD13; b) CD34; c) CD56; d) CD117; and e) a combination thereof. (b) the skeletal muscle mesenchymal stem cells do not express CD10, and / or (c) the skeletal muscle mesenchymal stem cells do not express one or more of CD2, CD5, CD14, CD19, CD33, CD45, and DRII; The composition of claim 6.

16. (a) the subepithelial umbilical cord-derived mesenchymal stem cells express markers selected from the group consisting of: a) CD29; b) CD73; c) CD90; d) CD166; e) SSEA4; f) CD9; g) CD44; h) CD146; i) CD105; and j) combinations thereof. (b) the subepithelial umbilical cord-derived mesenchymal stem cells do not express a marker selected from the group consisting of: a) CD45; b) CD34; c) CD14; d) CD79; e) CD106; f) CD86; g) CD80; h) CD19; i) CD117; j) Stro-1; k) HLA-DR; and l) combinations thereof. (c) the subepithelial umbilical cord-derived mesenchymal stem cells express one or more of CD29, CD73, CD90, CD166, SSEA4, CD9, CD44, CD146, and CD105; (d) the subepithelial umbilical cord-derived mesenchymal stem cells do not express one or more of CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1, and HLA-DR; (e) the subepithelial umbilical cord-derived mesenchymal stem cells are positive for SOX2; (f) the subepithelial umbilical cord-derived mesenchymal stem cells are positive for OCT4, and / or (g) the subepithelial umbilical cord-derived mesenchymal stem cells are positive for OCT4 and SOX2; The composition of claim 6.

17. 10. The composition of claim 1, wherein the one or more products obtained from the regenerative cells include cell lysates, apoptotic bodies, exosomes, and / or microvesicles.

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