Treatment of cerebral hypoxia including stroke, chronic traumatic encephalopathy, and traumatic brain injury

Abstract

To provide methods, compositions, and means for treatment or prophylaxis using fibroblasts possessing regenerative properties for the treatment of brain injuries including stroke, transient ischemic injuries, chronic traumatic encephalopathy, traumatic brain injury, and tauopathies.SOLUTION: Embodiments of the disclosure administer fibroblasts with regenerative properties, systemically, locally, or a combination of the two, concurrent with or subsequent to a brain injury. In some embodiments of the disclosure, fibroblasts or products thereof are administered intranasally, intrathecally, and / or intravenously.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 755,553, filed November 4, 2018. No. 6,396,433, filed on Oct. 23, 2003, which is incorporated by reference in its entirety.

[0002] (Technical field) The disclosed embodiments relate to at least the fields of cell biology, molecular biology, immunology, and medicine. This is what we do. [Background technology]

[0003] Stroke is classically defined as an acute focal injury to the central nervous system (CNS) due to vascular causes. The resulting neurological deficits have been described as either hemorrhagic or infarct-related. is known to be a leading cause of disability and death worldwide[1], with stroke-related mortality at 1.5% and The number of strokes in Japan is increasing. [2] Between 1990 and 2010, the global incidence of stroke remained stable. However, the incidence of first stroke, prevalence of stroke, and disability due to stroke were not significantly different. Other parameters such as harm-adjusted life years and stroke-related deaths were 68, 84, and 89, respectively. The difference between the percentages and the figures is due to demographic changes, increased life expectancy, and increased This could reflect improvements in global healthcare services. By 2030, the number of 1 million deaths are expected from stroke [1]. Moreover, it is the leading cause of long-term disability. [3]

[0004] There are two main types of stroke: ischemic stroke and hemorrhagic stroke. Thrombolysis and / or thrombectomy are used to treat ischemic stroke, accounting for more than 80% of the total number of strokes. It is the only validated treatment strategy among the 100 patients with glaucoma [4,5]. Unfortunately, the inclusion criteria are limited. As a result, fewer than 20% of patients receive such treatment. In addition to direct brain damage, rapidly progressive sterile inflammation contributes to the infarct size during the first 1–3 days after stroke. There is growing evidence that it worsens occlusion size and therefore clinical outcome [6 ]. In the art, there are many indications for improving clinical outcomes and being widely used in patients suffering from stroke. New treatments that may be of benefit are needed.

[0005] Traumatic brain injury (TBI) is a disruption of brain function resulting from a percussive or penetrating injury to the skull. TBI is heterogeneous in its causes and can be viewed as a two-stage event:1 ) mechanical shock, which results in primary pathological events such as hemorrhage and ischemia, tissue rupture and axonal injury; 2) Primary damage caused by diffuse inflammation, cell death, and Secondary damage as a result of the primary injury, such as gliosis and neuropathy. This secondary damage begins immediately after the injury and It can last for several weeks and is thought to involve active inhibition of neural stem cell activity. Together, these events lead to neurodegeneration.[7]

[0006] Head injuries are described as open or closed. Open head injuries are caused by bullets, sharp objects, etc. Penetration of the scalp and skull by a dangerous object, or skull fracture, resulting in laceration of brain tissue. Closed injuries occur when rapid brain acceleration or deceleration occurs from a shock, bump, fall, or other sudden attack. This rapid acceleration or deceleration occurs when the contact point (coupe) or its opposite point ( [8] The temporal and frontal lobes are the most vulnerable to damage. This may involve axonal and / or vascular severing. Twisted blood vessels may leak and cause hematomas. , contusion, or even intracerebral or subarachnoid hemorrhage.[9]

[0007] TBI can be acute and recent, or it can involve inflammation and scarring effects on brain tissue. Concussion can also be a chronic condition resulting from the long-term consequences of such acute brain injuries, including It is described as an immediate but temporary loss of consciousness accompanied by a brief period of amnesia. TBI victims may appear dazed, disappointed, or confused. A concussion may be accompanied by convulsions, low blood pressure, fainting, and paleness. Symptoms are usually short-lived in uncomplicated cases of isolated concussion.

[0008] Soccer players and boxers suffer significantly more concussions than athletes in other sports. He appears to be suffering from the National Football League (NFL) Athletes have recently brought to the public's attention the long-term consequences of receiving multiple blows during their playing days. Frequently reported symptoms include loss of cognition, communication skills, and Decreased mental health, decreased emotional stability, decreased coordination, memory loss, dementia and postmortem changes in the microstructure of the brain [10-12] Interestingly, studies have not linked the position of soccer players to brain injury.

[13] have shown a correlation between enhancement markers.

[0009] An increasingly prevalent subset of TBI is blastogenic or blastogenic TBI (bTBI). ) with the increasing use of explosives, including improvised explosive devices (IEDs), in the fight against terrorism. However, bTBI is also on the rise. Such injuries often occur in the Iraq and Afghanistan conflicts. These are called the salient injuries of war in the United States and affect both military and civilian workers in combat zones. Blast injuries are the most common cause of TBI in U.S. soldiers during combat. It is the leading cause of disability among combat service members. Related to TBI, but not limited to: A hematoma in the brain. A hematoma is broadly defined as an accumulation of blood outside of a blood vessel. When it occurs in the brain, , and is associated with the activation and migration of microglial cells to the site of injury.

[0010] Approximately 1.5 million people in the United States suffer traumatic brain injuries (TBI) each year, which affect a portion of brain function. It reflects physical damage to the brain that can result in temporary or permanent impairment. Of these, approximately 50,000 die and another 80,000 suffer some degree of disability. These include accidents (motor vehicle, bicycle, pedestrian), assault, and sports-related injuries.

[0011] A significant amount of patients who suffer from TBI have mild injuries and therefore are not diagnosed immediately after the injury. Undiagnosed and untreated TBI can cause some signs and symptoms to be undetectable. If left untreated, it can have a debilitating effect on the patient's physical, emotional, behavioral, and social It may have a significant impact on the social or family situation and cause functional impairment. Secondary damage from the injury continues after the initial impact, so early treatment (if any) is necessary. Therefore, rapid diagnosis, and especially point-of-care treatment, is desirable.

[0012] Reduce infarct size in injuries, including at least TBI, as well as limit secondary inflammatory responses There is a need in the art for treatments for brain injuries or diseases that result from Summary of the Invention

[0013] The present disclosure is directed to the treatment or prevention of brain injury or medical conditions in an individual in need thereof. In certain embodiments, the treatment comprises administering to an individual Induce regeneration in the brain of the body. Certain embodiments are directed to treating or preventing brain injury or medical conditions. In certain aspects, the fibroblasts are administered to an individual to prevent The regenerative activity may be intrinsic to the fibroblasts or may be expressed by the fibroblasts themselves. Can be engineered to increase

[0014] In some embodiments of the present disclosure, the individual has brain injury or is at risk of having brain injury. Administration of fibroblasts may be given after an individual has been diagnosed with brain injury, or For example, one of skill in the art may reasonably predict that an individual will have brain damage or may have a particular future It may be given to an individual prior to being diagnosed with brain injury if they are at the time suspected of having brain injury. In the latter instance, fibroblasts can be administered prophylactically to the individual.

[0015] In some embodiments, the brain injury is, for example, a traumatic brain injury (TBI), stroke, or ischemia. Examples of traumatic brain injury include chronic myocardial infarction, hemorrhagic stroke, or tauopathy. traumatic brain injury, one or more concussions, blast-induced traumatic brain injury, backlash brain injury ( coup-contrecoup brain injury, cerebral contusion, shaking child syndrome, penetrating injury, diffuse axonal injury, second impact injury, locked-in syndrome , or a combination of these. Individuals may ); Exposure to explosives (e.g., military or demolition personnel); Recreation sports for the sake of, or as a profession, such as football or boxing or the occurrence (directly or indirectly) of a mechanical force on an individual's head, such as in a vehicle accident; Brain injury may have occurred from a traumatic event that caused a TBI, for example. Brain pathology can result from any event that causes trauma to the head to cause the brain to swell. There may be cases where this does not match the actual situation.

[0016] In some embodiments, the individual is suffering from a medical condition, such as having suffered a stroke or a transient ischemic attack. A stroke can be ischemic or hemorrhagic. Individuals who suffer a transient ischemic attack may have a loss of function in certain areas of the brain. The loss of function may be from the primary event and / or may be induced by the primary event. It may also be from inflammation caused by

[0017] In certain embodiments, the individual has either an acute tauopathy or a chronic tauopathy. Acute tauopathies and other medical conditions that may be causing the Examples of chi include traumatic brain injury, stroke, or transient ischemic attack. Chronic tauopathy Examples of dementia include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinson-Dementia Complex, and Argyro. Phyllic-Grane Dementia, British Amyloid Angiopathy, Cerebral Amyloid Angiopathy, Cortico-cerebrovascular Degeneration, Creutzfeldt-Jakob disease, diffuse neurofibrillary tangles with calcification, Down's syndrome syndrome, frontotemporal dementia with parkinsonism linked to chromosome 17, Gerstmann -Gerstmann-Straussler-Sch Einker's disease, Hallervorden-Spatz ) disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motor neurons with neurofibrillary tangles or neuron) disease, Pick's disease, postencephalitic parkinsonism, prion protein encephalopathy Amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, These conditions include single-infarct dementia, and multi-infarct dementia. Tau levels can be measured in acute or chronic To aid in the diagnosis of an individual with a tauopathy, This is also fine.

[0018] In certain embodiments of the present disclosure, an individual receives an effective amount of Fibroblasts are administered. Fibroblasts may be derived from, by way of non-limiting example, skin, foreskin, fat, peripheral Blood, cerebrospinal fluid, bone marrow, placenta, umbilical cord blood, Wharton jelly, or other tissues, or combinations thereof Fibroblasts may be derived from any source, including the ability to produce growth factors, neural stem cells, and the like. the ability to induce cell proliferation, the ability to induce a healing response, or a combination thereof; The ability to produce growth factors may be, for example, VEG. Produces growth factors including F, VDNF, NGF, PDGF-BB, or combinations thereof Fibroblasts may express CD105, CD73, CXCR-4, CD90, CD34, NANOG, OCT-4, SSEA-4, stem cell factor receptor, or a combination thereof The present invention may express one or more specific markers or proteins, including at least one of the following combinations: In some embodiments, the fibroblasts may lack the CD45 cell marker.

[0019] The fibroblasts may be administered to an individual using any suitable method or route of administration. The cells can be administered by injection, such as by syringe. Administration can be systemic or Fibroblasts may also be administered intranasally, intrathecally, or locally to a specific site in the brain. The administration of fibroblasts may be performed intravenously, intravenously, or / and intravenously. The optimum route of administration and dosage can be determined.

[0020] Any individual receiving the treatment or prevention of the present disclosure may be of any age, sex, race, etc. The individual may be an adult, a child, an infant, or in utero.

[0021] Embodiments of the present disclosure include methods of inducing a regenerative and / or healing response in the brain of an individual. The method includes administering a therapeutically effective amount of fibroblasts to the individual. Traumatic brain injury, e.g., chronic traumatic brain injury, one or more concussions, blast-induced traumatic brain injury, Backlash brain injury (coup-contrecoup brain injury) , cerebral contusion, shaken baby syndrome, penetrating injury, diffuse axonal injury, second impact injury, locked-in syndrome, and combinations thereof; The individual may or may not have had a stroke (ischemic or hemorrhagic stroke) and / or Patients may or may not have suffered from a transient ischemic attack. In certain embodiments, total tau, truncated microtubule associations, Circulating levels of linked tau, phosphorylated tau, and / or tau-A are measured in the individual.

[0022] Fibroblasts may have regenerative activity and may be administered prophylactically to an individual. In an embodiment, the regenerative activity is VEGF, BDNF, NGF, PDGF-BB, and the like. The regenerative activity includes the ability to produce a factor selected from the group consisting of a combination of the above. In certain embodiments, the fibroblasts may be capable of stimulating proliferation of dermal stem cells. , foreskin, fat, peripheral blood, cerebrospinal fluid, bone marrow, placenta, umbilical cord blood, Wharton jelly, and other The fibroblasts are derived from a tissue selected from the group consisting of CD105 , CD73, CXCR-4, CD90, and / or C73. NANOG, OCT-4, SSEA-4, stem cell factor receptor, and their combinations The fibroblasts may express at least one of the markers selected from the group consisting of: The mammalian cell may be isolated by a method comprising isolating a population of mammalian cells and enriching for a subpopulation, Here, the subpopulation is CD45 - express a phenotypic profile and thereby possess regenerative properties Fibroblasts are isolated. The fibroblasts may be administered intranasally, intrathecally, and / or intravenously. These can be administered locally to the brain or systemically. Add-on treatment for stroke and / or transient ischemic attack or chronic or acute tauopathy The additional treatment may include at least one medical procedure, at least one drug, at least one In certain embodiments, the method may include, at least one supportive care regimen, or a combination thereof. , medical procedures include surgery to remove clotted blood, surgery to repair skull fractures, brain Surgery to reduce bleeding within the skull, surgery to relieve pressure within the skull, or a combination of both. Medications include anxiolytics, anticoagulants, anticonvulsants, antidepressants, diuretics, muscle relaxants, stimulants, drugs to medically induce coma, and combinations thereof. The agent may be at least one agent for treating tau pathology, a vaccine for tau or a tau Antibodies against related proteins, microtubule stabilizers, tau aggregation inhibitors, and inhibitors of post-translational modifications of tau At least one selected from the group consisting of drugs that inhibit the proliferation and proliferation of the tumor, Hsp90 inhibitors, and combinations thereof. In certain embodiments, the supportive care includes at least one physical therapy. Law, occupational therapy, recreational therapy, speech or communication therapy, psychological counseling These include cognitive therapy, brain stimulation therapy, or a combination of these.

[0023] The foregoing describes the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages which form the subject of the claims of this specification. The disclosed concepts and specific embodiments are presented below to carry out the same objectives of the present design. It will be appreciated by those skilled in the art that the present invention may be readily utilized as a basis for modifying or designing other structures for purposes of this invention. It should be understood that such equivalent constructions are within the scope of the appended claims. It should be understood by those skilled in the art that there is no need to depart from the spirit and scope of what is described. Further objects and advantages are set forth in the novel features believed to be characteristic of the design disclosed herein. The following, both as to organization and method of operation, when considered in conjunction with the accompanying drawings, It will be better understood from the following description. However, each of the figures is for purposes of illustration and explanation. It should be expressly understood that these are provided for purposes of illustration only and are not intended as a definition of the limits of the present disclosure. do. [Brief description of the drawings]

[0024] 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:

[0025] [Figure 1] Figure 1 shows the improvement in neurological deficit scores when fibroblasts were substituted for mesenchymal stem cells (MSCs). In the four bar groupings, from left to right, the bars are: control; stroke; stroke + MSCs; and stroke + fibroblasts. Detailed Description of the Invention

[0026] For purposes of understanding and illustrating the practice of the present invention, the present invention is not limited to the specific embodiments illustrated. It should be understood that the above definitions are not exhaustive and may, of course, be varied. It is also to be understood that the terminology used is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0027] I. Definition

[0028] As used herein, "a" or "an" can mean one or more. As used herein, when used in conjunction with the word "comprising," "a" or " The term "an" can mean one or more, and as used herein, "another" can mean one or more. "of" can mean at least two or more, and in certain embodiments, aspects of the disclosure For example, the term "consisting essentially of" or "consisting of" one or more elements of the disclosure. Some embodiments of the disclosure may include one or more elements, method steps, and / or methods of the disclosure. It may consist of, or consist essentially of, any of the components described herein. Any method or composition described herein may be used with respect to any other method or composition described herein. It is intended that the present application can be used in the process described herein. to be limited to particular embodiments of machines, manufacture, compositions of matter, means, methods, or steps; As used herein, "or" and / or "and" or "and" are not intended to The term "and / or" is used to describe several elements in combination with one another or exclusively. For example, "x, y, and / or z" can be used with "x" alone, "y" alone, or "z" alone. , "x, y, and z", "(x and y) or z", "(x or (y and z)" or "x or x, y, or z can mean "y or z." x, y, or z are specifically excluded from the embodiments. It is specifically contemplated that the

[0029] Throughout this specification, the terms "one embodiment," "embodiment," "particular embodiment," "related embodiment," "relevant embodiment," "relevant" and "relevant" are used interchangeably. "particular embodiment", "additional embodiment", or "further embodiment" References to specific features, configurations, or combinations thereof described in connection with the embodiments are not intended to be construed as limiting the present invention. or features are included in at least one embodiment of the present invention. The appearances of the above phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Moreover, any particular feature, structure or characteristic may be incorporated into one or more embodiments. may be combined in any suitable manner.

[0030] As used herein, the terms "about" or "approximately" refer to a reference amount, level, value, 30, 25, 20 for a number, frequency, percentage, dimension, size, amount, weight or length Amount, level, value, or number that changes by 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% , frequency, percentage, size, measure, amount, weight or length, and in certain embodiments , a value that pluses or minus 15%, 10%, 5%, or 1% before the number. With respect to a biological system or process, the term refers to a value within an order of magnitude, preferably within 5-fold, or more. Preferably, it means within a factor of 2. Unless otherwise specified, the term "about" refers to a particular numerical value. within the acceptable error range.

[0031] As used herein, the term "administer" or "administration" refers to the administration of a therapeutically effective amount of a composition. The term "provision of a therapeutic agent to an individual at a site where the therapeutic agent is needed" refers to providing a therapeutic agent to an individual at a site where the therapeutic agent is needed. Methods of administration include injection, such as by using a syringe; Alternatively, other delivery routes may be used, such as implantation of the composition at the desired site. The pathways are encompassed by this disclosure and are detailed elsewhere herein.

[0032] As used herein, "fibroblasts" are cells that (1) adhere to plastic and (2) grow in culture. They express the CD73, CD90, CD105, and CXCR-4 antigens, whereas CD14, CD3 4, CD45 and HLA-DR negative, and (3) three lineages: osteogenic, chondroblastic, and A collection of one or more cells capable of differentiating into at least one of the adipogenic and adipogenic lineages. Other cells with fibroblast-like properties are included within the definition of fibroblasts. The cells have a) regenerative activity, b) production of growth factors, c) direct or mediated by endogenous host repair mechanisms, The present invention has at least one of the following conditions: the ability to induce a healing response through induction of a mechanism of action of the Fibroblasts are found in bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissue, umbilical cord blood, and Wha These include, but are not limited to, rton jelly, placenta, amniotic tissue, pluripotent stem cell derived tissue, and teeth. In some embodiments, fibroblasts can be derived from any tissue that is not , which are CD34 positive upon initial isolation from tissue, have not been described phenotypically or functionally. Fibroblasts contain cells that are similar to cells that express NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD 106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO -1 and STRO-3, or a combination thereof. Fibroblasts may include cells isolated from tissue using a method such as the steps of: may later express the marker.

[0033] As used herein, a "healing response" refers to the incorporation of a regenerative composition (e.g., at least one The molecular, cellular, or tissue responses of an individual to the administration of regenerative fibroblasts The healing response occurs at the site of injury resulting from brain injury, including brain injury as encompassed herein. This reaction can either restore damaged brain tissue or destroy it. Function may be partially or completely restored. The response may be directly activated by the regenerative composition. The regenerative composition may induce an individual's endogenous repair mechanisms (e.g., the immune system) to repair the damage. Can repair damage and / or restore function partially or completely in damaged brain tissue possible.

[0034] As used herein, the term "pharmacologically acceptable" refers to an animal or does not produce adverse, allergic, or other untoward reactions when administered to humans; It refers to the fruiting body and composition.

[0035] As used herein, the term "pharmaceutical acceptable carrier" refers to any suitable carrier, including water, ethanol, polyvinyl chloride, ethyl acetate ... Polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) oils, coatings, isotonic and absorption delaying agents, etc. Any suitable stimulant, including, but not limited to, stimulants, liposomes, commercially available detergents, etc. and any solvent or dispersion medium. Supplementary biologically active ingredients may also be used in such carriers. It can be incorporated into the

[0036] As used herein, the term "stroke" or "cerebrovascular accident" refers to a stroke involving the blood vessels of the CNS. Refers to neurological symptoms and signs, usually focal and acute, resulting from a disease. The middle age group comprises approximately 80% of diagnosed stroke patients and ischemic cerebral infarction caused by thrombosis or embolism. Stroke, or hemorrhagic stroke, caused by rupture of blood vessels in the brain, constitutes approximately 20% of diagnosed stroke cases. Stroke can occur in either the anterior circulation (branches of the internal carotid artery) or the posterior circulation (vertebral artery). Infarctions occurring in the territory of the middle cerebral artery are the most common. This often results in a stroke syndrome accompanied by contralateral muscle weakness, sensory loss, and visual field defects. Depending on the hemisphere involved, either language or spatial perception disorders may be observed. Infarction of the cerebral artery territory can cause weakness, loss of sensation, and urinary incontinence. Damage to the supplementary motor area can cause speech disorders. Bilateral anterior cerebral artery territory infarction, for example, occurs when both arteries are connected to a single trunk. Abuse occurs when it occurs abnormally and consists of severe emotional blunting, motor inertia and mutism. These symptoms are associated with the inferior anterior orbitofrontal cortex. Infarction of the posterior cerebral artery territory is countered by destruction of the cerebral cortex. Anterior choroidal artery occlusion causes contralateral hemiplegia and sensory loss. Infarction limited to structures supplied by the deep perforator branches of the anterior cerebral artery may result in psychomotor slowing. Various combinations of speech impediments, dysarthria, agitation, contralateral neglect, and, in the case of the left hemisphere, language impairment. Atherothrombotic vascular occlusion can also occur in the internal carotid artery rather than in the intracranial vessels. The infarction involves both the middle and anterior cerebral arteries and is accompanied by weakness and loss of sensation in the arms and legs. Alternatively, the infarction may be limited to the distal shared territory (the border zone of these vessels). Destruction of the motor cortex results in weakness limited to the arms or legs.

[0037] As used herein, the term "tauopathy" refers to a condition characterized by the development of neurofibrillary tangles or glomerulonephropathy in the brain. This means that many neurodegenerative diseases result from the accumulation of tau in the brain, including retinal fibrillary tangles. Tauopathies are characterized by the excessive accumulation of tau, misfolding of the tau protein, and tau sealing. These may be due to the formation of inclusion bodies, post-translational modifications on tau, or other tau protein-related pathologies. Tauopathies involve traumatic brain injury, stroke, or the acute accumulation of tau protein in the brain. As used herein, the term "tauopathy" refers to any of a number of acute tauopathies, including other diseases such as tauopathy, cerebrovascular disease, and other conditions. When tauopathy is diagnosed, it is generally characterized by a relatively long-term course of CSF, ISF, and blood tauopathy. and / or extracellular fluid, including blood fractions (e.g., plasma). This condition is associated with the gradual onset of elevated tau in the extracellular fluid of the brain. Chronic tauopathies include Alzheimer's disease, Amyotrophic lateral sclerosis / Parkinson-Dementia Complex, Argyrophilic Grain Dementia, British amyloid angiopathy, cortico-encephalo-muscular degeneration, Creutzfeldt-Jakob disease, calcification-associated Diffuse neurofibrillary tangles, Down syndrome, Parkinsonism linked to chromosome 17 Frontotemporal dementia with frontotemporal lobar degeneration, Gerstmann-Straussler-Shea Brinker's disease, Hall-Baden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy Pfeiffer, Niemann-Pick disease type C, non-guanine motor neuron disease with neurofibrillary tangles, Pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive Subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, and Including, but not limited to, multi-infarct dementia.

[0038] The term "traumatic brain injury," also known as "TBI," is a form of acquired brain injury. TBI is a condition that results from trauma-induced damage to the brain. For example, TBI can occur as a result of a fall, a car accident, a sporting event, or Occurs when the head strikes an object suddenly and violently, during any number of different ways. TBI can also result from a subject puncturing the skull and entering brain tissue. These types of TBI can also result in contused brain tissue, bleeding in the brain, large or small lacerations in the brain, and This may result in nerve damage due to shear forces and / or swelling of the brain. Many secondary disorders occur, including spasms, neurochemical imbalances, etc. Symptoms of TBI include: Depending on the level of damage to the brain, it can be classified as mild, moderate, or severe. The individual may remain conscious or may experience loss of consciousness for a few seconds or minutes. Other symptoms of traumatic brain injury include headache, confusion, dizziness, blurred vision and fatigue. ringing in the ears, bad taste in the mouth, fatigue or lethargy, changes in sleep patterns, difficulty concentrating or paying attention , changes in thinking ability; people who have suffered moderate or severe traumatic brain injuries may show these same symptoms. However, the headache worsens or does not go away, and vomiting, nausea, and repeated convulsions and seizures occur. sleep, inability to wake up, dilated pupils in one or both eyes, slurred speech Symptoms include muscle weakness or numbness in the limbs, decreased coordination, confusion, restlessness, and agitation. Examples of TBI include diffuse axonal injury, concussion, contusion, backlash brain injury, and secondary Impact syndrome, penetrating injury, shaken baby syndrome, chronic traumatic encephalopathy (CTE), blastocyst-induced These include, but are not limited to, traumatic brain injury, and locked-in syndrome.

[0039] As used herein, the term "transient ischemic attack" (TIA) refers to a sudden neurological deficit. This refers to focal cerebral ischemia that causes cerebral infarction and is not associated with permanent cerebral infarction (e.g., The diagnosis of TIA may be clinical. The anticoagulant, warfarin, may reduce the risk of stroke after some types of TIA. An IA is defined as symptoms lasting less than an hour; most TIAs last less than 5 minutes. If the damage resolves within an hour, the possibility of an infarction is very high. Defects that resolve spontaneously within 1 to 24 hours are often not detectable by diffusion-weighted MRI and other studies. It is no longer considered a TIA because it has been shown to be frequently accompanied by infarction. TIAs are most common in older adults, with a TIA significantly increasing the risk of stroke in the first 24 hours. It will start within hours.

[0040] "Treatment," "treatment," or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment can refer to a method of reducing the disease or condition itself, rather than just the symptoms. Treatment can be any decrease from pre-treatment levels and can be used to treat a disease, condition, or It cannot be limited to complete ablation of the symptoms of a disease or condition. The methods provided herein include administering to a patient a therapeutically effective amount of ... medicament for treating an established disease, including a reduction in the severity of at least one symptom of the disease. or 10%, 20%, 30%, 40%, 50%, 60%, 70% or greater in severity of disease progression. %, 80%, 90%, or 100% reduction in the immunogenicity of a cell. The disclosed methods for determining the level of A subject is considered to be therapeutic if there is a detectable decrease in the immunogenicity of the cells. Thus, the decrease can be as low as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 100, 200, 300, 400, 500, 600, 150, 10 ...200, 300, 400, 500, , 70, 80, 90, 100%, or any amount in between. Does not necessarily refer to a cure for a disease or condition, but rather to an improvement in the outlook for a disease or condition. It is understood and contemplated herein that the treatment may be an effective treatment for at least one of the following conditions: Alternatively or additionally, a reduction in the severity or extent of at least one symptom. This can refer to a delay in

[0041] II. Fibroblasts

[0042] Embodiments of the present disclosure include fibroblasts that may be derived from autologous, allogeneic, syngeneic, and / or xenogeneic sources. The disclosed methods and compositions are directed to systems and methods for the use of cells in the brain of an individual. The present invention includes specific engineered cells for the treatment of inflammatory degenerative conditions, including those in In particular, the cells include at least any type of fibroblast. Also disclosed are fibroblasts of various tissue origin that actively inhibit degenerative processes in the brain. In one embodiment of the present disclosure, fibroblasts are used to treat various conditions, including those that are caused by immune responses. and also for the treatment of degenerative brain conditions (TBI, stroke, transient ischemic attacks, and / or as a cell therapy for the prevention and / or treatment of acute or chronic tauopathies In at least one embodiment, fibroblasts are used to treat brain injury directly or indirectly. One or more specific drugs and / or conditions can be treated and / or prevented indirectly. In certain embodiments, the agent is interferon gamma and / or platelet lipase. and optionally at least interferon gamma and / or platelet-rich plasma. Plasma (and / or platelet-rich lysate) activates fibroblasts to directly or indirectly stimulate the immune response. The route of administration, dosage and frequency of administration can be tailored to suit the disease process, if any. The dose is determined as a function of the severity and stage of the disease and can be optimized for routine medical practice. Cut.

[0043] In some embodiments of the present disclosure, the fibroblasts administered to an individual can be of any origin. , and any tissue type (e.g., skin, foreskin, fat, peripheral blood, cerebrospinal fluid, bone marrow, placenta, umbilicus The fibroblasts may be derived from various sources, including cord blood, Wharton's jelly, or a combination thereof. Methods for isolating cells are well known in the art, and any such method may be used in In certain embodiments, fibroblasts can be obtained by, for example, NANOG, OCT-4, SSEA-4, stem cell factor receptor, NGF-R, EGF-R, CD29, CD49a, CD73, CD105, CD140b, CD146, MSCA- 1, SSEA4, STRO-3, telomerase, NANOG, beta-III-tubule Phosphorin, NF-M, APP, GLUT, NCAM, Nurr1, GFAP, NG2, Oligo 1. Alkaline phosphatase, vimentin, osteoprotegerin, osterix, Dipsin, erythropoietin, SM22-α, c-1-antrypsin, ceruloplus Min, AFP, BDNF, NT-4 / 5, TrkA, BMP2, FGF2, FGF4, P DGF, TGFalpha, TGFbeta, VEGF or a combination thereof The fibroblasts may be cultured prior to use in the methods of the present disclosure. Any suitable method and culture medium for culturing may be used.

[0044] In certain embodiments, allogeneic or autologous fibroblasts are grown in an unmanipulated manner ( For example, the level of one or more particular factors (e.g., interferon gamma, and / or status) without prior exposure to, for example, placental fibroblasts or adipose tissue-associated fibroblasts. The disclosed nucleic acid is selected from sources that are naturally characterized by their immunomodulatory activity, such as erythrocytes. In another embodiment, any fibroblast can be transformed to confer an immature phenotype to the fibroblast. These cells are cultured under conditions that can induce post-differentiation, where the immature phenotype is mediated by regeneration and and / or correlates with enhanced anti-inflammatory and / or immunomodulatory capabilities to induce a healing response. For example, fibroblasts may be transformed with the presence of one or more histone deacetylase inhibitors, such as valproic acid. The cells can be cultured in the presence of HDAC inhibitors (Moon et al., 2008; Huang et al., 2011). In addition to the agents, 8-Br-cAMP (Wang et al., 2011); M-CSF treatment (Li et al., 2016); exposure to reverecin (Li et al., 2016); and / or exposure to stem cell extracts. Other means of inducing dedifferentiation of fibroblasts, such as dew (Xiong et al., 2014), have also been shown to , can be utilized in the context of the present invention. Extracellular markers such as EGFR-2, CD34, and / or CD133, as well as SOX- This can be achieved by assessing intracellular markers such as 2, NANOG, and / or OCT-4. In other words, fibroblasts with regenerative properties possess SOX, NANOG, and OCT4. however, upon dedifferentiation they have high levels.

[0045] In some embodiments of the disclosure, the dedifferentiated fibroblasts are selected from the group consisting of NANOG, OCT, and the like. -4, SSEA-R, EGF-R, CD29, CD49a, CD73, CD105, CD 140b, CD146, MSCA-1, SSEA4, STRO-3, telomerase, NA NOG, β-III-tubulin, NF-M, APP, GLUT, NCAM, Nurr 1, GFAP, NG2, Oligo1, Alkaline phosphatase, Vimentin, Osteoproteinase Tegrin, Osterix, Adipsin, Erythropoietin, SM22-α, HGF , c-MET, α-1-antrypsin, ceruloplasmin, AFP, PEPCK 1, BDNF, NT-4 / 5, TrkA, BMP2, BMP4, FGF2, FGF4, PDGF, PGF, TGFalpha, TGFbeta, VEGF, and combinations of these The present invention may be utilized in a method for expressing one or more markers selected from the group consisting of:

[0046] In certain embodiments of the present disclosure, the method is used to induce a regenerative and / or healing response. The fibroblasts may, for example, bind to a) one or more autoantigens; and / or b) one or more immunomodulatory The fibroblasts are genetically engineered to express a protein. In at least some cases, the gene used for the study is determined. The polynucleotide form is transfected into fibroblasts, which are then cultured and immunocompetent. The gene that allows regulation or allows immune regulation is transfected. Transfection to induce nodes and induce a regenerative and / or healing response in the brain Genes of particular interest include at least the following: Fas ligand, TGF -β, IL-4, IL-10, HLA-G, indoleamine 2,3 deoxygenase, Galectin family members, galectin 3, arginase, and / or IL-20 ( de Jesus et al., 2016;Wang et al., 2011;Zhao et al., 2010;Min (Cancedda et al., 2001; Cancedda et al., 2001). or an active portion thereof, which allows expression in fibroblasts, such as the CMV promoter. The gene can be cloned into a mammalian expression construct containing a promoter sequence that encodes the (E. et al., Exp. Dermatol. 1995, 4:317-21). Examples of suitable constructs include For pcDNA3, pcDNA3.1(+ / -), pGL3, PzeoSV2(+ / -) , pDisplay, pEF / myc / cyto, pCMV / myc / cyto (respectively (e.g., commercially available from vendors such as Invitrogen) or human foreskin cells. The pSH expression vector (Vent ura and Villa, 1993, Biochem.Biophys.Commun.1 92:867-9), but are not limited to these. Retroviral vectors and Examples of such packaging systems are commercially available from Clontech, San Diego, CA, USA. The Retro-X vectors pLNCX and pLXSN are commercially available. These allow cloning into multiple cloning sites, and transgenes are delivered via the CMV promoter. Vectors derived from Mo-MuLV include pBabe. , where the transcribed gene would be transcribed from the 5'LTR promoter. After completing the transfection, the fibroblasts can be detached from the tissue culture plate. The cells are harvested by a suitable means, for example by trypsinization, and then suitable for expansion. Approximately 3 days after transfection, the cell culture medium is transferred to a suitable container or vessel. The medium is replaced with one suitable for the growth and proliferation of blast cells. One example is Neurobasal-A (I (Invitrogen), 1% D-glucose (Sigma Aldrich), 1% Niacin / Streptomycin / Glutamine (Invitrogen), 2% B27 Thioic acid (Invitrogen), 0.2% EGF (Peprotech, USA ), 0.08% FGF-2 (Peprotech), 0.2% heparin (Sigma Aldrich, USA) and valproic acid (Sigma Aldrich) at a concentration of 1 μM. The medium is a Neurobasal A (NBA) growth medium containing up to 100% ATP. For example, replace the medium three times a week, and re-seed the cells regularly (for example, up to a maximum of once a week). 2–8 times, becoming more regular as colonies begin to develop), and widespread colonization The practitioner of the present invention will remove cells that have not been reprogrammed until the reprogramming is achieved. Various quality control measures for performing implantation are known in the art. Exemplary criteria for determining the identity of a particular individual include by means of flow cytometry, viability, endotoxin content, etc. These include marker identification using ELISA and assessment of microbial and mycoplasma contamination.

[0047] In certain embodiments of the invention, the fibroblasts are characterized by their viability and proliferation capacity. To preserve the cells, the cells are cultured ex vivo using means known in the art. The present invention relates to a method for the preparation of fibroblasts comprising the steps of: In one embodiment, the fibroblasts are cultured in conditions lacking xenogeneic components (e.g., The xenogeneic components are cultured in fetal bovine serum (FBS). are known to induce a psychiatric response (Selvaggi et al., 1997; Mackens En et al., 2000; Kadri et al., 2007; Forni et al., 1976; Lauer et al. In many individuals, natural antibodies of the IgM isotype are associated with fetal bovine serum-associated compounds. (Irie et al., 1974) and the administration of cells grown in the presence of fetal bovine serum Ingestion of certain substances can cause rejection, inflammation, or anaphylaxis (Macy et al., 1989). In embodiments, the present disclosure provides an additional feature for reducing the immunogenicity of fibroblasts, comprising: Fetal bovine serum was replaced with human platelet-rich plasma, platelet lysate, umbilical cord serum, autologous serum, and / or standard serum. This involves replacing the tissue in xeno-free medium with a defined cytokine mixture. Procedures for doing so are known in the art for other cell types and are incorporated by reference (R iordan et al., 2015).

[0048] In one embodiment of the present disclosure, fibroblasts are administered by injection (intramuscular injection), including to hypoxic areas. The compound may be administered to a subject by any suitable route, including by injection. Suitable routes include , intravenous, subcutaneous, intrathecal, oral, rectal, intrathecal, intrareticulum, intraventricular, intrahepatic, and intrarenal. .

[0049] In some embodiments, fibroblasts are derived from skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, in tissues including cerebrospinal fluid, adipose tissue, foreskin, placenta, Wharton's jelly, and / or umbilical cord In certain embodiments, the fibroblasts can be derived from placenta, fetus, neonatal, or adult tissue. A human, or a mixture of both.

[0050] The number of administrations of cells to an individual will depend, at least in part, on the nature of the conditions described herein, such as the type and severity of the injury. It depends on factors described herein and can be optimized using routine methods in the art. In certain embodiments, a single administration is required. In other embodiments, multiple administrations of cells are required. Of course, this system requires individual input that can change over time and circumstances. specific needs, the rate of loss of cell activity as a result of cell loss or activity of individual cells, etc. Thus, each individual can be monitored for appropriate dosage, and It is expected that such practices of monitoring individuals with

[0051] Embodiments of the present disclosure provide methods for reducing the immunogenicity of certain types of fibroblasts. Fibroblasts are found in the skin, heart, blood vessels, bone marrow, skeletal muscles, liver, pancreas, brain, cerebral fluid, fat, and peripheral blood. in various tissues or organs, such as peripheral blood, placenta, umbilical cord blood, Wharton's jelly, and / or foreskin These may be derived from biopsy (as appropriate) or autopsy. In such a case, the cells may be fibroblasts, which may be derived from fetal, neonatal, or adult origin, or a combination thereof. Contains cells.

[0052] In certain embodiments, the isolation procedure utilizes an enzymatic digestion process. The tissue is dissociated and the cell population is extracted, which is then harvested and expanded for isolation of fibroblasts. Many enzymes are required to separate individual cells from the complex tissue matrix to facilitate growth in culture. It is known in the art that such techniques are useful for isolating individual cells. A wide range of digestive enzymes are available to those of skill in the art for use in isolation, including weakly digestive (e.g., deoxyribonucleic acid) and ribozyme-dependent (e.g., ribozyme-dependent) enzymes. xylose, neutral protease, dispase) to strong digestive (e.g., These enzymes range from ribozyme A (IL-1) to trypsin (IL-2) and such enzymes are commercially available. A non-exhaustive list of enzymes that are compatible with this specification includes enzymes that exhibit mucolytic activity, metalloproteases, and the like. proteases, neutral proteases, serine proteases (trypsin, chymotrypsin, or ela For example, collagenase is a It is known that deoxyribonucleic acid is useful for isolating various cells from tissues. ATPase is capable of digesting single-stranded DNA and minimizes cell clumping during isolation. The enzymes can be used alone or in combination. Serine proteases are Since it can degrade the other enzymes used, it can be used in sequences subsequent to the use of other enzymes. The temperature and time of contact with the serine protease can be monitored. Protease can be inhibited by α2-microglobulin in serum, so it is not used for digestion. The media used are often serum-free. EDTA and DNase are commonly used to improve yield or Several methods can improve the efficiency, for example, collagenase and dispersase. enzymatic treatment with hyaluronidase, collagenase, dispase, and hyaluronidase; Such methods are provided and in certain preferred embodiments, include the use of collagenase and neutral protease. A mixture of protease and dispase is used in the dissociation step. At least one collagenase from Clostridium histolyticum in the presence of either the protease activity, dispase or thermolysin A particular method for this disclosure uses collagenase and dispase enzyme activity. In addition to collagenase and dispase activity, digestion with both hyaluronan and hyaluronan is also used. Other methods involve digestion with uronidase activity. Those skilled in the art can readily culture cells from a variety of tissue sources. It will be appreciated that many such enzymatic treatments for isolation are known in the art. For example, collagenase from the LIBERASE BLENDZYME (Roche) series and Enzyme combinations of neutral proteases are useful and can be used in the methods of the present invention. Other sources of enzymes are known, and those skilled in the art can also obtain such enzymes directly from their natural sources. Those skilled in the art will also be able to readily identify novel methods for isolating the cells of the present invention. The system is well equipped to evaluate the enzymes, or additional enzymes or enzyme combinations. The treatment may be for 0.5, 1, 1.5, or 2 hours or more in length. The tissue is incubated at approximately 37°C during the enzymatic treatment of the dissociation step. This may also improve cell yield since the cells may be trapped within the viscous digest.

[0053] The use of enzymatically active materials is disclosed but is not required for the isolation methods provided herein. Methods based solely on mechanical separation have not been successful in isolating the cells from the disclosed tissues. obtain.

[0054] The isolated cells can be cultured after the tissue has been dissociated in any culture medium as described herein above. The cells may be resuspended in a centrifugation step to separate the cells from tissue or other debris. Resuspension can be achieved by mechanical methods of resuspension or simply by adding culture medium to the cells. The growth conditions provided herein may include the addition of culture media, supplements, atmospheric conditions, etc. The optimal incubation temperature is approximately 37 °C. °C, but the temperature can be about 35 °C to 40 °C depending on other culture conditions and the desired use of the cells or culture. The cells may be maintained in a temperature range of about 5% to about 20% oxygen in their atmosphere. Also provided is a method for obtaining cells that require L-valine, the method comprising the steps of: To achieve this, the cells must be cultured in the presence of L-valine. The requirement for valine was tested by growing the strain on a D-valine-containing medium lacking the L-isomer. It can be confirmed.

[0055] Before reaching the senescent state, cells undergo at least 25, 30, 35, or 40 doublings. A method is provided for determining whether a 1×10 14 Can double to reach more than 100 cells Methods are provided for inducing cells capable of reproducing, preferably at least about 1x10 14 , 1x10 15 , 1x10 16 , or 1x10 17The cells were cultured at approximately 1x1 0 3 From about 1x10 6 Cells / cm 2 can be doubled enough to produce when seeded to Preferably, these cells are derived within 80, 70, or 60 days. is produced in

[0056] In certain embodiments, tissue fibroblasts are isolated, expanded, and immunoreacted with CD10, CD13, CD 44, CD73, CD90, CD141, PDGFr-α, CD105, CDXC-4, One or more markers selected from the group including HLA-A, HLA-B, or HLA-C In addition, the cells express CD31, CD45, CD117, CD141, HLA-DR , HLA-DP, or HLA-DQ.

[0057] In some embodiments of the present disclosure, the genetic modification of the fibroblasts is One method is to reduce the immunogenicity of the cells by using immunogens such as immature dendritic cells. In another embodiment, the genetic modification comprises cytoplasmic transfer with reduced cell proliferation. Using gene editing, HLA or CD40, CD80, CD86, TNF-α , HMGB-1, IFN-γ, IL-1β, IL-17, FAP, IL-18, IL-3 3, or combinations thereof.

[0058] In certain embodiments of the present disclosure, the one or more immunomodulatory agents are operable in eukaryotic cells. The antibodies are expressed in universal donor fibroblasts via recombinant expression vectors and then used for immune regulation. Expression of the promoter may be controlled by a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In certain embodiments, the vector is a retrovirus, a lentivirus, or Viral vectors such as adenovirus, adeno-associated virus, or herpes simplex virus The vector may be naked DNA, plasmid DNA, minicircle DNA, etc. It is a non-viral vector of interest for transfection into fibroblasts. The oligonucleotides include at least the following: Fas ligand, TGF-β, IL- 4. IL-10, HLA-G, indoramin 2,3 deoxygenase (IDO), galactamycin tin family members, galectin 3, arginase, IL-20, HGF, PDGF -BB, EGF, IGF, GDF-5, GDF-11, angiopoietin, FGF-1 , FGF-2, FGF-5, FGF-15, or combinations thereof. Recombinantly expressed angiogenic agents include FAS ligand, IL-2, IL-4, IL-10, and IL-20 , IL-35, HLA-G, I-309, IDO, iNOS, CD200, Galectin 3 , arginase, PGE-2, TGF-β, CTLA-4, PD-L1, IFN-γ, or may include combinations thereof.

[0059] In certain embodiments, a therapeutically effective amount of the modified cells is administered to an individual along with one or more immunomodulatory agents. Exemplary immunomodulatory agents include FAS ligand, IL-2R, IL-2, IL- 10, IL-20, HLA-G, PD-L1, IDO, iNOS, CD200, Galacti 3, arginase, PGE-2, atorvastatin, fluvastatin, pravastatin , Rosuvastatin, Pitavastatin, Acetylcysteine, Rapamycin, IVIGF- β, TGF, PDGF, CTLA-4, anti-CD45RB antibody, hydroxychloroquine, Flunomide, Dicyanogold, Sulfasalazine, Methotrexate, Etanercept Abatacept, anakinra, certolizumab, golimumab, which may include adalimumab , infliximab, tocilizumab, cyclosporine, IFN-γ, everolimus, rapamycin mycin, or a combination of these.

[0060] In certain embodiments, at least in some cases, the growth factor is The cells do not require exogenous growth factors, except for the supplemented serum provided with A method is used that does not require the use of specific growth factors. Methods for inducing cells are also provided herein. These methods are similar to those described above. However, these contain specific augmenting agents in the culture medium in which the cells are ultimately resuspended and grown. This may require the absence of growth factors (for which cells are not required). In practice, this method selects for cells that are able to divide in the absence of a particular growth factor. The specific cells are, in some embodiments, cultured in a serum-free, chemically defined culture medium. The cells can be grown and expanded in a growth medium. In such a case, the cells can be supported and Growth in serum-free medium may require specific growth factors that can be added to the medium to maintain cell viability. Factors that can be added for proliferation include one or more of FGF, EGF, IGF, and PDGF. In some embodiments, two, three, or all four factors are present in a serum-free medium. or added to a chemically defined medium. In certain embodiments, leukemia inhibitory factor, LIF is added to serum-free media to support or improve cell growth.

[0061] According to certain embodiments, the present disclosure provides a pharmaceutical composition comprising fibroblasts for treating brain injury. The present invention teaches the preparation of a method for the preparation of a fibroblast-derived fibroblast matrix (FCC) comprising: Fibroblasts may also be enriched for CD73 and / or CXCR-4 expression. According to some embodiments, the CD105 expressing cells have CXCR-4 mediated chemotactic activity. It further contains a subpopulation of strongly SDF-1 mobilized CD105 / CXCR-4+ cells.

[0062] In certain embodiments, the chemotactic fibroblasts are derived from bone marrow, umbilical cord blood, umbilical cord tissue, or tissue samples obtained from a subject. CD105+ fibroblasts were isolated from a population of fibroblasts isolated from the spleen, placenta, or skin. According to some embodiments, the chemotactic fibroblasts are prepared by purifying the Isolating or purifying CD105-expressing cells from a population of mononuclear cells isolated from an individual's skin biopsy It is prepared by:

[0063] According to certain embodiments, chemotactic fibroblasts are derived from the subject following treatment with a hematopoietic stem cell mobilizing agent. CD105-expressing fibroblasts were isolated from a population of cells isolated from peripheral blood collected from According to some such embodiments, the hematopoietic stem cells are prepared by purifying the hematopoietic stem cells. The mobilizing agent may be G-CSF, GM-CSF, or a pharma- ceutically acceptable analog or derivative thereof. According to some embodiments, the hematopoietic stem cell mobilizing agent comprises a combination of colony stimulating factors. According to some embodiments, the hematopoietic stem cell mobilizing agent is a recombinant analog or derivative. According to some embodiments, the CD105 The chemotactic activity of + / CXCR-4+ cells was enhanced by SDF-1, VEGF, and / or CXCR- Further, according to another embodiment, the expression of at least one of CD105+ cells is mediated by About 1% to at least about 95% are at least about 24 hours, at least about 48 hours after acquisition, Or is viable for at least about 72 hours.

[0064] III. Fibroblast Administration

[0065] Certain embodiments of the present disclosure relate to administration of fibroblast cells to an individual. The cells are pharma- ceutical acceptable. The pharmaceutical composition may be prepared for administration in a suitable carrier, for example, a sterile isotonic saline solution. In embodiments, the individual has suffered a traumatic brain injury, stroke, transient ischemic attack, and / or a tauopathy. The brain injury may include one or more of the following: The brain injury is improved by administration of fibroblasts. In certain embodiments, the fibroblasts may induce neurodegenerative effects that are associated with systemic The therapeutic agent is administered locally to the site of injury, but may be administered locally to the site of injury using any suitable delivery method; It promotes regeneration and inhibits axonal dysfunction after diffuse injury.

[0066] In certain embodiments, fibroblasts are administered to an individual by injection, including intrathecal and intravenous injection. Fibroblasts may also be administered intranasally. Cells are administered intranasally via aerosolized The fibroblasts may also be administered locally to the site of injury, e.g., to the brain. Access for direct administration may occur during surgery on an individual suffering from TBI. Administration in such instances may be by delivering the cells directly to brain tissue.

[0067] The appropriate dose of fibroblasts for use in accordance with various embodiments of the present disclosure depends on a number of factors. It may vary considerably for different situations. For primary and adjuvant therapy The parameters that determine the optimal dose of placental fibroblasts to be administered to the subject generally include some of the following: may include any or all of: the disease being treated and its stage; the species of the subject, its health, sex, age age, weight, and metabolic rate; the subject's immune status; other treatments being administered; the subject's medical history or genetic makeup; Complications expected by genotype. Parameters also include whether fibroblasts are syngeneic, autologous, allogeneic, or their potency (specific activity); the specific activity of the fibroblasts they target; and the location and / or distribution of fibroblasts that must be Other parameters may include characteristics of the site such as blast cell engraftment. Co-administration of factors (such as growth factors and cytokines) with fibroblasts is included. The optimal dose in also depends on the manner in which the cells are formulated, the manner in which they are administered, and the manner in which the cells are administered. Consider the degree to which the drug will be localized at the target site after administration. Finally, determining the optimal dose necessarily involves: There was no suboptimal threshold for maximum beneficial effect and no increase in fibroblast dose-related adverse effects. This would provide an effective dose above a threshold that outweighs the benefit of the reduced dose.

[0068] Optimal dose of fibroblasts for some embodiments used for autologous mononuclear bone marrow transplantation A relatively pure preparation of placental fibroblasts has been investigated in various experiments. The optimal dose in this form is 1 x 10 per kg of recipient mass per administration. 4 ~1×1 0 8 fibroblasts / kg. In particular cases, 1x10 4 ~1x10 8 , 1x10 4 ~ 1x10 7 , 1x10 4 ~1x10 6 , 1x10 4 ~1x105、 1x10 5 ~1x10 8 , 1x10 5 ~1x10 8 , 1x10 6 ~1x10 8 , or1x10 7 ~1x10 8 , In some embodiments, the effective amount per administration is 1×10 5 ~1×10 7 In many embodiments, the effective amount per dose is , 5×10 5 ~5×10 6 fibroblasts / kg. For reference, the higher doses mentioned above are similar to the doses of nucleated cells used in autologous mononuclear bone marrow transplantation. , CD34 used in autologous mononuclear bone marrow transplantation + Similar to cells / kg.

[0069] A single dose may be delivered all at once, partially, or continuously over a period of time. It should be understood that the entire dose may also be delivered to a single location or may be delivered to several locations. In various embodiments, the fibroblasts may partially extend over the initial doses, followed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more The fibroblasts may be administered initially by one method, and then maintained by administration of Thereafter, the subject may be administered the same or one or more different methods. The level can be maintained by continued administration of cells. , initially, or to maintain levels of fibroblasts in the subject, or both. In various embodiments, the fibroblasts are administered to the patient by intravenous injection. Other dosage forms may be used depending on the patient's condition and other factors, as discussed elsewhere. .

[0070] Human subjects generally receive longer treatments than experimental animals, but treatments generally do not delay the progression of the disease process. It is noted that the length of the antibody is proportional to the therapeutic efficacy. using the results of other procedures performed in animals such as rats, mice, and non-human primates. This should be taken into consideration when determining appropriate doses for humans. Based on this discussion, and taking into account the guidance provided by this disclosure and the prior art, The present invention provides a method for administering a therapeutically effective amount of a medicament for a patient of ordinary skill in the art without undue experimentation. Suitable regimens for single or sequential administration may all be the same or may be variable. Suitable regimens can be determined by one of skill in the art from this disclosure, the references cited herein, and the like. , and can be ascertained from knowledge in the art.

[0071] The dosage, frequency and duration of treatment will depend on the nature of the disease, the subject and any other medications that may be administered. It will depend on many factors, including the treatment. A wide variety of regimens can be used. In some embodiments, fibroblasts are Other fibroblasts are administered to the subject in two or more consecutive doses. The fibroblasts may be administered in a single dose, two doses, and / or three or more doses. In some other embodiments, the doses may be the same or different, They may be administered with equal or unequal intervals between them.

[0072] The fibroblasts can be administered at any frequency over a wide range of times. In other embodiments, the fibroblasts are administered for a period of less than one day. In some embodiments, they are administered for 2, 3, 4, 5, or 6 days. Fibroblasts were cultured at least once a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 The compound is administered for a period of 0, 11, 12 weeks or more. ,They are 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, number In various embodiments, they are administered for 1, 2, 3, 4, 5, It may be administered for a period of 6, 7, 8, 9, 10, 11, or 12 months. These are invested for periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. Generally, the length of treatment will depend on the length of the disease process, the effectiveness of the treatment being administered, etc. and proportional to the condition and response of the subject being treated.

[0073] In certain embodiments of the present disclosure, the circulating levels of one or more biomarkers are indicative of brain injury. The amount of fibroblasts to be administered for treatment of One such biomarker is the tau protein. Tau is classified into six types in humans: It is a structural protein with four isoforms and is a normal component of axons. Different isoforms may be applicable for biomarker measurement; total tau (t‐tau tau), truncated microtubule-associated tau (c-tau), phosphorylated tau (p-tau), and tau- A. Tau is associated with axonal damage after TBI. Specifically, the levels of c-tau in the CSF Its presence is a sensitive indicator of axonal damage [14,15]. It has also been shown that measuring the level of tau in an individual or The amount can be determined by, for example, ELISA, mass spectrometry, HPLC, LC / MS, Western blotting, etc. Any method known in the art, including immunoblotting, immunoprecipitation, immunostaining, and immunoelectrophoresis, may be used to detect the presence of the antibody. Tau can be measured in blood, plasma, CSF, ISF, or a combination thereof. , can be measured in any biological sample from an individual.

[0074] In certain embodiments, the fibroblasts are used to treat a medical condition, such as a stroke or acute ischemic attack. The fibroblasts may be administered in combination with another treatment to treat such conditions. such as cognitive therapy, communication therapy, brain stimulation therapy, or a combination of these. It may be administered during supportive care.

[0075] In certain embodiments, the fibroblasts are administered in one or more The fibroblasts may be administered in combination with at least one medical treatment, For example, removing blood clots, repairing skull fractures, reducing bleeding in the brain, and relieving pressure inside the skull. These drugs may be administered during surgery to induce coma. Fibroblasts may be administered to patients with at least one other treatment modality, and may be used to treat stroke during recovery. Fibroblasts may be administered to reduce pain and / or swelling. coagulants, anticonvulsants, antidepressants, diuretics, muscle relaxants, stimulants, or combinations thereof; The fibroblasts may be administered along with one or more other drugs useful in recovering from brain injury. , at least one supportive therapy, such as physical therapy, occupational therapy, recreational therapy, or speech therapy Or communication therapy, psychological counseling, vocational counseling, cognitive therapy, or may be administered in combination with a combination thereof.

[0076] In certain embodiments, the fibroblasts are used to treat a medical condition, such as a tauopathy. The fibroblasts may be administered in combination with another therapy. In combination with an agent that inhibits tau folding and / or the formation of tau inclusions. The agent may be a vaccine for tau pathology (e.g., AADvac-1, ACI -35), antibodies against tau or tau-related proteins (e.g., BMS -986168, ABBV-8E12), microtubule stabilizers (e.g., TP I-287), tau aggregation inhibitors (for example, TRx0237) ), drugs that inhibit post-translational modifications of tau (e.g., drugs that inhibit tau phosphorylation (chloramines) Lithium, tideglusib, etc.), drugs that inhibit tau-linked glycosylation (thiamet-G, etc.) ), drugs that inhibit tau acetylation (e.g., salate), Hsp90 inhibitors, or combinations thereof Examples of agents that modulate tau or tau-related mechanisms include: may have non-tau-related activities, and therefore their classification and mechanism of action may be either alone or In combination with the administration of the fibroblasts disclosed herein, These are provided only as non-limiting examples of possible uses.

[0077] IV. Brain Damage

[0078] Certain embodiments of the present disclosure are directed to individuals with brain injury (e.g., where the brain injury is a traumatic brain injury (TBI)). The present invention relates to a method for administering fibroblasts to a patient suffering from TBI. Examples of TBI include chronic traumatic injury (TCI) and chronic pulmonary artery disease (TCD). Encephalopathy, one or more concussions, blast-induced traumatic brain injury, backlash brain injury, cerebral contusion, shaking Bruised baby syndrome, penetrating injury, diffuse axonal injury, second impact injury, lock TBI can range in severity, A variety of symptoms may be present. In some embodiments, an individual may experience a range of symptoms, even if they are not all at the same severity level. may or may not have two or more TBIs. The injury may or may not be undiagnosed. The adverse events that cause a TBI are It can be from a direct or indirect impact to the body. An individual may have experienced, for example, one or more blunt impacts and One or more injuries from at least one impact to the head, including one or more stab impacts Individuals may or may not have received a TBI. Individuals may also suffer from: or suffered one or more TBIs from a whiplash injury, such as those resulting from shaken baby syndrome Maybe.

[0079] Certain embodiments relate to administration of fibroblasts to an individual having brain injury, wherein the brain injury A stroke can be either an ischemic stroke or a transient ischemic attack (TIA). For example, hemorrhagic stroke. Administration of fibroblasts may be initiated at the time of diagnosis of stroke or TIA, or The time of subsequent administration of fibroblasts may be at the time of subsequent diagnosis of stroke (or TIA). Or it may be minutes, hours, days, weeks, or years after initial diagnosis. The fibroblasts are then co-cultured with at least one other brain tumor suppressor or treatment to act additively or synergistically with the treatment or treatment. It may be administered simultaneously with stroke or TIA treatment. In some embodiments, the individual is fibroblasts, wherein the individual is determined to be at risk for having a stroke or TIA. An individual suspected of having a stroke may be provided with an effective amount of fibroblasts. .

[0080] In some embodiments of the present disclosure, fibroblasts are administered to an individual, wherein the individual have a tauopathy. Tauopathy can be acute or chronic. Acute tauopathy can be TBI, diffuse axonal injury, tremor Concussion, contusion, backlash brain injury, second impact injury, penetrating injury, shaking injury Any of the embodiments described above or herein may include, for example, the following: In any of these, the acute tauopathy may be stroke. In any of the embodiments, the acute tauopathy may be chronic traumatic encephalopathy. The tauopathy may be a chronic tauopathy, as further defined herein. - If an individual is suspected of having taopause and has a personal or family history, It may or may not be diagnosed, as it may not be present. EXAMPLES

[0081] The following examples are included to demonstrate certain non-limiting aspects of the disclosure. The techniques disclosed in the examples are believed to work well in the practice of the disclosed subject matter. It should be understood by those skilled in the art that it represents technology discovered by the inventors. However, those of skill in the art will appreciate that in light of this disclosure, many of the specific embodiments disclosed may be Modifications may be made, similar or implied, without departing from the spirit and scope of the disclosed subject matter. It should be understood that similar results can still be obtained. Example 1 Fibroblasts are superior to MSCs in reducing stroke-induced neurological deficits

[0082] For the study, female Sprague-Dawley rats (Ch) weighing 260–310 g were used. Arles River Laboratories, Wilmington, MA) The animals were randomly assigned to the experimental groups. The investigators were The animals were blinded to dose and treatment group assignment during the clinical evaluation. They underwent recurrent cerebral artery occlusion (rMCAo). Anesthesia was achieved with 3% isofluorane and 70% nitrous oxide. Rats were intubated and induced with 1–0.5% isofluorane, 70% nitrous oxide, and The patient was mechanically ventilated with a mixture of oxygen and the remainder of the oxygen. A catheter was inserted into the right femoral artery and vein to measure blood pressure. Ar was monitored and arterial samples were taken for blood gas and glucose assessment. Terial PCO2 and PO2 were maintained within normal limits by adjusting the ventilator. For the purpose of immunization, rats were administered 0.75 mg / kg panchromium bromide intravenously. Temperature was measured continuously and maintained at 37-37.5°C by a heating pad under the rat's body. Head temperature was monitored separately throughout the experiment using a 29-gauge thermocouple implanted in the right temporal muscle. The temperature was then maintained at 36-36.5°C by a heating lamp placed above the rat's head.

[0083] The common carotid artery (CCA) was exposed through a midline incision in the ventral neck and bluntly dissected using microsurgery. The vagus nerve was carefully separated from the surrounding tissues, including the adjacent vagus nerve, by a surgical microscope. the ECA, branches of the superior thyroid and ascending pharyngeal arteries, as well as branches of the terminal lingual and maxillary arteries The ECA was then incised and coagulated. Two long 5-0 silk sutures were used to secure the ECA to the carotid bifurcation. Two short (3 cm) 5-0 silk sutures were tied as distally as possible to the closed ECA at the bifurcation. A loose tie was tied around the segment, exposing the ICA and visualizing the origin of the pterygopalatine artery (PPA). The intraluminal 3.0 nylon suture has a bulbous, enlarged tip and is heated near a flame. and introduced into the distal segment of the ECA via a small incision on the vessel. The catheter is then advanced into the ECA lumen to reach the bifurcation. Two short silk sutures are then tied to The ECA was cut at the distal segment between the two long sutures. The CA stump was held toward the surgeon so that the stump and the ICA were in a straight line. Then, the suture was placed into the ICA, taking special care not to enter the PPA. The ICA was advanced until a sudden drop in cerebral blood flow was confirmed by LDF. The length of the suture inserted into the ICA ranged from 19 to 25 mm. A sudden sharp decrease in was interpreted as indicating successful MCA occlusion. The suture was then left for 90 min of occlusion. During the procedure and the 2-hour recirculation, blood pressure and blood gases remained within normal ranges. The patient was kept in a podiatric tether and cranial and rectal temperatures were maintained at normal levels.

[0084] A standardized neurobehavioral test battery was performed as previously described (Ley et al. JJ, Vigdorchik A, Belayev L, Zhao W, Busto. R et al. (2005) Second generation azulenyl nitrone antioxidants, stilbazulenyl nitrones, Conferring sustained neuroprotection in experimental focal cerebral ischemia in rats: neurobehavioral and histopathological studies and pharmacokinetics. J Pharmacol Exp Ther 313:1090- 1100), including tests of postural reflexes, sensorimotor integration, and proprioception. The total neurological score is Normal scores ranged from 0 to a maximum possible score of 12.

[0085] Forleskin fibroblasts were obtained from AllCells, and CD73-expressing fibroblasts were The CD401 cells were cultured according to the manufacturer's instructions, except that purification was performed prior to administration to animals. Selection of 73-expressing cells was performed using magnetic activated sorting (MACS) (Milteny). Mesenchymal stem cells were obtained from AllCells and were prepared according to the manufacturer's instructions. Both MSCs and fibroblasts were cultured at 1 million cells per animal. The drug was administered intravenously at a concentration of 0.01 mg / kg / day, 24 hours after middle cerebral artery ligation.

[0086] As seen in Figure 1, fibroblast administration was associated with superior neurological recovery compared to MSCs. It was related. (References)

[0087] The following references provide exemplary procedural or other information supplementary to those set forth herein: To the extent that details are provided, they are expressly incorporated herein by reference.

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[0099] 12.Alosco,M.L.,ら,Repetitive head impact exposure and later-life plasma total ta u in former National Football League pla yers.Alzheimers Dement (Amst),2017.7:p.3 3-40.

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[0102] 15. Ost, M., et al., Initial CSF total tau corre. lates with 1-year outcome in patients wi th traumatic brain injury.Neurology,2006 .67(9):p.1600-4.

[0103] 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 disclosure is not intended to be limited to particular 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 does not intend to perform substantially the same function or achieve substantially the same result as the corresponding embodiment(s) disclosed herein. Any now existing or later developed process, machine, manufacture, composition of matter, means, Methods or processes may be utilized in accordance with the present disclosure. The scope of the present invention includes within its scope such processes, machines, manufacture, compositions of matter, means, methods, or processes. It is intended to include processes.

Claims

[Claim 1] A composition for inducing a regenerative and / or healing response in the brain of an individual, comprising a therapeutically effective amount of fibroblasts administered to the individual.

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