UA109265C2 - APPLICATION OF PROGRAMMED NURSING STEM CELLS IN THE PREPARATION OF A MEDICAMENT OR PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

The invention relates to the use of one or more reprogrammed stem nerve cells in the preparation of a medicament or pharmaceutical composition for the renewal or replacement of tissues or cells of a patient suffering from a neurological disease, in which reprogrammed stem nerve cells obtained by reprogramming the lithic cells , in which reprogramming involves contacting these murdered cells with antibodies that cleave the mediator receptor capture, recognition or indication of the antigen on the surface of the commuted cells.

Description

progenitor cells of the first cell line from the patient, in which the reprogramming includes contacting these committed cells with antibodies that bind to a receptor that mediates the capture, recognition, or indication of an antigen on the surface of the committed cells.

Inclusion by reference

All documents cited or referred to in the documents cited herein, together with any product instructions, descriptions, product specifications and process maps for any product referred to herein or in any document included by reference, are hereby incorporated into this description by reference, and may be used in the implementation of the invention.

The field of technology to which the invention belongs

A method of treating various diseases, disorders, or conditions in a patient that uses reprogrammed cells, such as retrodifferentiated, transdifferentiated, or predifferentiated cells. The method involves obtaining committed progenitor cells of a patient (patient), retrodifferentiation of committed progenitor cells to obtain retrodifferentiated target cells, and administration of retrodifferentiated cells to the patient. In certain embodiments, the method involves obtaining committed progenitor cells of the patient, transdifferentiation of committed progenitor cells to obtain transdifferentiated target cells, and administration of transdifferentiated target cells to the patient. Retrodifferentiated or transdifferentiated target cells restore or renew the patient's tissues or cells.

Technical level

Stem cells are characterized by their ability to renew themselves through mitotic cell division and to differentiate into various lineages of specialized cell types. The two major types of mammalian stem cells are embryonic stem cells, which are released from the inner cell mass of the blastocyst, and differentiated stem cells, which are found in mature tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells renew specialized cells and support the normal renewal of organs capable of repair, such as blood, skin, or intestinal tissue.

Stem cells are widely distributed in developing embryos, although the number of stem cells decreases as development progresses. On the contrary, the adult body contains a limited number of stem cells, which are limited to certain compartments of the body.

The therapeutic use of stem cells has the potential to revolutionize the treatment of many diseases or disorders. While some mature stem cell treatments, such as bone marrow transplants, already exist, medical researchers anticipate using stem cells to treat a wider array of diseases, including, among others, cancer, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis, multiple sclerosis and muscle damage. Such treatments can take advantage of stem cells' ability to differentiate into the cell types needed to treat a disease.

However, there is uncertainty about the success of treating such diseases using stem cells, as well as about the ease of obtaining stem cells. For example, hematopoietic stem cells are traditionally obtained from bone marrow, peripheral blood that mobilizes a growth factor, or umbilical cord blood (placenta). Hematopoietic stem cells can also be obtained from embryonic stem cells, which are extracted from embryos obtained using the technique of in vitro fertilization. However, obtaining from these sources is difficult and sometimes dangerous, and can be complicated by attendant ethical issues.

Further, the number of stem cells that can be obtained from these sources is limited. In addition, stem cells may have difficulty differentiating into the cells needed to treat a disease.

Disclosure of the invention

The present invention relates to the use of reprogrammed cells to restore or renew tissue or cells of a patient. For example, the present invention relates to the use of retrodifferentiation of differentiated or committed progenitor cells to restore tissue or renew tissues or cells of a patient. The present invention also relates to the use of transdifferentiated cells obtained by transdifferentiation of differentiated committed progenitor cells to restore tissues or renew tissues or cells of a patient.

The application is based, in part, on the applicant's discovery that committed progenitor cells or somatic cells obtained from a patient can be reprogrammed to produce cells of different lineages, and these reprogrammed cells can be injected into the patient to repair or renew tissues or cells. Examples of reprogramming processes include retrodifferentiation and transdifferentiation.

Consequently, the applicants have discovered that committed progenitor cells can undergo reprogramming to obtain different cell lines. For example, committed progenitor cells can undergo retrodifferentiation to produce retrodifferentiated cells, for example, cells that are less differentiated, such as pluripotent (pluripotent) stem cells, and that these retrodifferentiated cells can be administered to a patient to repair or renew tissues or cells. In another example, committed progenitor cells obtained from a patient can be transdifferentiated to produce transdifferentiated cells, e.g., cells of other cell lines than the committed progenitor cells, and that these transdifferentiated cells can be administered to the patient to repair or renew tissues or cells

The invention covers a method of restoring or renewing tissue or cells of a patient's cell lines by introducing reprogrammed cells into the patient. In particular, the invention covers a method of restoring or renewing tissue or cells of cell lines in a patient, which involves (Her) obtaining committed progenitor cells, (ii) retrodifferentiation of committed progenitor cells to obtain retrodifferentiated target cells and (ii) introducing retrodifferentiated target cells to the patient , in which retrodifferentiated target cells re-differentiate into cells of cell lines. These redifferentiated cells can belong to the same cell line or a different cell line, such as committed progenitor cells.

The invention also covers a method of restoring or renewing tissue or cells of a patient's cell line, which involves (І) obtaining committed progenitor cells, (К(« (ii) transdifferentiation of committed progenitor cells to obtain transdifferentiated target cells, and (ii) introducing transdifferentiated target cells to the patient.

In some embodiments, the patient may suffer from a disease, disorder, or condition, including, but not limited to, a bone marrow disorder, hematologic conditions, aplastic anemia, beta-thalassemia, diabetes, motor neurone disease, Parkinson's disease, spinal cord injury, muscle dystrophy, kidney disease, multiple sclerosis, congestive heart failure, hepatitis C virus, human immunodeficiency virus, head injury, spinal cord damage, lung disease, depression, non-obstructive azoospermia,

From andropause, menopause and infertility, rejuvenation, ulcerative scleroderma, psoriasis, wrinkles, liver cirrhosis, autoimmune disease, baldness, retinitis pigmentosa, crystalline dystrophy/blindness, diabetes and infertility. Thus, in some embodiments, the reprogrammed cells may be bone marrow cells that treat aplastic anemia, leukemia, lymphoma, or human immunodeficiency virus in a patient.

In some embodiments, reprogrammed target cells, such as retrodifferentiated cells, transdifferentiated target cells, or redifferentiated cells, may include, but are not limited to, pluripotent stem cells, pluripotent germ cells, hematopoietic stem cells, neural stem cells, epithelial stem cells, mesenchymal stem cells, endoderm and neuroectoderm stem cells, germ cells, extraembryonic, embryonic stem cells, kidney cells, alveolar epithelial cells, endoderm cells, neurons, ectoderm cells, islet cells, acinar cells, oocytes, sperm, hematopoietic cells, hepatocytes, skin /keratinocytes, melanocytes, bone/ osteocytes, hair/dermal papilla cells, cartilage/chondrocytes, fat cells/adipocytes, skeletal muscle cells, endothelial cells, cardiac muscle/cardiomyocytes and tropoblasts.

In certain embodiments, committed progenitor cells are obtained from blood or related tissues, including bone marrow. Committed progenitor cells can be obtained from whole blood, and/or can be obtained through apheresis. Blood can be mobilized or non-mobilized blood. Such committed progenitor cells include, but are not limited to, T cells, B cells, acidophilic granulocytes, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, lymphocytes, leukocytes, platelets, and red blood cells. Alternatively, committed progenitor cells can be derived from neuronal tissue of the central nervous system or peripheral nervous system, muscle tissue, or the epidermis and/or dermis of the skin.

In certain embodiments, committed progenitor cells are obtained from the blood or tissue of a patient. In some embodiments, the patient from whom committed progenitor cells are obtained and to whom reprogrammed target cells, such as retrodifferentiated target cells or transdifferentiated target cells, are administered is the same patient.

In some embodiments, committed progenitor cells retrodifferentiate 60 by contacting the committed progenitor cells with an agent. For example, committed cells-

precursors can be cultured with a medium (with an agent). In certain embodiments, the agent binds to a receptor that mediates antigen capture, recognition, or presentation on the surface of committed cells. The receptor can be an antisem of the MHC class | or MHC class II antigen. In some embodiments, the antigen class | is the NI A-A receptor, the NHA-B receptor, the NHA-C receptor, the NI A-E receptor, the NI A-E receptor or the NHA-C receptor, and the indicated class I antigen is the NHA-OM receptor, the NHA-OR receptor, NHA-BO receptor or NIG A-OB receptor.

In certain embodiments, the agent may be an antibody to the receptor, such as a monoclonal antibody to the receptor. In some embodiments, the antibody is a SK3/43 monoclonal antibody or a TAI monoclonal antibody. 185. In further embodiments, the agent modulates the expression of the MHC gene, such as the expression of MHC class I" and/or MHC class

Pk.

In some embodiments, the retrodifferentiated cells can be redifferentiated in a separate stage. For example, retrodifferentiated cells can be redifferentiated by contacting the retrodifferentiated cells with growth factors that include, but are not limited to, basic fibroblast growth factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, stem cell factor, interleukins-1-3, -6 and - 7, basic fibroblast growth factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, erythropoietin, stem cell factor, and bone morphogenetic proteins. The resulting redifferentiated target cells can then be administered to the patient.

In embodiments of the invention, committed progenitor cells can be transdifferentiated by culturing committed progenitor cells under specific culture conditions. For example, committed cells can be cultured in specific types of culture media in combination with retrodifferentiation agents. Examples of these culture media may include Eagle's modified Dulbecco's medium (OMEM) or IMOM medium and others. Tissue culture media may also include an agent that promotes differentiation, such as vitamins and/or mineral supplements, hydrocortisone, dexamethasone, beta-mercaptoethanol, and so on. In addition, additional cultivation conditions

These include chelating agents or antibiotics, cultivation at specific temperatures or levels of carbon dioxide or oxygen, and cultivation in special vessels. Cultivation conditions can determine the type of transdifferentiated target cells that arise.

Thus, one aspect of the invention is a method of obtaining target cells. The method may include obtaining committed progenitor cells and then reprogramming the committed cells. These methods are described in this application. In some embodiments, the method may include retrodifferentiation of committed cells. In other embodiments, the method may include transdifferentiation of committed cells. In other embodiments, the method may include retrodifferentiation of committed cells, and then redifferentiation of the retrodifferentiated cells.

Another aspect of the invention is the use of one or more reprogrammed target cells to obtain a drug to restore or renew tissues or cells of a patient's cell lines, or to treat disease or tissue damage.

Yet another aspect of the invention is a method of treating disease or tissue damage in a patient in need. In certain embodiments, the method involves obtaining committed progenitor cells, reprogramming the committed cells to obtain reprogrammed target cells, and administering the reprogrammed target cells to the patient. In some embodiments, target cells can be reprogrammed by retrodifferentiation, transdifferentiation, and/or redifferentiation. In certain embodiments, the target cells are retrodifferentiated target cells, transdifferentiated target cells, and/or redifferentiated target cells. Methods of obtaining committed progenitor cells and reprogramming committed cells are as described in this application.

In some embodiments, reprogrammed target cells, such as retrodifferentiated target cells, transdifferentiated target cells, or redifferentiated target cells can be administered by injection, implantation, or infusion. These cells can be administered by parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, transdermal injection, or spinal fluid injection. In certain embodiments, retrodifferentiated target cells or transdifferentiated target cells are administered in a pharmaceutical composition. for the pharmaceutical composition may include retrodifferentiated target cells or transdifferentiated target cells and at least one pharmaceutically acceptable excipient.

One aspect of the invention is a pharmaceutical composition for the administration of reprogrammed target cells, such as retrodifferentiated target cells or transdifferentiated target cells. The pharmaceutical composition may include one or more types of target cells and at least one pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may include excipients and/or other excipients suitable for administration to the patient.

Another variant of the invention is a method of obtaining a pharmaceutical composition or a medicinal product, which involves (i) obtaining committed progenitor cells, (ii) reprogramming the committed cells to obtain reprogrammed target cells, and (ii) optionally, combining the reprogrammed target cells with one or more pharmaceutical excipients. In some embodiments, the reprogrammed target cells are combined with one or more pharmaceutical excipients. In other embodiments, the reprogrammed target cells are not combined with one or more pharmaceutical excipients. Methods of obtaining committed progenitor cells and reprogramming committed cells are described in this application.

It should be understood that in this description and, in particular, in the claims, terms such as "comprising", "which contains", etc., may have the meaning defined for them in the US

Patent law; for example, they may mean, "includes", "which includes", etc.; and that terms such as "consisting of" and "consisting of" and "consisting of" have the meanings assigned to them in the United States Patent Law, i.e., that they include elements not expressly described, but exclude elements found in prior art, or which affect the essential or novel feature of the invention.

These and other embodiments are disclosed or apparent and encompassed in the following detailed description.

Brief description of the drawings

The following detailed description is provided by way of example, but is not intended to limit the invention solely to the specific embodiments described, and may be best understood

Zo in combination with the following drawings, on which:

Fig. 1 shows immunophenotyping of apheresized mononuclear cells before and after (lower panel) initiation of reprogramming. Cells were labeled with monoclonal antibodies bound to K-phycoerythrin (CRE) Su-5 or phycoerythrins (PE) (vertical symbols) for isotype control of immunoglobulin C1 (IDS) and CO34 or CO19, respectively. Cells were also stained for isotype controls CO45, COZ38, COb1, and IDOI monoclonal antibodies bound to fluorescein isothiocyanate (RITS) (horizontal markings). The lower panel shows an increase in hematopoietic stem cells, as shown by an increase in the relative number of СО34 and СО34С038-cells, which is accompanied by a decrease in mature leukocyte markers such as СУ45 and СО19;

Fig. 2A shows sequential immunophenotyping of peripheral blood samples from a patient with severe aplastic anemia followed by infusion of autologous NCSCs (after 1 day, 2 days,

From the day, 6 days and 14 days). Cells were labeled with monoclonal antibodies against COZ4 and CO45 (2nd horizontal panel), and CO34 and CO38 (3rd horizontal panel). The upper horizontal panel shows forward and side scatter flow cytometry, bone marrow smear, and cross section of autologous HE5Cs. Day 1 - Day 14 show an increase in cells that have large forward and side scatter indicative of granulocytes, and Day 17 - Day C show an increase in the relative number of circulating CO34 hematopoietic stem cells.

Fig. 265 shows sequential immunophenotyping of peripheral blood samples from a patient with severe aplastic anemia followed by autologous reprogrammed human stem cell (HRC) infusion (after day 1, day 2, day 3, day 6 and day 14). Cells were labeled with monoclonal antibodies against COZ34 and CO61 (1st horizontal panel), CO19 and COZ (2nd panel), and CO33813 and CO07 (3rd horizontal panel). The graph of RGAS5sap shows an increase in the number of myeloid cells, as shown by a gradual increase in cells that express CO3813, including progenitor cells, which is shown by a gradual increase in the relative number of cells that co-express CO33813 with CO7. In addition, there was a gradual increase in the relative number of lymphocytes, as shown by the increase in the relative number of SХО19 and OZ lymphocytes;

Fig. C shows the analysis of the bone marrow of a patient with severe aplastic anemia before and after the infusion of autologous NAS. A bone marrow smear before and after therapy (a and Y) shows an increase in red blood cells; cross-section of the Iegrpipe before and after therapy (c and 4) shows an increase in the saturation of bone marrow cells; clonal analysis of bone marrow followed by NK5C infusion shows increased growth of the megakaryocyte colony-forming unit (ie); colony-forming monocytes (Y); colony-forming granulocytes-macrophages (49); of colony-forming myelocytes-erythroids (Y); and explosive erythroids (i);

Fig. 4 shows karyotyping and C-banding of a peripheral blood sample of a patient with severe aplastic anemia, followed by 4 years of infusion of autologous NK5C, demonstrating the absence of genetic abnormalities;

Fig. 5 shows an increase in the absolute mean levels of fetal hemoglobin in patients with thalassemia following treatment with autologous reprogrammed cells;

Fig. b shows an increase in the average hematocrit, which is the average size of red blood cells (erythrocytes), presented in femtoliters, in patients with thalassemia following treatment with autologous reprogrammed cells;

Fig. 7 shows the increase in mean cellular hemoglobin, which is the weight of hemoglobin per cell presented in picograms, in thalassemia patients after treatment with autologous reprogrammed cells;

Fig. 8 shows a decrease in serum ferritin, expressed in nanograms per milliliter, in thalassemia patients after treatment with autologous reprogrammed cells;

Fig. 9 shows an increase in C-peptide levels in the fasting state and after the assimilation of a mixed flour meal in diabetic patients after treatment with autologous reprogrammed cells;

Fig. 10 shows the reduction of levels of glycosylated hemoglobin (GBH) in diabetic patients after treatment with autologous reprogrammed cells;

Fig. 11 shows a decrease in the levels of creatine phosphokinase (CPC) and lactate dehydrogenase (LDH) in patients with muscular dystrophy after treatment with autologous reprogrammed cells;

Fig. 12 shows a decrease in the levels of liver enzymes, alanine aminotransferase (AL) and aspartate aminotransferase (A5T), in patients with muscular dystrophy after treatment with autologous reprogrammed cells;

Fig. 13 shows the reduction of microalbumin-urea levels in 12 patients with kidney disease after treatment with autologous reprogrammed cells;

From Fig. 14 shows the reduction of levels of glycosylated hemoglobin (GNAI) in 12 patients with kidney disease due to diabetes after treatment with autologous reprogrammed cells;

Fig. 15a shows magnetic resonance imaging (MRI) scans of the brain of a multiple sclerosis patient before (left scan) and three months after (right scan) treatment with autologous reprogrammed cells and demonstrates a reduction in damage enhancement after stem cell therapy;

Fig. 150 shows MRI scans of various areas of the patient's brain before (left scan) and three months after (right scan) treatment with autologous reprogrammed cells.

In pre-treatment scans, arrows indicate damage in the brain, while in post-treatment scans, arrows show reduced damage;

Fig. 1ba shows a magnetic resonance imaging (MRI) scan of the brain of a multiple sclerosis patient before (upper scans) and six months after (lower scans) treatment with autologous reprogrammed cells.

Fig. 16p shows additional MRI scans of the patient's brain before (upper scans) and six months after (lower scans) treatment with autologous reprogrammed cells.

In scans showing the pre-treatment state, the arrows indicate damage in the brain, while in the scans showing the post-treatment state, the arrows indicate reduction in damage with reduced brain atrophy, as shown by ventricular shrinkage and gyrus enlargement;

Fig. 17a shows sagittal magnetic resonance imaging scans of a multiple sclerosis patient before (left scan) and six months after (right scan) treatment with autologous reprogrammed cells.

Fig. 17p shows transverse MRI scans of the patient's spinal cord before (left scan) and six months after (right scan) treatment with autologous reprogrammed cells. In pre-treatment scans, arrows indicate spinal cord damage, while in post-treatment scans, arrows indicate reduced damage;

Fig. 182 shows a decrease in the levels of the liver enzyme alanine aminotransferase (AT), a

Fig. 18p shows a decrease in the levels of aspartate transferase (A5T) in patients infected with hepatitis C followed by treatment with autologous reprogrammed cells;

Fig. 192 shows magnetic resonance imaging (MRI) scans of the brain of a patient with a head injury from a car accident. Before treatment (upper scans), the ventricles show dilation and displacement with a wide, acute hematoma. After treatment with autologous reprogrammed cells (bottom scans), the ventricles show a reduction in parameters of brain atrophy, such as ventricular shrinkage and gyrus enlargement with hematoma reduction.

Fig. 19 shows additional MK scanograms! brain of the patient before and after (bottom scanograms) treatment;

Fig. 20 shows an X-ray image of a lung disease patient before and after (right image) treatment with autologous reprogrammed cells. After the treatment of the patient, an improvement in the volume of the lungs and a reduction in the size of the damage is shown, as shown by a reduction in the areas of hypo-density;

Fig. 21 shows the levels of the sex hormone - follicle-stimulating hormone (FH), luteinizing hormone (HH), progesterone (rho) and testosterone (h1e5h) in patients with obstructive azoospermia treated with autologous reprogrammed cells. Levels show a significant increase in free testosterone in parallel with the increase in testicular size (data not shown), determined by ultrasound;

Fig. 22 shows retinal sensitivity and vision loss in a low vision patient before and after (lower panel) treatment with autologous reprogrammed cells.

From the retinal sensitivity data, it follows that the orange area indicates reduced retinal sensitivity. From the visual impairment data, it follows that white areas indicate normal vision, while pink, orange, and black areas indicate increased vision loss. After the treatment, the patient experienced an improvement in his field of vision, as the orange areas according to the retinal sensitivity results before the treatment turned green and white after the treatment, and the black areas with vision loss before the treatment turned white after the treatment.

Implementation of the invention

Definition

The term "committed progenitor cells" as used herein refers to cells that exhibit a differentiated nature. These cells are often considered mature and

With specialized Examples include white blood cells, red blood cells, epithelial cells, neurons, and chondrocytes.

The term "uncommitted cells" as used herein refers to cells that do not exhibit a mature differentiated character. These cells are often considered immature and unspecialized. An example of an uncommitted progenitor cell is a stem cell, which is an immature cell that is capable of self-renewal (dividing without restriction) and differentiation (specialization). The term "reprogramming" as used herein refers to the process in which a committed cell of the first line of differentiation is transformed into a cell of another cell type. This different cell type may have a different lineage of cell differentiation. Reprogramming can occur through such processes as retrodifferentiation, transdifferentiation, redifferentiation.

The term "reprogrammed cell" as used herein refers to a cell that is a committed cell that has undergone reprogramming. Reprogrammed cells may include retrodifferentiated cells, transdifferentiated cells, or redifferentiated cells.

The term "retrodifferentiation" as used herein refers to the process by which a committed, ie, mature, specialized cell reverts back to a more primitive cell stage. A "retrodifferentiated cell" is a cell that results from the retrodifferentiation of a committed cell.

The term "transdifferentiation" as used herein refers to the process by which a committed cell of the first line of differentiation is transformed into another cell of a different cell type. In some embodiments, transdifferentiation may be a combination of retrodifferentiation and redifferentiation. A "transdifferentiated cell" is a cell , which is formed from a transdifferentiated committed cell.For example, a committed cell such as a whole blood cell can be transdifferentiated into a neuron.

The term "redifferentiation" as used herein refers to the process by which an uncommitted cell or a retrodifferentiated cell differentiates into a more mature, specialized cell. The term "redifferentiated cell" refers to a cell derived from a predifferentiated uncommitted cell or a retrodifferentiated cell.

If the redifferentiated cell is obtained as a result of redifferentiation of a retrodifferentiated cell, the redifferentiated cell may have the same or a different lineage of differentiation as the committed retrodifferentiated cell.

For example, a committed cell, such as a white blood cell, can be retrodifferentiated to produce a retrodifferentiated cell, such as a pluripotent stem cell, and then the retrodifferentiated cell can be redifferentiated to produce a lymphocyte of the same cell lineage as the white blood cell (committed cell), or redifferentiated to obtain a neuron that has a different line of differentiation than a white blood cell (committed cell).

The term "target cell" as used herein refers to a cell that is obtained for administration to a patient for the purpose of restoring or renewing tissue or cells. For example, the target cell can be a reprogrammed target cell, such as a retrodifferentiated target cell or a transdifferentiated target cell, thus retrodifferentiated or transdifferentiated target cells are administered to the patient.

Committed cells

As described above, the committed cells proposed by the present invention are cells that exhibit a differentiated nature. The committed cell may include any components that relate to antigen display, capture, or recognition. For example, a committed cell can be a cell of MHC class I: and/or MHC class II".

A committed cell may also be any cell derived or derived from an undifferentiated cell. Thus, in one embodiment, an uncommitted cell is also an undifferentiated cell. As an example, the committed cell can be a lymphoid stem cell or a myeloid stem cell that is differentiated into a pluripotent stem cell. Committed cells can be derived from biological material such as blood or related tissues, including bone marrow or umbilical cord blood, neuronal tissue from the central nervous system or peripheral nervous system, muscle tissue, or epidermal and/or skin tissue (ie, e.g. , scrapings from the oral cavity).

Biological material can be of postnatal origin.

Biological material can be obtained using methods known in the art that are appropriate for the type of tissue. Examples include, but are not limited to, excision, needle extraction, swabbing, and apheresis.

In certain embodiments, committed cells are obtained from whole blood or processed blood products, such as plasma or the light layer of a blood clot, because their removal from subjects can be performed under minimal medical supervision. Blood samples are usually treated with anticoagulants such as heparin or citrate. Cells in a biological sample can be processed to enrich certain cell types, remove certain cell types, or separate cells from the bulk of the tissue. Suitable methods for cell purification and separation are centrifugation (such as density gradient centrifugation), flow cytometry, and affinity chromatography (such as the use of magnetic beads that incorporate monoclonal antibodies to cell surface markers or penning) (see Meyese-JuOadeu, Te Zsiepievivi 1999 , 13: 21).

For example, ficoll-gypsum gradient separation is suitable for removing erythrocytes and granulocytes to leave mononuclear cells such as lymphocytes and monocytes.

Examples of committed cells that can be derived from blood include, but are not limited to, SES-T cells, SES-B cells, SES-eosin cells, SES-Bav cells, SES-Ba:x cells, SES-CM cells , SES-M, SES-MES cells, VES-E cells, SES-E cells, T-cells, B-cells, eosinophils, granulocytes, basophils, neutrophils, monocytes, megakaryocytes and erythrocytes.

The resulting committed blood cells can be identified by the expression of certain antigens. For example, B-cells are cells СО19:, СО21:, bО22: and OKU. T cells are cells

Ср2г», С03», and either СО4», or СОВ8». Immature lymphocytes are CO4: and CO8: cells. Activated

T-cells are OK cells. Natural killer cells (NK) are CO56 and CO165 cells. T-lymphocytes are CO7 cells. Leukocytes are cells of СО457". Granulocytes are cells of СО13: i

СО33". Cells of monocyte macrophage are CO14" and OK" cells.

In certain embodiments, the committed cell can be a B-lymphocyte (activated or non-activated), a T-lymphocyte (activated or non-activated), a cell of the monocyte-macrophage cell line, a nucleated cell that is capable of expressing antigens of the class | or class II, a cell that can stimulate the expression of class | antigens or class II or an enucleated cell (ie, a cell that does not contain a nucleus, such as a red blood cell).

In alternative embodiments, the committed cell may be selected from any group of cells that includes large granular lymphocytes, null lymphocytes, and natural killer cells, each of which is capable of expressing CO56 and/or CO16 cell surface receptors.

Since committed cells are actually primary cultures, it may be necessary to supplement the cell populations with appropriate nutrients to maintain viability. Appropriate cultivation conditions are known to a specialist in technology. However, treatment of the cell population is preferably initiated as soon as possible after the removal of biological samples from patients, usually within 12 hours, preferably within 2-4 hours. Cell viability can be checked using known techniques such as trypan blue or propidium iodide exclusion.

Retrodifferentiated cells

Retrodifferentiation is a type of reprogramming method in which cell structures and functions are progressively altered to give rise to less specialized cells.

Retrodifferentiation can occur naturally, and cells can undergo limited retrodifferentiation ip mimo in response to tissue damage.

Alternatively, retrodifferentiation can be induced using methods described in application O5 08/594,164, now patent O5 6,090,625; application 05 09/742,520, now patent 05 7 112 440; application 05 10/140,978, now patent 05 7 220 412; application 05 10/150,789, now patent 05 7 410 773; and application O5 Mo. 09/853,188, all of which are incorporated herein by reference.

Retrodifferentiated cells of the invention may include, but are not limited to, pluripotent stem cells, lymphoid stem cells, myeloid stem cells, neural stem cells, skeletal muscle satellite cells, epithelial stem cells, endodermal stem cells, mesenchymal stem cells, and embryonic stem cells. cells

In certain embodiments, committed cells are obtained from blood and retrodifferentiated to obtain retrodifferentiated cells of the hematopoietic cell line.

Examples of these retrodifferentiated cells include, but are not limited to, pluripotent stem cells, lymphoid stem cells, and myeloid stem cells.

Committed cells can be retrodifferentiated by contacting the cells with an agent that actually contacts the cells. The cells are then cultured to allow these cells that were actually exposed to the agent to develop through the process of retrodifferentiation and eventually become undifferentiated.

The contact step may include a means of binding to surface antigens on committed cells. The tool can act in direct coupling or in indirect coupling with the committed cell. An example of direct binding is when a committed cell has at least one cell surface receptor on its surface, such as a B chain, that has homologous regions (regions that usually have the same or similar sequence), such as sequences that can be found on B cells, and in which the agent directly engages the cell surface receptor. Another example is when the committed cell has a cell surface receptor on its cell surface, such as an α-chain that has homologous regions, such as regions that may be found on T cells, and wherein the agent directly engages the cell surface receptor .

An example of indirect binding is that the committed cell has at least two cell surface receptors on its cell surface and binding of the agent to one of the receptors affects the other receptor, which then causes retrodifferentiation of the committed cell.

The agent for retrodifferentiation of the committed cell may be a chemical compound or composition. For example, the agent may be capable of binding to a cell surface receptor on the surface of a committed cell. In certain embodiments, the agent actually engages a receptor present on the surface of the committed cell --- this receptor may be expressed by the committed cell.

For example, agents may include, but are not limited to, any one or more cyclic adenosine monophosphate (CAMP), a CO4 molecule, a CO8 molecule, part or all of a T-cell receptor, a ligand (anchored or free), a peptide, a T-receptor cells (TOC), antibody, cross-reacting antibody, monoclonal antibody or polyclonal antibody.

Growth factors such as hematopoietic growth factors such as erythropoietin and granulocyte-monocyte colony-stimulating factor (ZM-C5BE) may also be used.

If the agent is an antibody, cross-reactive antibody, monoclonal antibody, or polyclonal antibody, then the agent may be one or more of the agents such as an antibody, cross-reactive antibody, monoclonal antibody, or polyclonal antibody of any one or more of: beta-chain of MHC antigen class II, beta-chain of MHC antigen NO A-OK, a-chain of MHC antigen class I or class II, a-chain of antigen NO A-

ORE, a - and B-chain of MHC class II antigen or MHC class I antigen. An example of an antibody is SZ/43 (supplied by Deko (Bako)).

The term "antibody" may include various fragments (whether obtained by proteolytic cleavage or recombinant technology) and derivatives that retain binding activity, such as Rab, E(ab)2 and 5cEm antibodies, as well as mimetics or bioisosteres thereof .

Antibodies also include genetically engineered variants in which some of the amino acid sequences have been altered, for example by replacing amino acid residues to enhance binding, or in which the antibodies have been produced in different types of cells desired by the organism to treat according to the methods of the invention. to reduce the likelihood of adverse immune reactions (an example is humanized mouse monoclonal antibodies).

Agents used to induce the transformation of a committed cell into a retrodifferentiated cell can preferably act extracellularly relative to the committed cell.

For example, the committed cell may include a receptor that actually binds to the agent, and the agent actually binds to the receptor.

For example, the receptor may be a cell surface receptor. Specific examples of cell surface receptors include, but are not limited to, MHC class I and MHC class II receptors. The receptor may include an α-component and/or an RD-component, as is the case for MHC class I and MHC class II receptors.

The receptor may include a B-chain that has homologous regions, for example, at least regions homologous to the B-chain of NI A-OB.

Alternatively, or in addition, the receptor may include an α-chain that has homologous regions, for example, at least homologous regions to the α-chain of NO A-OK. The receptor can be a class I or class II antigen of the major histocompatibility complex (MHC). In certain embodiments, the cell surface receptor may include, but is not limited to, the NHA-OK receptor, the OM receptor, the OR receptor, the BO receptor, the NHA-A receptor, the

NHA-B, NIA-C receptor, NIHA-E receptor, NIH A-E receptor, or NI A-b receptor. In some embodiments, the cell surface receptor may be a NIG A-OB receptor.

The agent can be an antibody to the receptor, such as a monoclonal antibody to the receptor.

An example of an agent may be an agent that modulates MHC gene expression, such as expression

Class I emergency and/or class I emergency.

In certain embodiments, the agent may be used in combination with a modifier

From a biological reaction. Examples of biological response modifiers include, but are not limited to, an alkylating agent, immunomodulator, growth factor, cytokine, cell surface receptor, hormone, nucleic acid, nucleotide sequence, antigen, or peptide. For example, the alkylating agent may be or may include cyclophosphoamide.

Other biological response modifiers may include compounds capable of up-regulating MHC class I and/or class II antigen expression, which, in some embodiments, may allow the agent that binds to the MHC receptor to work more effectively.

Since any cell type can be made to express MHC class I and/or class II antigens, this may provide a method to retrodifferentiate different cell types that either primarily express MHC class I and/or class I antigens! or not

Committed cells are usually cultured with the agent for at least two hours, usually from 2 to 24 hours, preferably from 2 to 12 hours. Cultivation is usually performed at temperatures from about room temperature or, for example, about 22°C to about 37°C, including 33°C. The progress of the retrodifferentiation procedure can be checked periodically by removing a small aliquot of the sample and examining the cells using microscopy and/or flow cytometry. Alternatively, the device can include tracking means for on-line monitoring of the progress of the retrodifferentiation procedure.

In addition to the use of retrodifferentiation agents, committed cells can be cultured in autologous plasma or serum, or in embryonic blood serum or horse serum. Optionally, committed cells can be cultured with anticoagulants, chelating agents or antibiotics. The temperature range for cell culture can be extended to 18-40 "C, and can also include 4-10 95 CO" mM or 10-35 95 OO". In addition, culture can take place in blood bags, tissue dilution bags, or plastic vessels, both covered and uncovered.

Certain types of retrodifferentiated cells can be obtained by retrodifferentiation using certain culture conditions. For example, committed cells can be retrodifferentiated into pluripotent cells by culturing the committed cells in Dulbecco's modified Eagle's medium (MEM), non-essential amino acids (MEAA), I-glutamine (I-dish), and B-mercaptoethanol (2B-ME), in combination with retrodifferentiating agents. Committed cells may also be initially exposed to chelating agents. for As another example, to obtain mesenchymal cells, committed cells can be cultured - in combination with retrodifferentiation agent(s) - using

OMEM (low glucose) and I-dish, or OMEM (low glucose), Y-dish, 2 B-ME and MEAA. Further, the antibiotic gentamicin can also be used in cell culture.

Transdifferentiated cells

Transdifferentiated cells are obtained by culturing committed cells with tissue culture medium in combination with retrodifferentiation agents.

Committed cells thus undergo transdifferentiation, in which committed cells are transformed into cells of a different cell type; in some embodiments, the committed cells will become cells of another differentiation lineage.

The type of target cell obtained by transdifferentiation depends on the culture conditions.

These conditions vary according to the type of tissue culture medium, the presence/absence of different differentiation-promoting agents, the presence/absence of different sera, the culture temperature, the presence/absence of oxygen or carbon dioxide, and the type of container or vessel used for culture.

Examples of tissue culture media used for transdifferentiation include, but are not limited to, IMOM medium, UOMEM medium, Needle Minimum Essential Medium (EME), o0-MEM, KRMI, where the medium was developed), 1640, Nat- E-12, E199, MSOV, I viromii: I-15, medium E

Williams, or any commercially prepared tissue culture medium.

Agents that promote differentiation include anticoagulants, chelating agents, and antibiotics. Examples of such agents may be one or more of the following: vitamins and minerals or their derivatives, such as A, BZ, C (ascorbate), ascorbate-2-phosphate, 02, 03, K, retinoic acid, nicotinamide, zinc or compounds zinc, and calcium or calcium compounds; natural or synthetic hormones such as hydrocortisone and dexamethasone; amino acids or their derivatives, such as I-glutamine (I-diI0), ethylene glycol tetraacetic acid (ESTA), proline and non-essential amino acids (MEAA); compounds or their derivatives, such as B-mercaptoethyl, dibutyl cyclic adenosine monophosphate (ar-cCAMP), monothioglycerol (MTS), putrescine, dimethylsulfoxide (OM50O), hypoxanthine, adenine, forskolin, cilostamide and 3-isobutyl-1-methylxanthine; nucleosides and their analogues, such as 5-azacytidine; acids or salts thereof, such as ascorbic acid, pyruvate, oxalic acid, linoleic acid, ethylenediaminetetraacetic acid (EOTA), citrate dextrose anticoagulant of formula A, EOTA disodium, sodium butyrate, and glycerophosphate; antibiotics or drugs such as 5418, gentamicin, pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine) and indomethacin and proteins such as tissue plasminogen activator (TPA).

These differentiation-promoting agents can be used to target specific types of cells. For example, vitamin B3 can be used to obtain acinar cells, such as islet cells, or hydrocortisone; dexamethasone can be used to obtain cells of mesenchymal nature or epithelial nature (for example, renal epithelial cells, skin and nearby structures, such as dermal papilla cells); and B-mercaptoethyl can be used to lead to ectodermal cells such as neuronal cells, including antigen-presenting cells of the CNS.

The culture medium may contain autologous plasma; platelets; serum, such as fetal blood collection; or serum derived from mammals, such as horse serum. In addition, the conversion process can occur inside a blood bag, a base, a tissue culture bag, or a plastic tissue culture vessel. Tissue culture vessels may be attached or unattached tissue culture vessels, or may be coated or uncoated with agents such as gelatin, collagen, matrigels, or extracellular matrices that support attachment or buoyancy depending on the type of tissue or particular cells to be obtained .

Additional cultivation conditions include a temperature that can be from about 10 to about 60 "C, or from about 18 to about 40 "C; the level of carbon dioxide (CO''), which can be from about 0 to about 20 95 , or from about 4 to about 10 95 ; and oxygen (O2), which can be from about 0 to about 50 95, or from about 10 to about 35 9.

Examples of methods for obtaining target cells used in combination with retrodifferentiation agents are discussed in Table 1.

Table 1

Methods of obtaining different types of target cells in combination with retrodifferentiation agents

What are the cultivation conditions

The type of target cells are used in combination with Additional conditions of cultivation with retrodifferentiation agents "Preliminary action on committed

Pluripotent cells of chelating agents and stem cells or X OMEM, MEAA, 1-diy anticoagulants. heterogeneous population 28-ME For pluripotent progenitor cells removal of retrodifferentiating progenitor cells means by dilution only with culture medium 2, |" OMEM (low glucose) I -diy; or

Mesenchymal trunk |, z .

I-dish, 28-ME, MEAA vas "cultivation can be at "VERMI 1640, not necessarily with MEAA, | approximately 30-32 "C for

I-dimu, 28-ME of spermatozoa and approximately 38- for oocytes, or 39 "C for oocytes. " medium EME, retinol, I -dIy |" cells can be exposed to sodium pyruvate chelating agents sodium lactate, MEAA such as EOTA and EST for oocytes; or before retrodifferentiation and " OMEM, Natz E12 predifferentiation. vitamin C, vitamin E " For spermatozoa "Pluripotency retinoic acid, retinol can include germ cells pyruvate, optionally with the addition of okada "Oocytes Pentoxifylline for acid, OM5O and zinc "Spermatozoa spermatozoa; or or zinc compounds; "Cultivation conditions Gamete 100 can be used for pluripotent stem cells as a basal medium instead of those listed above, and with x For oocytes, an additional condition can include the addition of the cultivation conditions given in dr-cAMP, disodium EOTA, the next column for forskolin, cilostamide and hypoxanthine spermatozoa or oocytes. "For oocytes, medium 199 may be used as basal medium instead of "OMEM, Na!tz E12, and hydrocortisone optionally with vitamin K; or "Culture conditions

Kidney still for pluripotent stem cells listed above, with hydrocortisone

Continuation of table 1

Methods of obtaining different types of target cells in combination with retrodifferentiation agents

What are the cultivation conditions

The type of target cells are used in combination with Additional conditions of cultivation with retrodifferentiation agents x OMEM, MEAA, I -dIm optionally with 28-ME nicotinamide, or "Cultivation conditions same 00000000 | for pluripotent " With antibiotic 418 and

Alveolar epithelium stem cells x Endoderm listed above, from vessel cultivation

And and , tissues covered with matrigel. nicotinamide optionally with dexamethasone, retinoic acid, B-CAMP; or x MOM, I -din, ascorbic acid, MTS x OMEM with Na!tzh E12, MEAA 28-ME, optionally with putrescine, retinoic acid, I -aYshi hydrocortisone ascorbate; or x Neuron "Culturing conditions x Ectoderm for pluripotent stem cells are listed above optionally with putrescine, retinoic acid hydrocortisone, ascorbate "OMEM, Natvh E12 vitamin BZ; or x ERMI 1640 with vitamin

VZ; or

Islet cells "Culturing conditions for pluripotent cells

Acinar stem cells are listed above, with vitamin BZ (nicotinamide), optionally with dexamethasone

Continuation of table 1

Methods of obtaining different types of target cells in combination with retrodifferentiation agents

What are the cultivation conditions

The type of target cells are used in combination with Additional conditions of cultivation with retrodifferentiation agents x Cultivation can be performed at 33 C x Cultivation can be performed at room temperature to increase megakaryocytes in culture x"differentiated" IMOM, optionally from cells can be hydrocortisone; or exposed to "IMOM, I-glutamine and MTO; or chelating agents "Cultivation conditions before conversion to

Hematopoietic cells for pluripotent to increase the number of progenitors of erythroid stem cells in the culture listed above, with x KERMI 1640 can be 28-ME, replaced by MTO, used as a basal medium optional with vitamins for enrichment of lymphoid progenitors "sodium butyrate and/or

B-azacytidine can be added to the culture to promote primitive erythroid differentiation "ROMEM or IMOM or a-minimum essential medium, I -diy, optionally with dexamethasone

I -ascorbic acid-2-phosphate and nicotyrnamide; . or

Hepatocytes of the liver. .

E Williams medium, sodium pyruvate, dexamethasone; or "Culture conditions for pluripotent stem cells are listed above, with dexamethasone

Continuation of table 1

Methods of obtaining different types of target cells in combination with retrodifferentiation agents

What are the cultivation conditions

The type of target cells are used in combination with Additional conditions of cultivation with retrodifferentiating agents xNatz E12, OMEM (media ratio 3: 1), hydrocortisone, and optionally with adenine; or "E199 or OMEM with 1 -ai, not necessarily with .. can be with hydrocortisone and: .

Skin/keratinocytes with adenine; or gentamicin as an antibiotic "Culturing conditions can be cultured at 36.4 70 for pluripotent stem cells listed above, with hydrocortisone optionally with calcium or calcium compounds or ascorbate

C: M optionally with 3-isobutyl-1-

Melanocytes I methylxanthine; or x Medium 199 and hydrocortisone x OMEM, β-glycerophosphate, dexamethasone, ascorbate and

II-actions, optionally with vitamin 03; or "Cultivation conditions

Osteocytes/bone for pluripotent stem cells are listed above, with dexamethasone glycerophosphate and ascorbate, optionally with vitamin 03. "Williams medium E, I-di, hydrocortisone and/or vitamin 02, adenine and linoleic acid; or x OMEM, Na! tz5x E12 as a basal environment, I-actions ... hydrocortisone and/or

Cells of papillae of the dermis: and hair vitamin p2, adenine and linoleic acid; or "Culture conditions for pluripotent stem cells are listed above hydrocortisone, vitamin 02 adenine and linoleic acid

Continuation of table 1

Methods of obtaining different types of target cells in combination with retrodifferentiation agents

What are the cultivation conditions

The type of target cells are used in combination with Additional cultivation conditions with retrodifferentiation agents "OMEM, pyruvate, ascorbate 2-phosphate, dexamethasone and proline; or "Cultivation conditions for pluripotent

Chondrocytes/cartilage d r nc stem cells listed above ascorbate 2-phosphate dexamethasone and proline x OMEM, dexamethasone and

Adipocyte/fat cell) indomethacin; or "dexamethasone and indomethacin x OMEM, low glucose not necessarily with hydrocortisone "may be with dexamethasone gentamicin as an antibiotic

And -glutamine and sodium pyruvate; " can be with low or serum concentration x OMEM, Na!tz" E12 or E10 as 7" can be in

Skeletal muscles basal environment or OMEM and | culture vessels with environment 199; or gelatin "Culturing conditions 7 can be with increased for pluripotent temperature up to 397 stem cells "can include the above with glucose, addition of 5-azacytidine gentamicin and low serum

Blood vessels x OMEM, MEAA and 28-ME; or (endothelium) x MOM and dexamethasone x A: 1 OMEM and medium M199 or OMEM (low glucose)

Yi-diyu, MEAA; or OMEM. .

Culture vessels are covered (low glucose) with gelatin, for completeness with ascorbic acid. . . or DMSO; or differentiation and autologous

Cardiac muscle/ x ' serum or plasma depleted at. Conditions for cultivation of cardiomyocytes and platelets for pluripotent women. . Complete differentiation of stem cells is possible: observe the above, with . on a glass slide by antigravity cultivation or in a vibrating environment x OMEM, G-aim, 28-ME can also be used to

MEAA with continuous 2. carry out autologous factors by diluting the same: neck. growth and hormones are necessary for the culture medium minus the differentiation of cells on

Tropoblast retrodifferentiating agent of mesoderm and ectoderm; or including embryonic " ERMI 1640, 28-ME t. nai . TE . stem cells and sodium pyruvate and I-glutamine precursor cells

Characteristically, for the cultivation conditions described for each cell type in Table 1, removal of the retrodifferentiation agent by serial dilution of the culture medium with the appropriate culture medium leads to stronger transdifferentiation. During cultivation, the culture medium, which optionally contains various promoters of differentiation, can be diluted by adding more medium, but without the retrodifferentiation agent. Without being bound by theory, dilution appears to increase differentiation because cells become less dense and proliferation-stimulating factors become less concentrated. Therefore, the addition of medium can further enhance transdifferentiation and will affect what type of cells within the cells of the cell line are obtained. For example, if the target cell is a neuron, the addition of culture medium may result in a developmental shift toward a more mature neuron rather than a neuron progenitor cell (both are from the same cell line). As another example, direct differentiation of skeletal muscle progenitor cells will differentiate only by serial dilution of the culture medium, which is achieved by gradually reducing the serum concentration.

Cell redifferentiation

Retrodifferentiated cells can be used to obtain target cells by recommitting or redifferentiating retrodifferentiated cells into a target cell type. This can be done by contacting retrodifferentiated cells with growth factors.

For example, retinoic acid has been used to differentiate stem cells into neural cells. Methylcellulose followed by co-cultivation with a bone marrow stromal line, and I--7 was used to differentiate stem cells into lymphocyte precursors (Mizynapi ei ai., Ipi Ittipo 1994, 6: 909-916). Her e Rade (Mem 5siepiiv YuOes. 16, 2000) claims that stem cells can be differentiated into epithelial cells of the lung.

Vizspoii (Oem Vioi! 1986, 115: 129-39), describes how to differentiate muscle satellite cells into mature muscle fibers. Neural progenitor cells can develop with basic fibroblast growth factor and epidermal growth factor (MaKaishKy apa Makkatiga, U Meshigozsi Kez 1995, 41: 153-168). Hematopoietic stem cells can develop using a number of growth factors, including ZM-C5E, erythropoietin, stem cell factor, and interleukins (II -1, II -3,

Zo I -6) - look Meisai! (Mayige 1989, 339: 27-30) for an overview of these various factors.

Roijuspik et al., (EMVO in 1994, 13: 5274-83), even demonstrated the differentiation of stem cells into hematopoietic cells using low-oxygen (5 95) conditions.

The predifferentiated cell may belong to the same cell line as the committed cell from which the retrodifferentiated cell was derived. Alternatively, the predifferentiated cell may belong to a different cell line than the committed cell from which the retrodifferentiated cell was derived. For example, a B lymphocyte can be retrodifferentiated into a stem cell СО34-С2038 NO A-ЮОВ:. This stem cell can then be redifferentiated or committed towards a B cell lineage (same cell line) or a lymphoid cell line (different cell line).

Target cells

Target cells according to the invention are reprogrammed cells that can be obtained by retrodifferentiation, transdifferentiation or redifferentiation as described above. According to the invention, target cells may include, but are not limited to, pluripotent stem cells, lymphoid stem cells, myeloid stem cells, neural stem cells, skeletal muscle satellite cells, epithelial stem cells, endodermal and neuroectodermal stem cells, germ cells cells, extraembryonic and embryonic stem cells, mesenchymal stem cells, kidney cells, alveolar epithelial cells, endoderm cells, neurons, ectoderm cells, islet cells, acinar cells, oocytes, spermatozoa, hematopoietic cells, hepatocytes, skin/keratinocytes, melanocytes, bone/ osteocytes, hair/skin papilla cells, cartilage/chondrocytes, fat cells/adipocytes, skeletal muscle cells, endothelial cells, cardiac muscle/cardiomyocytes and tropoblasts.

As described above, committed cells and/or retrodifferentiated cells are cultured under certain conditions to induce retrodifferentiation and/or transdifferentiation and/or redifferentiation and obtain target cells. The duration during which committed cells or retrodifferentiated cells are cultivated is not limited to a certain period of time, but rather to the fact that target cells have been produced.

Determination of production or changes in the number of retrodifferentiated, transdifferentiated, or redifferentiated target cells can be accomplished by monitoring changes in the relative number of committed cells that down-regulated the expression of cell lineage-associated markers or transcription factors, and/or changes in the relative number of cells. which have cell surface markers that are characteristic of target cells.

Alternatively, or in addition, there may be a controlled reduction in the number of cells that have cell surface markers typical of committed cells rather than target cells. For example, the target cell can be an embryonic stem cell that is characterized by multistage-specific markers, such as ROSHBE1 (OST-4), TEVT, KI E4, TE, 5OX2, Mapod, or stage-specific embryonic markers Z and 4 (Z5EA-3 and 55EA-4), high-molecular glycoproteins TKA-1-60 and TKA-1-81 and alkaline phosphatase (Apagem/5 ei a|I., Nouaghidota 1984, 3: 347-361; Kappadi ei. I., EMVO U 1983, 2 : 2355-2361; Eoh vi ai., Oem Vio! 1984, 103: 263-266; O7aucha eyi a!., Seii!! Oineg 1985, 16: 169-173). They also do not express ZZEA-1, the presence of which is an indicator of differentiation. Other markers are known for other types of stem cells, such as nestein (Mezivip) for neuroepithelial stem cells (in

Meigovzsi 985, 5: 3310). Mesenchymal stem cells are positive for 5H2, ZNZ, СО029, СО44,

Ср71, ср90, сб0106, Ср120а и 20124, for example, and negative for СО34, СО45 and СО14.

Pluripotent stem cells are СО34:ОВ8 tat" cells (other useful markers are СОЗ38: and СО36). Lymphoid stem cells are OK", СОЗ4» and tat cells (also СО38). Myeloid stem cells are cells СО34-, ОВУ, СО137, СО33, СО077 and tat».

Additional cellular markers for target cells can be detected by microarray analysis. The analysis may include isolation of RNA from target cells that have been retrodifferentiated and/or transdifferentiated and/or redifferentiated, labeling the isolated RNA with a dye, and hybridizing the isolated RNA with a microarray.

A microarray may include genes or oligonucleotides that represent an entire genome, or may include genes or oligonucleotides associated with a particular organ system, tissue system, disease, pathology, or the like. Cellular markers can be identified by the fact that genes/oligonucleotides exhibit high signal intensity and are thus up-regulated or down-regulated in target cells. This information can then be used to identify target cells, based on the presence of markers, or even groups or characteristic regions of markers that have been identified by microarray analysis.

Confirmation of target cells can also be performed using a number of immunoassays, such as SES assays (see also Examples). Very primitive hematopoietic stem cells are often measured using the long-term dilution cell initiation assay (I TS-IS) (Eames et al., ) Tiz5 Si Meii 1991, 13: 55-62). And TO-IS supports hematopoiesis for 5-12 weeks.

For other cell types, such as cells of the central nervous system, pancreas, liver, kidney, skin, etc., cell culture can be continued until target cells appear, as characterized by immunohistochemistry, flow cytometry, microarray analysis, or polymerase chain reaction with reverse transcription (RT-PCR), which are techniques known in the art. This may also include functional assays, for example by vaccinating an immunocompromised recipient or correcting or ameliorating an underlying clinical condition as described herein.

Target cells can be identified by microarray analysis or CT-RSE, which demonstrate the acquisition of new cell line-specific transcription factors, proteins, and signals in target cells. For example, retrodifferentiated stem cells transformed into target cells of an ectodermal cell lineage can express genes such as Mesiip,

Stirioi, ije11, Y/NH1 and/or EMI, and if further differentiated into neurons, will express neurofilaments (ME). On the other hand, cells transformed into target cells of the endodermal cell line can express genes such as 5х7, 5х17, Мода!І, РОХ1 and/or ГОХА?2; but target cells, further differentiated into pancreatic islet cells, can express genes such as insulin (IM5) and pepigod3 (MOM3). Separately, conversion to desired target cells can be accompanied by down-regulation of mature transcription factors associated with the primary starting population that underwent transformation.

In addition, determining the production of target cells can occur by recognizing certain structural and/or morphological characteristics of the target cells, for example, cell shape, size, etc. These characteristics are known in the technology for target cells according to the invention.

Once the relative amounts of the desired cell type have increased to an appropriate level, which may, for example, be as low as 0.195 or as high as 5 95, the resulting altered cell populations can be used in many ways. Regarding the number of target cells formed, for example, pluripotent stem cells, and this is important to assess the proliferative capacity of stem cells. Although in some circumstances the number of stem cells or other retrodifferentiated cells generated may be low, studies have shown that as few as 50 pluripotent hematopoietic stem cells can recreate the entire hematopoietic system in a murine donor.

Thus, therapeutic utility does not require the generation of large numbers of cells.

Conversion of committed cells into retrodifferentiated, transdifferentiated or redifferentiated target cells can also be performed ip by injecting the agent, mixed with a pharmaceutical carrier or solvent, into the patient. However, it is desirable in many cases for retrodifferentiation, transdifferentiation, or redifferentiation to be performed ex vivo (in vitro, outside the body).

Treated cell populations obtained by IP can be used subsequently with minimal processing. For example, they can simply be combined with a pharmaceutically acceptable carrier or diluent and administered to a patient in need of stem cells.

However, it may be desirable to enrich a population of cells for retrodifferentiation, transdifferentiation, or redifferentiation of target cells or to purify cells from a population of cells. This can usually be done using a number of methods (see Matseve-

Radeu--Tpe Zsiepiivyi 1999, 13). For example, cells can be purified based on cell surface markers using chromatography and/or flow cytometry. However, it is often neither necessary nor desirable to extensively purify retrodifferentiated, transdifferentiated, or redifferentiated target cells from the cell population, as other cells present in the population (eg, stromal cells) can support stem cell viability and function. Flow cytometry is a well-known, reliable and effective technique for the characterization of cells within mixed populations, as well as for cell sorting. Thus, means of purification or isolation may include flow cytometry. Flow cytometry functions on the basis of the physical properties of particles in a liquid suspension, which can be distinguished when examined by a beam of light. Of course, such particles can be cells. Physical properties include cell size and structure or, as has become very popular in recent years, cell surface markers linked by monoclonal antibodies linked to fluorescent molecules.

Zo Kteivzed ei ai., (U NetaiyuSheg! 1994, 3: 263-89) states that "due to the availability of monoclonal antibodies api-5O34, multiparametric flow cytometry has become the tool of choice for the determination of hematopoietic stem cells and progenitor cells." Kgei55ed goes on to describe general techniques for subgrouping and flow cytometry studies of cells expressing POP34.

Tgaperiapi 1994, 13: 6649-54) describes the purification of SHOZ34" cells by immunoadsorption followed by flow cytometry based on NHA-OK expression. As discussed above, CO34: is a useful marker in combination with stem/progenitor cells. Flow cytometry techniques cytometry to sort stem cells on the basis of other physical properties are also available. For example, Mizb5eg et al., (Vioa Seijie 1980, 6:391-407) state that stem cells can be isolated on the basis of their size and degree of structuring. et al., (Wioo4 Seeley 1980, 6: 625-44), also state that "viable stem cells can be sorted from simple hematopoietic tissues with a high purity that can be verified."

Just like selection for cells based on the presence of a cell surface marker or other physical property (positive selection), cell populations can be enriched, purified, using negative criteria. For example, cells that have markers of specific cell lines, such as CO4, CO8, CO42, and CO3, can be removed from the cell population by flow cytometry or affinity chromatography.

A very useful technique for cell purification involves the use of antibodies or other affinity ligands bound to magnetic beads. The pellets are cultured with a population of cells and cells that have a cell surface marker, such as SOZ34, to which the affinity ligand binds, are captured. A sample tube containing cells is placed in a magnetic sample concentrator, where the beads are attracted to the periphery of the tube. After one or more washing steps, the target cells are partially or mostly completely cleared of other cells. When using a negative selection format, instead of washing cells associated with the granules by eliminating the liquid phase, the liquid phase is retained, and therefore cells associated with the granules are effectively removed from the cell population.

These purification methods based on affinity ligands can be used with any cell type for which appropriate markers have been characterized or can be characterized. Ograpkoma ei ai., (9 Spgotaiyodg V Viotei Arri 1996, 687: 449-52) describe the microretrieval of hematopoietic stem cells from mouse bone marrow suspension by flow fractionation in a gravity field. OgrapKkoma et al further comment that the method was used to study mouse bone marrow stem cells because these cells are larger than other bone marrow cells and therefore possible to separate from the mixture.

Thus, physical parameters other than a cell surface marker can be used to purify/enrich stem cells.

Cell populations that include reprogrammed target cells, such as retrodifferentiated, transdifferentiated, or redifferentiated target cells, and/or purified reprogrammed target cells, such as retrodifferentiated, transdifferentiated, or redifferentiated target cells, obtained by the methods of the present invention can be maintained ip migo, using known techniques. As a rule, minimal growth media such as Napk5, KERMI 1640, OMEM or

IMOs supplemented with serum derived from a mammal such as EB5 and optionally autologous plasma to provide a suitable growth environment for the cells. Stem cells can be cultured on feeder nutrient layers, such as stromal cell layers (see

Oegutsdipa eyi aiYi., Stiy Vem Ittypoiodu 1993, 13: 115-150). Stromal cells are believed to secrete factors that maintain progenitor cells in an undifferentiated state.

Long-term culture systems for stem cells are described in Yu.

RPuzioi 1977, 91: 335) and Oeeehhieg ei a!., (Asia Naeeatsoi! 1979, 62: 299).

For example, IerkomuzKi ei ai., (Taperiapiyop 1992, 53: 1011-93) claim that human hematopoietic cells CO34- can be purified using a technology based on the use of monoclonal antibodies that are covalently immobilized on the surface of polystyrene, and that cells COZ34 -, purified in this way, can be maintained with a viability greater than 85 95. I erkomubKki ei aY., (U Netaiyeg 1993, 2: 339-42) also describe how to isolate and culture human cells SOZ34-. See also NauioskK ei ai., (iptipoteiivoaz 1994, 5: 217-25) for an overview of different methods.

Cell populations that include stem cells and purified preparations that include stem cells can be frozen/freeze-preserved for future use. Appropriate methods of freezing cells and their subsequent thawing are known in the technology.

In one embodiment, the retrodifferentiation, transdifferentiation, or redifferentiation occurs with cells from or in leukocyte film samples. The term "leukocyte film" refers to the layer of white cells that forms between the layer of red cells and the plasma when uncoagulated blood is centrifuged or settled.

Methods of treatment

Reprogrammed target cells proposed by the present invention, such as retrodifferentiated target cells, transdifferentiated target cells and redifferentiated target cells can be combined with various components to produce compositions proposed by the invention. The compositions may be combined with one or more pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition (which may be for human or veterinary use).

Suitable carriers and diluents include, but are not limited to, isotonic saline solutions, for example, phosphate buffered saline solutions. The composition proposed by the invention can be administered by direct injection. The composition can be prepared for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral and transdermal administration, or by injection into the cerebrospinal fluid. Compositions that include target cells can be administered by injection or implantation.

Cells can be provided in suspension or embedded in a carrier matrix, such as natural and/or synthetic biodegradable matrices. Natural matrices include, but are not limited to, collagen matrices. Biodegradable synthetic matrices include, but are not limited to, polyanhydrides and polylactic acid. These matrices can be a carrier for delicate cells in natural conditions.

The compositions may also include retrodifferentiated, transdifferentiated or redifferentiated target cells of the present invention and at least one pharmaceutically acceptable inert excipient, carrier or excipient.

Delivery can also be regulated, that is, delivered over a period of time, which can last from a few minutes to several hours or days. Delivery can be systemic (eg, intravenous injection) or targeted. Cells can be introduced under natural conditions using liposomal (516) transfer.

Target cells can be administered in doses from 1 x 105 to 1 x 107 cells per kg. For example, a 70-kilogram patient can be injected with 14 x 106 cells of SХО34: for tissue regeneration.

Dosages can be any combination of target cells listed in this application.

The methods of the present invention can be used to treat many diseases, conditions or disorders. Such conditions include, but are not limited to, bone marrow disorders, hematologic conditions, aplastic anemia, beta-thalassemia, diabetes, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney disease, liver disease, multiple sclerosis, congestive heart failure, hepatitis C virus, human immunodeficiency virus, head injury, lung disease, depression, non-obstructive azoospermia, andropause, menopause and infertility, rejuvenation, scleroderma ulcer, psoriasis, wrinkles, liver cirrhosis, autoimmune disease, baldness, retinitis and crystalline dystrophy /blindness or any impairment associated with tissue degeneration. Aplastic anemia is a rare but fatal bone marrow disorder characterized by pancytopenia and bone marrow cell insufficiency (Uoipd ei ai., Vioa 2006, 108: 2509-2519). The disorder can be caused by an immunopathological disease with activated cytotoxic T cells of the ! type, which express the cytokine TI, especially γ-interferon, targeting the hematopoietic compartment of stem cells, which leads to a disorder of the bone marrow and, therefore, an anhematopoiesis (Wasida|Ichro ei aiYi., , NetayoiIodu 2007, 23-28). The majority of patients with aplastic anemia can be treated with transplantation of stem cells obtained from the offspring of the same parents who are matched for NIGIA (Iosabzsitsii ei ai., NaeptaiioiIodisa. 2007; 92:11-18.), although the extension of this approach to older patients or patients, in which there are no family donors, remains a big question. Despite the acceptable survival rate after NI A-matched allogeneic stem cell transplantation, the procedure has some potential risks due to the immunosuppressive regimen used to prevent graft-versus-host disease (GVHD). For example, a large dose of cyclophosphamide with or without antithymocyte globulin (ATO) leads to a long period of immunodepression and makes the patient susceptible to opportunistic infections. Another potential risk is graft rejection, which can occur weeks or months after stem cell transplantation (Soyaiepeg ei ai., Agsp Ipiegp May 1981, 141: 758-763; Zapaegv ei ai., Zetip Netaiyu! 1991, 28: 244-249 ). In addition, the risk of graft failure increases with the number of blood transfusions received before stem cell transplantation.

Thalassemia is a hereditary autosomal recessive blood disease characterized by a reduced rate of synthesis of one of the globin chains that make up hemoglobin. Thus, there is an underproduction of normal globin proteins, which is often caused by mutations in regulatory genes that lead to the formation of abnormal hemoglobin molecules, causing anemia. The different types of thalassemias include alpha thalassemia, beta thalassemia, and delta thalassemia, which affect the production of alpha globin, beta globin, and delta globin, respectively. Treatment involves constant blood transfusion, iron chelation, removal of the spleen and allogeneic hematopoietic transplantation. However, permanent blood transfusion is not available for most patients due to the lack of bone marrow donors who are matched for NHA, while allogeneic hematopoietic transplantation is associated with many possible complications, such as infections and graft-versus-host disease.

Diabetes is a syndrome that leads to abnormally high blood sugar levels (hyperglycemia). Diabetes refers to a group of diseases that lead to high blood glucose levels caused by defects in either insulin secretion or insulin action in the body. Diabetes is usually divided into two types: type 1 diabetes, marked by reduced insulin production, or type 2 diabetes, marked by insulin resistance. Both types lead to hyperglycemia, which largely causes symptoms usually associated with diabetes, such as excessive urine production, subsequent compensatory thirst and increased fluid absorption, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. Diabetes is considered to be a chronic disease that cannot be cured. Treatment options are limited to insulin injections, exercise, appropriate diet, or, for type 2 diabetes patients, certain medications, such as medications that support insulin secretion by the pancreas, reduce the level of glucose produced by the liver, increase the sensitivity of cells to insulin, etc.

Motor neuron diseases belong to a group of neurological disorders that affect motor neurons. Such diseases include amyotrophic lateral sclerosis (AG 5), cephalic lateral sclerosis (RI 5) and progressive muscular atrophy (PMA). AI 5 is characterized by degeneration of both upper and lower motoneurons, which stops signal transmission to muscles and leads to their weakening and possible atrophy. RI 5 is a rare motor neuron disease that affects only the upper motor neurons, causing difficulty with balance, weakness and impaired mobility of the legs,

spasticity and language problems. PMA is a subtype of AI 5 that affects only the lower motor neurons, which can cause muscle atrophy, fasciculation, and weakness. Treatment of motor neuron diseases is unknown. Riluzole (rPy5oIe), which is believed to reduce motor neuron damage, has been approved as a treatment for type 5 AH, although it slows the development of type 5 AH rather than curing it. For RI 5, treatment is directed at syndromes only, eg baclofen (Basiotep) can reduce spasticity or quinine which can reduce convulsions.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of the nigrostriatal pathway resulting from the degeneration of dopaminergic neurons within the substantia nigra. The cause of PO is unknown, but is associated with the progressive death of dopaminergic (tyrosine hydroxylase (TH) positive) mesencephalic neurons, causing motor damage. So, PO is characterized by muscle immobility, tremors, bradykinesia and potential akinesia. Thus, there is currently no satisfactory treatment for Parkinson's disease or treatments for the prevention or treatment of Parkinson's disease or its symptoms. Symptomatic treatment of motor damage associated with the disease includes oral administration of dihydroxyphenylalanine ((-SORA), which can lead to real improvement in motor function, but its effect diminishes as the degeneration of dopaminergic neurons progresses. Alternative strategies include nerve tissue transplantation , which is based on the idea that dopamine supplied by cells integrated into the striatum can replace lost nigrostriatal cells, and gene therapy that can be used to replace dopamine in the affected striatum by introducing enzymes responsible for And -SORA or dopamine synthesis, such as the introduction of potentially neuroprotective molecules that can either prevent the extinction of TN-positive neurons, or stimulate regeneration and functional recovery in the damaged nigrostriatal system.

Spinal cord injury is characterized by damage to the spinal cord and, in particular, to a nerve fiber, which results in damage to part or all of the muscles or nerves below the site of damage. This damage can occur from spinal trauma that cracks, displaces, crushes, or compresses one or more vertebrae, or from non-traumatic damage that is caused by arthritis, cancer, inflammation, or disc degeneration. While treatment after a spinal cord injury may include medications such as methylprednisolone, which is a corticosteroid that reduces nerve cell damage and inflammation in the injured area, or medications that manage pain and muscle spasticity, such as spinal immobilization or surgery , to remove a herniated disc or any objects that can damage the spine, there is no known way to completely repair damage to the spinal cord.

Muscular dystrophy (MS) refers to a number of hereditary muscle diseases that weaken skeletal muscles. MO can be characterized by progressive muscle weakness, defects in muscle proteins, apoptosis of muscle cells, and tissue atrophy. There are more than 100 diseases that show characteristics of MO, although nine diseases, including: Duchenne disease (Schispeppe), Becker disease (WesKeg), pelvic girdle disease, brachio-scapulofacial myopathy, myotonic dystrophy, oculopharyngeal dystrophy, peripheral dystrophy and Emery-Dreyfus disease, classified as MO. There are no known treatments for

MO, and at the same time there are no special methods of treatment. Physical therapy can maintain muscle tone, and surgery can be used to improve quality of life.

Further, symptoms such as myotonia can be treated with medication, but there are no long-term treatments.

Kidney disease refers to conditions in which the kidneys are damaged and their ability to function is reduced, including removal of metabolites and excess water from the blood, stabilization of electrolytes, blood pressure, acid-base balance, and reabsorption of glucose and amino acids. The two leading causes of kidney disease are diabetes and high blood pressure, although other causes include glomerulonephritis, lupus, malformations, and blockages in the kidney. There is no cure for kidney disease, and thus therapy focuses on slowing the progression of the disease and treating the causes of the disease, such as controlling blood glucose and high blood pressure and diet; treat complications of the disease, for example, targeted fluid retention, anemia, bone disease; and replacement of lost kidney function, such as through dialysis or transplantation.

Multiple sclerosis (M5) is an autoimmune condition in which the immune system attacks the central nervous system, leading to demyelination. Mo affects the ability of nerve cells in the brain and spinal cord to communicate with each other because the body's own immune system 60 affects and damages the myelin that lines the neuron's axons. When myelin is lost, axons can no longer conduct signals effectively. This can lead to various neurological symptoms, which usually develop into physical and cognitive disability. There is no known cure for M5; during treatment, they try to restore function after an attack (sudden onset or worsening of M5 symptoms), prevent a new attack, and incapacity. For example, treatment with corticosteroids may help terminate an attack, whereas treatment with interferon during the initial attack has been shown to reduce the likelihood that clinical M5 will develop.

Human immunodeficiency virus (HIV) is a lentivirus that can lead to acquired immunodeficiency syndrome (AIDS), a condition in which the immune system begins to weaken. NIM primarily affects vital cells in the human immune system, such as helper T cells, macrophages, and dendritic cells. NIM infection leads to low levels of CO4- T cells by direct viral killing of infected cells, increased rate of apoptosis in infected cells, or destruction of infected T-cells by CO4- cytotoxic lymphocytes that recognize infected cells. Currently, there is no vaccine or treatment for NIM (HIV) or

AIDS (AIDS). Treatment of NIM (HIV) infection consists in very active antiretroviral therapy or

NAAKT (PpPidpiIu asiime apiigeigigomigy5 IShyegaru). Current NAACT options are combinations (or "cocktails") that consist of at least three drugs with at least two types of antiretrovirals. Typically, these classes are two nucleoside analogs of a reverse transcriptase inhibitor (MAKTI or METI, piseoside apaiodce gemegze Igapz5sgiriaze ippiryog) plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (MMATI).

Congestive heart failure refers to a condition in which the heart cannot pump enough blood to the rest of the body. This condition can be caused by coronary artery disease, scar tissue on the heart resulting from a myocardial infarction, high blood pressure, heart valve disease, heart defects, and heart valve infection.

Treatment programs usually consist of rest, appropriate diet, modification of daily activity, and medications such as angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, digitalis, diuretics, and vasodilators. However, a treatment program will not completely reverse the damage or condition of the heart.

Hepatitis C is an infectious liver disease caused by the hepatitis C virus. Hepatitis C can progress to scarring (fibrosis) and extensive scarring (cirrhosis). Cirrhosis of the liver can lead to liver failure and other complications such as liver cancer. Current treatment includes the use of a combination of pegylated alpha-interferon and the antiviral drug ribavirin. Success rates can vary from 50 to 80 95 depending on the viral genotype.

A head injury refers to injuries to the head that may not cause brain damage. Frequent causes of head injuries are road accidents, domestic and professional incidents, falls and attacks. Different types of problems can be caused by head injuries, including skull fractures, skull lacerations, subdural hematoma (bleeding below the dura mater), epidural hematoma (bleeding between the dura mater and the skull), brain contusion (blow to the brain), concussion (temporary loss of functions due to trauma), coma or even death. Treatment for a head injury will vary depending on the type of injury. If the brain is damaged, there is no quick fix to repair it, and often the damage can be irreversible with available treatments.

Pulmonary disease is a broad term that covers diseases of the respiratory system, which includes the lungs, pleural cavity, bronchioles, trachea, upper respiratory tract, nerves and respiratory muscles. Examples of lung diseases include obstructive pulmonary disease, in which the bronchioles become narrowed; restraining or fibrotic diseases of the lungs, in which the lungs lose compliance, which leads to incomplete expansion of the lungs and increased rigidity of the lungs; respiratory tract infections, which can be caused by a cold or pneumonia; respiratory tumors, in particular caused by cancer; diseases of the pleural cavity; pulmonary vascular diseases that affect pulmonary circulation. Treatment for lung disease varies depending on the type of disease, but may include treatments such as corticosteroids and antibiotics, oxygen, mechanical ventilation, radiation therapy, and surgery.

Depression is a mental disorder characterized by a low mood accompanied by a low sense of self-worth and loss of interest or pleasure in normally pleasant activities. Biologically, depression is accompanied by changes in activity in many parts of the brain, including the nucleus accumbens, which is a group of small nuclei in the upper brainstem that is the source of serotonin; the supracaesmatic nucleus, which controls biological rhythms, such as the cycle of sleep and activity; the hypothalamic-pituitary-adrenal axis, which is a chain of structures that are activated during the body's response to various stressors; the ventral tegmental area, which is believed to be responsible for the electrical circuit of the brain's "reinforcement of the conditioned reflex"; the nucleus accumbens, which is thought to play a role in stimulation, laughter, pleasure, affection, and fear; and the anterior cingulate cortex, which is activated by negative events. Treatment of depression involves the use of antidepressants that increase the amount of extracellular serotonin in the brain, exercise and psychotherapy. However, the effectiveness of this treatment continues to raise doubts.

Non-obstructive azoospermia is a disease of men in which there is no significant level of sperm in their ejaculate due to spermatogenesis. This disease is often caused by hormonal instability and can be treated using drugs that restore the former state.

Andropause is a menopause-like condition experienced by middle-aged men in which the production of the hormones testosterone and dehydroepiandrosterone decreases.

Treatment includes hormone replacement therapy and exercise.

Scleroderma is a chronic autoimmune disease that affects connective tissue.

Hardening of the skin is one of the visible manifestations of the disease, although it can affect connective tissue throughout the body. There is no known direct treatment for scleroderma.

Psoriasis is a chronic autoimmune disease that causes red, scaly patches on the skin. The cause of psoriasis is related to the excessive growth of skin cells. One hypothesis suggests that the cause is related to T cells that migrate to the skin and cause the release of cytokines that cause rapid production of skin cells. Treatment of psoriasis involves the use of drugs that act on T cells.

Retinitis is a type of progressive retinal dystrophy in which the photoreceptors or retinal pigment epithelium is abnormal and leads to vision loss. Treatment methods for retinitis are limited.

The conditions described herein may be treated with a specific type, or combination of types, of target cells. In preferred embodiments, the condition described herein can be treated by infusion of the cell types highlighted in Table 2.

Table 2

Treatment regimens for various conditions using reprogrammed, i.e., retrodifferentiated or transdifferentiated or redifferentiated target cells

State of (one type or a combination of) stem cells and/or islet cells

Motor neuron disease. born and epithelial cells, ectoderm cells and/or neurons

Muscular dystrophy: stem cells and/or skeletal muscle cells

Renal disease is the result of a wedge injury

Kidney disease in m pluripotent stem cells

Multiple sclerosis in stem cells and/or neurons

Cardiomyocytes, mesenchymal stem cells,

Congestive heart failure pluripotent stem cells and/or endothelial cells

Hematopoietic cells, pluripotent stem cells

Hepatitis C virus cells, mesenchymal stem cells and/or liver hepatocytes

Coo)

Continuation of table 2

Treatment regimens for various conditions using reprogrammed, i.e., retrodifferentiated or transdifferentiated or redifferentiated, target cells and both

State of (of one type or combination)

Pluripotent stem cells, mesenchymal

Lung disease stem cells, alveolar epithelial cells and/or endothelial cells andropause germ cells and/or sperm cells pluripotent germ cells

Pluripotent germ cells, pluripotent stem cells, skin papilla cells, neurons,

Rejuvenation of sperm, osteocytes, chondrocytes, cardiomyocytes, skeletal muscle cells, neurons, endothelial cells and/or melanocytes

Viramiscleroderma eratinocytiyor mesochymal stoveuroi wedges

Ulcers of scleroderma and nia 2. keratinocytes and/or mesenchymal stem cells and/or pluripotent stem cells pluripotent stem cells

Liver hepatocytes, mesenchymal stem cells,

Liver cirrhosis of endoderm cells and/or pluripotent stem cells

Autoimmune disease m. stem cells and I Neurons and/or pluripotent stem cells dystrophy/blindness

For example, a patient can be treated for the condition described above using the following stages: 1) a fistula-cannula is inserted into the patient's hand; 2) the harvest of white blood cells is collected by apheresis using an automated system, such as the SOVEF spectral device (Satygo PCT); 3) autologous retrodifferentiated stem cells are obtained from the patient's white blood cells; 4) autologous retrodifferentiated stem cells are washed and then administered intravenously to the patient; 5) monitoring the progress of the patient's condition includes taking blood tests and evaluating the injured areas.

The invention will be further described by means of the following non-limiting examples, which further illustrate the invention and are not intended, and should not be interpreted, as limiting the scope of the invention.

Examples

Example 1

Materials and methods

This clinical study made it possible to evaluate the safety of administration of a single dose of autologous reprogrammed Z h. cells with the subsequent action of the hematopoietic inductive culture medium for four patients with aplastic anemia.

This clinical study was approved by the ethics committee of King's Memorial Hospital

Edward (Kipd Edulaga Metogiai (KEM) NozryaiI) and was performed in collaboration with the Institute of Immunohematology (Ipbishcishie oi IttipoPetaiiodu (IPN)). Patients were required to fulfill the criteria set out in Table 3.

As a result, four patients with severe (3 men) and hypoplastic (1 woman) anemia participated in the study. These 4 patients were selected and observed by PN/KEM staff.

The clinical and medical history is described in Table 4, while the dosages of infusion of SHUOZ4- cells used for them are shown in Table 5.

Table C

Infusion criteria 1 more - Absolute number of neutrophils "0.5x105/|.

Each criterion ----- I 5 and M 2 - Number of platelets "20x109/. 5 is needed

C - Anemia with corrected reticulocyte "1 Fo 4 - Saturation of bone marrow cells "25 Fo and sh - saturation of bone marrow cells o with less than o 5 - Saturation of bone marrow "50 95 with less than 30

Only one of the hematopoietic cell criteria is required: i - 2 26-45 - - - b - The subject was evaluated within the first 3 months after diagnosis 7 - The subject did not receive previous immunosuppressive therapy

The patient was transfused with 2 units of irradiated packed red blood cells and 4 units of platelets to maintain a hemoglobin level above 8 g/dL and a platelet count above 50,000.

The patient was subjected to apheresis by processing 2-3 times the full volume of blood, using the Sobe 5resiga device for apheresis and a set for the separation of white blood cells (both from Satrgo VST). Apheresis included cervical and apical venous probing with a single-lumen catheter for venous catheterization.

An aliquot of cells was collected aseptically for CO34 analysis, followed by the collection of 150-200 ml of the light layer of the blood clot. After that, the light layer of the blood clot was subjected to reprogramming under the conditions of hematopoietic inductive cultivation. Briefly, the reprogramming technique involved the addition of 1000 μg of purified SKZ/43 (specifically obtained

BacoSuyutaiiop for Tgibiet Sogr.), diluted 30 ml of medium, IMOM, aseptically in a bag with white blood cells. The bag was then cultured in a sterile tissue culture thermostat, which was maintained at 37 C and 595 C for three hours. After the reprogramming process was completed, the transformed cells were analyzed for CO34 cell content. Then, the cells were washed twice with saline using a Sore 2991 cell processor.

After mixing and resuspending in saline, the cell suspension was administered to the patient through the jugular vein under the influence of gravity using an infusion set.

The main indicators of the body's condition, including the clinical blood analysis of the patients, were continuously monitored before and after the infusion of autologous reprogrammed cells.

Table 4

Clinical and medical history of patients with aplastic anemia before infusion of autologous reprogrammed human cells (NK5S)

Patient Clinical history before the infusion of NKZS Treatment before the infusion of KZS x"Diagnosed with TAA in 2002 "symptoms: complaints of weakness and shortness of breath. I have shortness of breath 2. - |Received trial frequent cases of rectal and gingival ...

Patient A, male, 25 years old, bleeding and vomiting and: her. . g. of anabolism during 8 severe aplastic anemia (TAA)) "received 4 and 2 units of blood and 2. o, . months without any platelets, respectively, each -: a significant improvement of the month "appearance is jaundiced with severe ocular hyperemia

Continuation of table 4

Clinical and medical history of patients with aplastic anemia before infusion of autologous reprogrammed human cells (NK5S)

Patient Clinical history before infusion of NKAZS Treatment before infusion of KZS xDiagnosed with hypoplastic anemia in January 2004. : y y Received symptoms: polymenorrhagia lasting . - You are a blood stimulant

Patient B, woman, 26 years, 6 months old, - and house. w drug from 3 months to hypoplastic anemia only before infusion of NK5S sick and in . / infusions of NEZS without which - received 4 and 6 units You and -: | any reaction of packed red blood cells and platelets, respectively "Diagnosed with very severe aplastic anemia in 2003 "symptoms: anemia, weakness, shortness of breath on exertion "heat and cold, fever that lasts: - . Received 10-15 days as a result of severe and . in cyclosporine therapy ... neutropenia 7.

Patient C, male, 19 years old, gh. and w during 6 months, which "multiple cases of infection, vomiting: and very severe aplastic anemia |. ended in March and the appearance of purple spots zhd M 2003, without any gluteal abscess effect "twice fainted due to cerebral hemorrhage "received 5 and 2 units of blood and platelets, respectively, every 2 weeks "anemia, weakness, shortness of breath "heat and cold, fever, as a result of severe neutropenia and multiple Received cyclosporine and

Patient 0, male, 35 years old, cases of infection and bleeding antituberculosis very severe aplastic anemia | "diagnosed with therapy years ago, no response to "received 5 and 2 units of blood and 6 months of platelet immunosuppression, respectively, every month

All clinical control was performed by PN/Chem staff. Transfusion requirements were determined pre- and post-infusion from transfusion reports received after any unit of blood product. Transfusion units are used only after obtaining consent from patients and immediate relatives as witnesses. Before the patient's transfusion, all blood products were irradiated at the Tata Memorial Hospital (Taia Metogia! Nozriia|). Patients were also asked questions about their well-being before and after the infusion of reprogrammed cells. All patients received a copy or original reports of their condition and all laboratory and clinical outcomes. The duration of follow-up of these patients was initially set at 2 years, but was increased. During the first month after the infusion, the patients were placed in the hospital in sterile rooms with pressure above atmospheric.

Table 5

Patient, date of infusion, weight and height, collected mononuclear cells and infused with SOZ34-/kg of infusion and dose/kg of cells

One million cells were stained according to the manufacturer's instructions with the following panels of monoclonal antibodies (all from bacoSuoptaiiop):

Panel 1 consisted of isotypic conjugates of the negative control Id1-RITS, IDa1-RE-Su5 and

Ids1-ARE;

Panel 2 consisted of human anti-СО45-RITS and СО34-ARE-SUu5;

Panel C consisted of human anti-SOZ38-RITS and СО34-ARE-SUu5;

Panel 4 consisted of СХО61-RITS and СО34-ARE-Су5;

Panel 5 consisted of SOZ33/13 ERE and SO7-RITS;

Panel 6 consisted of СО45 and Сиусорпогип-А-ВРЕ;

Panel 7 consisted of SOZ3-RITS and СО19-VRE.

Cell analysis was performed using the EASbZSaiyrig (VO bBiossiepse) system, using the software VO sei! Father's

For clonal microarray analysis, mononuclear cells (MMCs) of the patient's bone marrow before and after the infusion of reprogrammed cells were sown in the medium of methoculture CEN4A434, supplemented with recombinant growth factors according to the manufacturer's instructions (5et Seyi

Teppiodie5). Differentiation into hematopoietic cell colonies was assessed and counted over time using phase-contrast inverted microscopy.

Clinical blood analysis, liver enzymes and hemoglobin variants of the patients were continuously monitored before and after the procedure. After patients left the hospital, clinical blood analysis, liver enzymes, hemoglobin variants and peripheral blood karyotyping and range were monitored by an independent laboratory for reconfirmation purposes. These tests were often performed after infusion of autologous reprogrammed cells.

Peripheral blood samples and bone marrow cells were analyzed before and after infusion of autologous reprogrammed cells. This test was repeated at six-month intervals during the first year and annually for the next 2 years after starting therapy with autologous reprogrammed stem cells. In addition, reprogrammed cells were assayed before infusion to examine cell stability, assays were also performed after the 3-hour stage

From conversion, as well as after culturing converted cells for a maximum period of 1 month. Karyotyping and c banding were monitored by a third independent laboratory.

Bone marrow smears and trepanation sections were performed before and after infusion of autologous reprogrammed cells. This test was performed 14-20 days after infusion of autologous reprogrammed cells and annually thereafter.

The entire smear and trepanation sections were scanned using a microscope connected to a video camera before and after infusion of reprogrammed stem cells to assess and monitor graft engraftment.

The results

All patients tolerated apheresis and a single procedure of infusion of reprogrammed stem cells without adverse events. Patient A and patient O became transfusion independent after a single infusion of reprogrammed hematopoietic stem cells (HSCs) (see Tables 6 and 7). Engraftment of platelets, neutrophils and red blood cells followed after 3 and 2 days in patient A and patient 0, respectively. Embryonic hemoglobin incorporation was noted in patient A and patient O (see Tables 4 and 5), but not in patient B and patient C (data not shown). Before the infusion, liver enzymes increased in patient A and patient O, despite the absence of hepatitis C virus (HCV) (as determined by enzyme-linked immunosorbent assay (ELISA).

Liver enzymes began to normalize after KNSS infusion and reached normal levels 4 years after KNSS infusion. Patient B and patient C died 2 years and six months after infusion, respectively. The inclusion of embryonic Hb was noted in patients 001 and 004 (see tables b and

7). These two patients demonstrated long-term graft engraftment after a single infusion of autologous HE5C. Tab 7 4646

Table 6

Patient A: complete blood count, hemoglobin variant and liver enzymes of a patient with severe plastic anemia before and after infusion of autologous NKZS

The lowest

Blood analysis level to Ol Z/go04. 05/26/2005. 03/07/2006 01/05/2008. infusion after infusion | after infusion | after infusion | after infusion

Ministry of Internal Affairs O/mkli

NV h/dli) 726 17711771 11111719 11112 | 14

Air Force rOb/lmkl| 17717098 17777712 | 23 | 29 | 386

VETIS (5 14111111

MSM |fl 101.5 101.5 121.74 101.4

MeNIg/dl| 39,13

MES (g/dli 32.14

Platelets

Neutrophils 0.32 1.000 1.054 1184 1.994 103/μl

EZOOUMmkl| 17777717 11111062 | 074 | 065

WABOOZmkli | 77777777 17777171 171111011171110 | 0054

IMMRNIOS/MkClI | 0000000 1,500 1,922 2,220 2,214

MOMO Pbz/mklI | 0000000 0.500 0.062 0.222 0.324

NVA (g/dli 51111167 11192 | 2

NVA2 (g/dp | 11171103 1104 | 024 | 039

NVE (g/dl

OT (W/-

ZART (Ш/-

Notes: MIA - white blood cells; HB - hemoglobin; KVS: "red blood cells; KETIS - reticulocytes; MSM - average hematocrit number; MSN - average erythrocyte hemoglobin; 8) MCNS « concentration of average erythrocyte hemoglobin; EBO - eosinophils; VABO - basophils; GUMRN - lymphocytes; MOMO - monocytes; NVA - hemoglobin A;

NVAZ2 :- hemoglobin A2; HVE - hemoglobin E (fetal hemoglobin); ZSOT |O/L| - serum glutamatoxaloacetate transaminase (upper/lower); 19) BOARD (O/L. | - serum glutamatepyruvate transaminase (upper/lower)

Table 7

Patient 0: complete blood count, hemoglobin variant and liver enzymes of a patient with severe plastic anemia before and after infusion of autologous NARS dnali | The lowest | одв/уов/»2о04 | 05/30/2005 | 07/03/2006 | 01(05/2008 after blood analysis level before: - her: and with: . with. and infusion after infusion | after infusion | after infusion infusion

UUVS (105/mcli

HB g/dl) vvsSrOmkl| | 77777 177173.6 | 24 2 | 32 | 345

VETIS (5 11111124 | 134 | 77777111

MSM |fli 7777уЮ7/ЮК1 903 | 08.33 110.3

MES (g/dli 77771711 309 | 32 | 359 | 378

MmensSi/dlI | | 342 | 29.62

Platelets

Neutrophils

Continuation of table 7

Patient 0: complete blood count, hemoglobin variant and liver enzymes of a patient with severe plastic anemia before and after infusion of autologous NARS dnali | The lowest | одв/уов/»2о04 | 05/30/2005 | 07/03/2006 | 01(05/2008 after blood analysis level before : y m : y s : . y and infusion after infusion | after infusion | after infusion infusion

EBOPOUMkl! | 77777777 1771710990 | 022 | 054 | 064 2 sh

VABOPOUMKLI 7777777 17117010 1 0 11 0

NOISY! 77777171 201 1711167 | 759 | 7882 moMmo poumkli 77777771 177108 | 044 | 054 | 02 9 nvAI(/dlI! 17777771 17711706 | 73 | 71093 | 121 nvA2i/dli 77777777 17100339. | 09 | 028 | sh 036 2 sh nvETdl| 17 01 | be | 038 | 09 | hI 462 461 | 69 | 34

BaRTtII 1150 | 57 | 32 | 60 | 4

Notes: MIA - white blood cells; HB - hemoglobin; KVS: "red blood cells; KETIS - reticulocytes; MSM - average hematocrit number; MSN - average erythrocyte hemoglobin; 8) MCNS « concentration of average erythrocyte hemoglobin; EBO - eosinophils; VABO - basophils; GUMRN - lymphocytes; MOMO - monocytes; NVA - hemoglobin A;

NVAZ2 :- hemoglobin A2; HVE - hemoglobin E (fetal hemoglobin); ZSOT |O/L| - serum glutamatoxaloacetate transaminase (upper/lower); 19) BOARD IO/Ts| - serum glutamatepyruvate transaminase (upper/lower)

Flow cytometry of apherized mononuclear cells up to and from an hour after the start of hematopoietic reprogramming are shown in Fig. 1. The number of POP4 positive cells generated after hematopoietic reprogramming is listed in Table 4.

The presented flow cytometry of apherized mononuclear cells before and after hematopoietic reprogramming shows a significant increase in the number of positive cells

COZ34 with and without expression of CO45, CO38 and CO7 (Fig. 1). Upon infusion, CO34 cells circulated in the peripheral blood for 3-6 days and then differentiated at a viable level into myelocytes, as shown by a significant increase in cells expressing CO33813 with and without CO7, which have high forward and side scatter (see Fig. 2). . This pattern of reprogramming was observed in all patients.

Hardly any colonies were formed from the aspirate of the patient's bone marrow before the infusion of autologous NACPs with subsequent seeding in methylcellulose cell culture (see Fig. 3).

Clone micromatrix analysis only in patient B, who suffered from hypoplastic anemia, showed suppressed hematopoiesis. However, within 14-20 days after the infusion, all bone marrow aspirates obtained from patients gave rise to a normal level of many hematopoietic colonies with a moderate increase in the number of blast-forming units-erythroids (BPO-eguigoid). Bone marrow smear and trepanation section (see Fig. 3) 14-20 days after infusion of autologous

NAS showed a significant increase in the number of myelocytes at different stages of differentiation with mature and immature megakaryocytes in all patients when compared with the initial indicator.

Erythroid hyperplasia at different stages of differentiation was also noted in all samples.

There were no changes in karyotyping and the nature of the b range in peripheral blood or bone marrow samples obtained before and after the infusion (in patient A and patient O for more than 4 years) of autologous NES in all patients (see Fig. 4).

After infusion of autologous NK5C in patients with aplastic anemia, primitive (SOZ8 negative) and committed (SOZ38 positive) CO34 cells circulated in the peripheral circulation for three days before reprogramming into myelocytes (Fig. 2). Engraftment of the myeloid graft followed within 3 days after the infusion of NK5C in patient A, patient B and patient 0. On the other hand

On the other hand, engraftment of the myeloid graft followed on day 20 for patient 003 when analyzed by flow cytometry. A single infusion of NAC, without the use of any preconditioning regimen, leads to long-term graft survival in 2 out of 4 patients with aplastic anemia. Patient A and patient B demonstrated long-term graft survival without any transfusion after NK5C infusion. Engraftment of a transplant of neutrophils, red blood cells and platelets in such a patient was accompanied by the inclusion or increase in the level of hemoglobin E (tables 6 and 7). This was not observed in the other 2 patients who died. The inclusion of HVE in both of these patients confirms the ability to reprogram the infused NK5Cs into the juvenile phenotype of Hb, therefore, graft engraftment and recovery, as was observed with a transplant of umbilical cord blood stem cells (Eiyavia ei ai., GeiKetia 2000, 14; 931-934; I osaiyei ei AI., Vope Matom

Tkapzrapi 1996, 18: 1095-101).

The long-term engraftment of the transplant with preservation of the number of chromosomes and binding clearly reflects the safety of NK5C infusion in the hematopoietic state, where clonal evolution is not an isolated case in conventional therapy.

It is important that autologous NK5C are capable of long-term graft survival, and the survival rate in a subgroup of severe aplastic anemia patients without the use of any regimen that suppresses immunity is similar to the rates noted for syngeneic stem cells.

Thus, fourteen days after the infusion, analysis of the bone marrow of infused patients showed an increase in the saturation of bone marrow cells, and myeloid, erythroid and megakaryocyte cell lines at various stages of differentiation with a decrease in fat and stromal cells, which are the predominant inhabitants of the bone marrow of patients with severe aplastic anemia There was a significant increase in the mass of red blood cells in the bone marrow with a corresponding increase in the level of hemoglobin, as well as the number of reticulocytes. There was also a steady increase in fetal hemoglobin - an important compound in improving sickle cell anemia and beta thalassemia - and red blood cells that express fetal hemoglobin after the infusion. In addition, there was a significant improvement in red blood cell counts, as determined by red blood cell size, hemoglobin content, and concentration. The reprogrammed cells showed a normal karyotype and genetic stability after infusion. Finally, graft engraftment and long-term secondary colonization were observed in 3 patients suffering from aplastic anemia.

Example 2

Materials and methods

Autologous reprogrammed hematopoietic cells (target cells) were tested for 21 patients with beta-thalassemia. Nineteen patients had beta-thalassemia major and 2 had thalassemia intermedia. One of the patients with beta-thalassemia intermedia had the thalassemia/NOE variant (common in patients of Far Eastern and Indian origin) and the other had thalassemia/sickle cell disease.

Patients were apherized by 2-3 times processing of their entire blood volume. Autologous reprogrammed cells were generated by reprogramming white blood cells until target cells were obtained as indicated by their various characteristics as described above.

Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

The results

No toxic or adverse side effects were observed after the infusion of reprogrammed cells in beta-thalassemia patients, as shown by monitoring of vital signs of body condition, echocardiogram, bone density, liver and kidney enzymes, including karyotyping and with the range, when compared with the initial indicators. A statistically significant mean reduction (50 95) in blood transfusion requirements was recorded in patients with beta-thalassemia major immediately, almost 9 months after the infusion of reprogrammed cells, when compared with baseline. Two patients with thalassemia, which is beta-thalassemia intermedia (one had thalassemia/NEU and the other had thalassemia/sickle cell disease) were transfusion independent almost 9 months after infusion of reprogrammed cells.

Average weight and height after infusion of reprogrammed cells were significantly greater compared to the starting material, and organ size was normalized in thalassemia patients with enlarged spleen and/or liver. The absolute mean concentration of embryonic hemoglobin significantly increased in patients with thalassemia major and intermediate after infusion of reprogrammed cells, if compared with the initial values (Fig. 5).

In addition, mean red blood cell scores, as reflected by the improvement in hemoglobin content, red blood cell size (Fig. b), and concentration (Fig. 7) were also significantly improved compared to baseline.

Finally, mean serum ferritin (a biomarker for iron overload) was significantly reduced in thalassemia patients after infusion of reprogrammed cells (Figure 8). Excess iron is the main cause of mortality and morbidity in patients with thalassemia; sickle cell

From anemia; and any disorder caused by iron overload in the transfusion.

Example C

Materials and methods

Two patients with diabetes underwent apheresis by 2-3 times processing of their entire blood volume.

Autologous reprogrammed mesenchymal stem cells, pluripotent stem cells and islet cells (target cells) were obtained by reprogramming apherized white blood cells, until the target cells had not developed, as shown by their different characteristics as described above. Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

The results

After the infusion of autologous reprogrammed cells, the patients synthesized a normal level of insulin stimulated by c-peptide, which was determined on an empty stomach and during meals within 90 minutes. This normal level of c-peptide is maintained up to 3 months after the infusion of reprogrammed cells (Fig. 9). In addition, NBATS levels, which indicate glycemic control, were normalized after infusion of reprogrammed cells (Fig. 10). For example, patients with NIATS above 10 95 before infusion now have a NIATS of 5.8 95 after receiving reprogrammed cells.

In addition, the blood glucose levels of these patients were found to reach normal levels after the infusion, when compared with the initial values. In addition, a significant reduction in the intake/injection of insulin by a diabetic patient was noted after the infusion of reprogrammed cells.

Example 4

Materials and methods

Four patients with amyotrophic lateral sclerosis (AI5) received autologous reprogrammed cells. The diagnosis of this disease does not have a specific biomarker. This disease is diagnosed by clinical exclusion of other similar disorders.

Patients were apherized by 2-3 times processing of their entire blood volume. Autologous reprogrammed pluripotent stem cells, lung alveolar epithelial cells and neurons (target cells) were obtained by reprogramming apherized white blood cells until target cells were obtained as indicated by their various characteristics as described above. Patients were injected with autologous reprogrammed cells intravenously

From the infusion into the jugular vein or veins of the arm or thigh.

The results

In patients who suffered from AI 5 and were treated with autologous reprogrammed cells, there was a significant improvement according to the Pulmonary Function Test (PET).

Damage according to this lung function test is one of the reasons that lead to early mortality. Most patients experienced less complications in mobility of the limbs and neck, and some reported improvement in speech. Others showed improvement in the ability to walk as well as lift their head.

Example 5

Materials and methods

Four patients with Parkinson's disease were apherized by processing their whole blood volume 2-3 times. Autologous reprogrammed pluripotent stem cells and neurons (target cells) were obtained by reprogramming apherized white blood cells until the target cells had developed as indicated by their various characteristics as described above. Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

The results

Patients for whom such a symptom of the disease as tremors was pronounced, experienced a significant reduction in tremors, and for them, conventional drugs were used to treat this disorder. The first patient received 4 pills of Zipeteia (the Dopamine regulator Sinemet) every day, and after the transfusion for 4 months, only 1 pill per day.

Example 6

Materials and methods

Two patients with spinal cord damage were apherized by 2-3 times processing of their entire blood volume. Autologous reprogrammed pluripotent stem cells and neurons (target cells) were obtained by reprogramming apherized white blood cells until the target cells had developed as indicated by their various characteristics as described above. Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

Results One patient was lost to follow-up. Another patient was a paraplegic with a C5-C6 spinal cord injury. This patient was unable to sit or turn over in bed.

After treatment, he was able to sit up straight for very long periods of time and turn his body over in bed. It could also stand on its own after being placed against a wall. Next, he was able to move his finger and report sensations in his bladder.

ME analysis (magnetic resonance imaging) before and after the infusion of reprogrammed cells showed a slight reduction in the size of the lesion after the infusion of reprogrammed cells. The patient began to actively undergo physical therapy and generally felt much better than before.

Example 7

Materials and methods

Two patients with muscular dystrophy (MD) were treated with autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, and skeletal muscle cells (target cells) obtained by reprogramming apherized white blood cells until the target cells developed , as indicated by their various characteristics as described above.

The first patient was affected by MO of the muscles of the shoulder and pelvic girdle, which belongs to the class of MO, in which the muscles are most severely affected, usually the muscles of the thigh and shoulders, and the second patient was affected by nemaline MO.

Patients were apherized by 2-3 times processing of their entire blood volume. Autologous reprogrammed cells were obtained by reprogramming proposed by the invention. Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

The results

Muscle atrophy associated with MO can be determined by monitoring the level of the muscle enzyme creatine phosphokinase (CK). This enzyme is reduced in response to the infusion of retrodifferentiated stem cells (Fig. 11). Lactate dehydrogenase, an enzyme that increases during tissue rejection, also decreased (Figure 11). Patients also had decreased levels of the liver enzymes alanine aminotransferase (AIT) and aspartate aminotransferase (A5T), both of which are associated with inflammation and damage to liver cells and skeletal muscles.

In addition, patients demonstrated improved mobility, as determined by video camera recording before and after infusion of reprogrammed cells, and in a pulmonary function test after infusion of reprogrammed cells, when compared to baseline.

Example 8

Materials and methods

Kidney disease patients were treated with autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, and kidney cells (target cells) obtained by reprogramming apherized white blood cells until the target cells developed as indicated by their various characteristics as described above. Patients were subjected to apheresis by processing their entire blood volume 2-3 times. Autologous reprogrammed cells were generated by reprogramming according to the invention.

Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

The results

Patients who received infusions of autologous reprogrammed cells showed improvements in the levels of markers in various fluids that indicated healthier kidney function, such as urine output, serum creatinine, and blood urea nitrogen (BUM) or OKEA. For example, a 75-year-old diabetic man demonstrated significantly better renal function 24 months after treatment with autologous reprogrammed cells (see Table 8). The patient demonstrated increased levels of hemoglobin, which is a protein molecule in red blood cells that carry oxygen, and insulin-like growth factor 1 (IGF-1), a growth factor. The patient also showed decreases in urea, which is an organic compound, creatinine, which is a breakdown product of creatine phosphate in the muscles, uric acid, which is an organic compound excreted in the urine, and phosphorus, which is a mineral found in bones; a high number of these markers indicates insufficient kidney function. Further, the patient demonstrated a decrease in glycosylated hemoglobin (HbA1C), which is a form of hemoglobin commonly used as an indicator of plasma glucose concentration in people with diabetes.

Similarly, a 51-year-old diabetic man showed similar improvement 12 months after starting treatment (see Table 8). This patient had elevated hemoglobin levels and decreased levels of creatinine, NBA1C, and blood urea nitrogen (BUN), which is a measure of the amount of nitrogen in the blood in the form of urea.

Table 8

Levels of markers of kidney function in two men with diabetes

A 75-year-old man with diabetes A 51-year-old man with diabetes

Baseline 24 months post Baseline 12 months post infusion rate infusion rate

Hemoglobin |g/day 7787129 17712 1145 «

Urea |g/dl

Creatinine (mg/day

Uric acid |mg/dL

Phosphorus | mg/dli

IZE-1 (ng/dli 7144. | 61 1712 1 6

ISE-1 (ng/dL)

PTO (mg/dli

Notes: НБ ATS: "glycosylated hemoglobin; IOE-1 "insulin-like growth factor 1;

VIM - blood urea nitrogen

Analyzes of 12 patients with type II diabetes who were treated with autologous reprogrammed cells revealed reduced levels of microalbumin (Fig. 13), which is associated with the leakage of albumin into the urine and is an indicator of kidney disease, as well as dysfunction of the endothelium of vessels and heart - vascular disease. These 12 patients also had reduced levels of NAATS (Fig. 14).

In addition, a 45-year-old woman with autoimmune glomerulonephritis demonstrated improvement in kidney function within 18 months of treatment with autologous reprogrammed cells (see Table 9).

Table 9

Levels of kidney function markers in a 45-year-old woman with autoimmune glomerulonephritis

Hemoglobin |g/day 816

Creatinine I (mg/day

PTO (mg/dli nyshtninninnyshEnynyshshsh

Notes: PTO - blood urea nitrogen

In addition, a 59-year-old woman with end-stage chronic renal failure demonstrated improvement in creatinine, urea, hemoglobin, and parathyroid hormone (PTH) levels after treatment with reprogrammed cells (see Table 10). A marker of kidney function is leveled in a 45-year-old woman who suffered from an autoimmune (autoallergic) disease. The patient also reduced the number of hemodialysis sessions she attended per month from 12 to approximately 8 sessions.

Table 10

Levels of kidney function markers in a 59-year-old woman with end-stage renal disease before and after reprogrammed cell therapy

Marerfunction pre-therapy: |... post-therapy. 3... rker funkt y oz/02/2009 04/29/2009 05/23/2009 05/08/2009 08/11/2009

Hemoglobin (/dL| | 98 | 92 | 115

THOSE. SLMVS Other mkl) 3800 6800 5100 nin" PL

RI-T (per μl) 361,000 273,000 purchase price

N PBX (bo 561111

AZT (SHL- 25 | 16 | 14 her (Sh(

A.T O/. 749 | 7 HER 12 1 9 SHIE

Urea (mgidl | | (90 2 .BMc | 70 2 | ! (MB | g2iz

Continuation of table 10

Levels of kidney function markers in a 59-year-old woman with end-stage renal disease before and after reprogrammed cell therapy

S.Albumini/for | (Z | 34 | 93 | | Z

Rt(seeundi)i | 9312 | B (7 Her MA 11112 11

Phosphorimg/dL| | 8 | 92 | 59 | /

Kaliyimmoldl| | 59 | | 52 | 69 | 41

Sodium immol/ddl| | 133 (7/7 1136 her you | with the swarm of the moat river

Img/dli

RTNIpL/dLII HER 777717171171Я771717171717171717171111117195,6. | -.yuKYiY// 17 loveO

Note: TI C - total number of leukocytes; M/VS :- white blood cells; RIT - platelets;

H A1C - glycosylated hemoglobin; A5T - apartataminotransferase; AT - alanine aminotransferase; RT - prothrombin time; BMI - international normalized ratio (for blood coagulation); RTN - parathyroid hormone A 46-year-old patient with chronic renal failure due to diabetes also demonstrated improvement in creatinine, urea and hemoglobin levels after treatment with reprogrammed cells (see Table 11). The patient also had his hemodialysis sessions reduced from 12 to about 5 per month, and his subcutaneous epoetin alfa (ERVEHU) treatment was reduced from 4,000 units twice weekly to 4,000 units once every two weeks.

Table 11

Levels of kidney function markers in a 46-year-old woman with chronic renal failure before and after therapy with reprogrammed cells

ABIS Y177771717171717911111171171111117184117111111132

Calcium mg/dL 77771 Я77771711101Я777717117199 1111111

Phosphorimgdl| С І777777171717611111111 11111115 111115 SS

Note: TI C - total number of leukocytes; M/VS - white blood cells; RR :- after eating; RI TT - platelets; EB5 - fasting blood sugar; A5T - apartataminotransferase; AIT - alanine aminotransferase; RT - prothrombin time; BMI - international normalized ratio (for blood coagulation)

Example 9

Materials and methods

Multiple sclerosis patients were treated with autologous reprogrammed pluripotent stem cells, mesenchymal stem cells and neurons (target cells). Target cells were obtained by reprogramming apherized white blood cells until target cells developed as indicated by their various characteristics as described above. Patients were apherized by 2-3 times processing of their entire blood volume. Autologous reprogrammed cells were generated by reprogramming proposed by the invention. Autologous reprogrammed cells were administered to patients by intravenous infusion into the jugular vein or veins of the arm or thigh.

The results

Patients treated with autologous reprogrammed cells showed a reduction in brain and spinal cord damage. Reduction of the lesion can occur within three months from the start of treatment (Fig. 15a-b). Reduction of brain tissue damage occurred within six months (Fig. 1ba-b).

Patients treated with autologous reprogrammed cells also demonstrated resolution of spinal cord lesions (Fig. 17a-B). Further, these patients demonstrated improvement in disability assessment according to the extended Kurtzke Disability Assessment Scale (EDS5), demonstrated remission, and did not participate in conventional therapy up to four years after receiving a single infusion.

Example 10

Materials and methods

A woman with NIM was treated with autologous reprogrammed hematopoietic stem cells. The patient underwent apheresis with 2-3 times processing of her entire blood volume. Target cells were obtained by reprogramming apherized white blood cells, until the target cells did not develop, as indicated by their various characteristics, as described above. Autologous reprogrammed cells were administered to the patient by intravenous infusion into the jugular vein or veins of the arm or thigh.

From Results

Before treatment, the screening test for antibodies to NIM-1 and NIM-2 showed a value of 3.68. Values of 1.0 or greater are considered positive.

Two months after the start of treatment with autologous reprogrammed cells, the value of the test for antibodies to NIM-1 and NIM-2 was 0.46, indicating that the patient did not show a positive result for NIM-1 and NIM-2. Six months after initiation of treatment, the screening test value for NIM-1 and NIM-2 was 0.48, further indicating that the patient did not test positive for NIM.

Example 11

The effects of treatment with autologous reprogrammed cells have been demonstrated in patients suffering from other conditions and diseases. The target cells listed here were obtained by reprogramming apherized white blood cells until the target cells had developed as indicated by their various characteristics as described above.

Congestive heart failure A 1-year-old man with congestive heart failure was injected with autologous reprogrammed cardiomyocytes, pluripotent stem cells, mesenchymal stem cells, and endothelial cells.

Treatment resulted in an improvement in ejection fraction (EE); levels of brain natriuretic peptide precursor (BNP), which is a prognostic factor associated with coronary artery disease; fasting blood glucose levels; and NBATS levels (see Table 12). In addition, the heart, which was previously dilated, returned to normal size, as shown in Table 12 by a decrease in end-diastolic left ventricular internal size (LVDI), end-systolic left ventricular internal size (LVDI/5), and finally -diastolic thickness of the intraventricular septum (IM50), determined by echocardiogram.

Table 12

Markers of coronary artery disease in 61-year-old patients with congestive heart failure before and after therapy with reprogrammed cells

Disease markers 1 month. to | 1 month after | 2 months after | 4 months after | 6 months after /10 months after the coronary artery | treatment | treatment | treatment | treatment | treatment | treatment

GMO (cm) 65 | 63 | 59 | 69 | 65 | 57

IMIS/5 (cm) иУ5О | сми 13 | 13 | those2 | 72 | 06 1 10

IMRUMO (cm) 07 1 06 | 06 | 08 | 06 | 10

Mass index 283 251 209.5 167 151.5 of the left ventricle

Igm/mi 141.5 133.5 111.4 88.8 81

Rgo BMR (pg/ml) 2969 800 | 600 | 600 | 60 | 497

Fasting blood sugar (mg/day 244 118 145 87 95

But AIS (be 92 | pi | pa | 72 | 65 | 6

Note: ГМИО/О - end-diastolic internal size of the left ventricle; And MIB/5 - end-systolic internal size of the left ventricle; EE - emission fraction; ЮМ5О - end-diastolic thickness of the intraventricular septum; And MRM/O - end-diastolic thickness of the back wall of the left ventricle; Rgo VMR is a precursor of brain natriuretic peptide; HN A1C - glycosylated hemoglobin

Hepatitis C

Thirteen patients with hepatitis C virus (HCV) and beta-thalassemia received an infusion of autologous reprogrammed hematopoietic cells, pluripotent stem cells, mesenchymal stem cells, and hepatocytes. Treatment effects were shown to decrease or eliminate HCM burden (see Table 13) and improved blood markers (see Table 14) such as liver enzymes, bilirubin, albumin, prothrombin time, and international normalized ratio (for blood coagulation). In particular, patients noted the normalization of liver enzymes alanine aminotransferase (AGT) and aspartate aminotransferase (A5T), which are associated with inflammation and damage to liver cells (Fig. 18a-br).

Table 13

Viral load in 12 patients infected with hepatitis C virus treated with autologous reprogrammed stem cells

Identification number Initial level of viral Z month after infusion, viral load of the patient x 1000 load x 1000 of that 81118110 61116110 61112110 61110110 10287771 t.iimillions///// | 7.7 million../7/7/:ZO shish now: write 21101110 nnshEzhnnynnn write six saw

Table 14

Blood markers in a hepatitis C patient with cirrhosis at baseline and after reprogrammed cell therapy

Hemoglobin

Total bilirubin|mgidl| ////77с17111111111169111111Ї11111111засСсСС (Sechovinaim/idl! ///777777771111111111111111160111111111 11111136

Note: MIA - white blood cells; AT (O/L) 5 alanine aminotransferase (upper/lower);

AT (Ш/) - aspartate aminotransferase (upper/lower); BMI - international normalized ratio (for blood coagulation); RT - prothrombin time

For example, a 44-year-old man with cirrhosis of the liver caused by hepatitis C virus infection also demonstrated positive dynamics of the liver enzymes alanine aminotransferase (AT) and aspartate aminotransferase (A5T), improvement of the international normalized ratio (for blood coagulation), bilirubin and albumin, and incidentally detected sugar in the blood (see table 15). In fact, this patient was treated with albumin before the reprogrammed cell therapy, but was not treated with albumin after the therapy.

Table 15

Blood markers in a 44-year-old man with liver cirrhosis caused by hepatitis C virus infection at baseline and after therapy with reprogrammed cells. - After therapy

Blood marker level oomeneeyuyun 00 nor r r oVUtvya

Alkaline phosphatase (O/.) 7196. | ..YuuYUYYYYYY.| 147 | (| 58 matchmaker 777777777717171717171771717117149 17771771 1170 | 63 | from

Bilirubin O|mgdl| /////////7/17771717168 | 54 | 47 | 49 | 4

Creatinine mg/dli 1777/7007. | 07 | 08 | 07 | 07

Kaliyimmolli 77777711 1132 | 38 | 44 (Sodium 77771717 11134 | 141 | 137

Continuation of table 15

Blood markers in a 44-year-old man with liver cirrhosis caused by hepatitis C virus infection at baseline and after therapy with reprogrammed cells. - After therapy

Blood marker level oomeyaeeyuno 00 Nord r r oVUtvya (Uric acid 17777771 35 | 77771171 124 | Z oalpha-fetoprotein.////////////177777717177l/l | norm | HER (croglobulins. 77777771 fnegativiiid//:/ ОИ 7 / Y77771

Note: TI C - total number of leukocytes; RI.T - platelets; A5T - apartataminotransferase; AH! T - alanine aminotransferase; SOT - gamma-glutamyltransferase; KV5 - random blood sugar; BMI - international normalized ratios (for blood coagulation)

Head injury

A patient with a head injury after a car accident was treated with an infusion of autologous reprogrammed pluripotent stem cells and neurons. The treatment led to the restoration of the damaged brain tissue (Fig. 19a-c), improvement of the ejection fraction (EC); levels of brain natriuretic peptide precursor (BNP), which is a prognostic factor associated with coronary artery disease; fasting blood glucose level; and NBA1C levels (see Table 10).

Lung disease

A patient with limited lung disease associated with motor neuron disease was treated with infusion of autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, alveolar epithelial cells, and endothelial cells. Six months after the start of treatment, the forced vital capacity (FVC), which corresponds to the volume of air that can be forcefully exhaled after a full inhalation, increased from 5095 to 7195, while the forced expiratory volume in 1 second (HEMI1), which corresponds to the volume of air that can be forcefully exhaled in one second, increased from 64 95 to 68 95. Next, the ratio of REMI1 to

The EMC, which is approximately 75-80,956 in healthy adults, decreased from 100,95 to 82,965. A chest x-ray showed less darkening of the lung cavity after treatment (Figure 20).

Menopause

A 1-year-old patient in menopause was given an infusion of autologous reprogrammed pluripotent stem cells, pluripotent embryonic cells and oocytes. After treatment, the patient had an increase in various hormones and protein levels, including insulin-like growth factor 1 (IGF-1), ethegdiaoi, and low-density lipoprotein (I0) (see Table 16).

Table 16

Effects of administration of autologous reprogrammed stem cells to a menopausal patient

Adrenocortipmol/l| | 21777170 Y71117o Her (CalcitoninIn/ml!d | 7028 | (40 | 2 sh(5Bo | ZK F

Continuation of table 16

Effects of administration of autologous reprogrammed stem cells to a menopausal patient

EZN (IME/U 77/11 11111412 7111155. | 388 sh -

VN (ImMEL. (77777171 11111181 11111660

Prolactin in kg/ml 1 162 | 7 | 7105 | 06 (Free test Ipmol/l! | 8 | 64 | 75

RrimoidlI 777 1777711794Ї7771711790 77111102 | 103

Inhibinod ////77771711111740Ї111111111111111Ї1111130 11111111

OsteocalcinIn/ml! (77777771 1111171680 tZNipod/lI 7 177771084. | 1111107 1111052 Zh z Kz MA.

CalcitoninIng/lL ///17777110128 77111140 Y111150 111111

Notes: ISE-1 - insulin-like growth factor 1; ISE-1-turnip - insulin-like binding growth factor 1; CH - growth hormone; 8) ONEA-5 - dehydroepiandrosterone sulfate; 9) adrenocort - adrenocortical hormone; E5H - follicle-stimulating hormone; IN - luteinizing hormone; NOAH - high-density lipoprotein; I.O. - low-density lipoprotein; НЬ A1С « glycosylated hemoglobin; EbBz - selection of fetal blood; TZN - thyroid-stimulating hormone; 25) ETZ - free triiodothyronine; ET-4 - free thyroxine

Depression

A patient with depression was treated with an infusion of autologous reprogrammed pluripotent stem cells and neurons. After treatment, the patient showed increases in various hormones and protein levels, including insulin-like growth factor-1 (IGF-1), cortisol, and testosterone (see Table 17).

Table 17

The effect of introducing autologous reprogrammed cells to a menopausal patient.

Continuation of table 1 7

The effect of introducing autologous reprogrammed cells to a menopausal patient

Notes: CH - growth hormone; ISOE-1 - insulin-like growth factor 1; ISE-Br - insulin-like growth factor 1, which binds protein; adrenocort - adrenocortical hormone; 5-59 - globulin that binds the sex hormone; EZN - follicle-stimulating hormone;

IN - luteinizing hormone; e2 - estradiol; ONEA-5 - dehydroepiandrosterone sulfate; TZN - thyroid-stimulating hormone; ETZ - free triiodothyronine; ET-4 - free thyroxine

Non-obstructive azoospermia

A patient with non-obstructive azoospermia was treated with infusion of autologous reprogrammed pluripotent stem cells, pluripotent embryonic cells and sperm. After treatment, an increase in testosterone was observed in the patient for eight months (Fig. 21).

Loss of vision

A patient suffering from vision loss due to a benign tumor that was removed was treated with an infusion of autologous reprogrammed pluripotent stem cells and neurons. After the treatment, the patient showed increased sensitivity of the retina and improved vision (Fig. 22).

Thus, having described in detail the preferred embodiments of the present invention, it is to be understood that the invention defined by the foregoing paragraphs should not be limited to the specific details set forth in the foregoing description, as many obvious changes are possible without departing from the spirit or scope of the present invention.

Claims (47)

FORMULA OF THE INVENTION 1. The use of one or more reprogrammed neural stem cells in the preparation of a medicine or pharmaceutical composition for the renewal or replacement of tissues or cells of a patient suffering from a neurological disease, in which the reprogrammed neural stem cells are obtained by reprogramming the committed progenitor cells of the first cell line from the patient, wherein the reprogramming involves contacting these committed cells with antibodies that bind to a receptor that mediates capture, recognition, or display of antigen on the surface of the committed cells. 2. Application according to claim 1, characterized in that the patient suffers from diseases or disorders selected from the group consisting of motor neuron disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, multiple sclerosis, head injury, depression, deterioration vision 3. Application according to claim 1, which is characterized by the fact that committed cells are obtained from whole blood, bone marrow, neural tissue, muscle tissue, epidermis or skin. 4. Application according to item C, which differs in that the committed cells are obtained from whole blood. 5. Application according to claim 4, which differs in that committed cells are obtained by apheresis. b. Application according to claim 4, which differs in that committed cells are obtained from mobilized or non-mobilized blood. 7. Application according to claim 4, which is characterized by the fact that committed cells are selected from the group including T-cells, B-cells, eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, lymphocytes, leukocytes, platelets and red blood cells. 8. Application according to claim 1, which is characterized by the fact that the reprogramming includes retrodifferentiation, transdifferentiation, redifferentiation of committed cells or their combination. 9. Application according to claim 8, characterized in that the reprogramming includes retrodifferentiation of committed cells to obtain retrodifferentiated cells. 10. Application according to claim 1, which is characterized by the fact that the receptor is an antigen of the major histocompatibility complex (MHC) of class I or an antigen of MHC of class III. 11. Application according to claim 1, which differs in that the agent is a monoclonal antibody to the receptor. 12. Application according to claim 11, which is characterized by the fact that the antibody is selected from the group that includes the monoclonal antibody SKZ/43 and the monoclonal antibody TAG. 185. 13. Application according to claim 1, which is characterized by the fact that the medicine or pharmaceutical composition is administered by injection or implantation. 14. Application according to claim 13, which is characterized by the fact that the medicine or pharmaceutical composition is administered parenterally, intramuscularly, intravenously, subcutaneously, intraocularly, orally, by transdermal injection or by injection into the spinal fluid. 15. Use according to claim 8, characterized in that the reprogramming includes transdifferentiation of committed cells to obtain transdifferentiated cells. 16. The application according to claim 15, which is characterized by the fact that the committed cells are transdifferentiated by culturing the committed cells in a tissue culture medium that includes one or more retrodifferentiation agents and one or more differentiation promoters. 17. Application according to claim 1, which is characterized by the fact that the tissue culture medium is selected from the group that includes the medium of IMOM, OMEM, EME, a-MEM, KRMI 1640, Nat-B-12, E199, MSOV, Ieromiy 1-15 and MUmMiiate EE medium or any commercially available culture media. 18. Application according to claim 16, which is characterized by the fact that the promoter of differentiation is an anticoagulant, a chelating agent or an antibiotic. 19. Application according to claim 16, which differs in that the promoter of differentiation is a vitamin, mineral or their derivative. 20. Application according to claim 19, which is characterized by the fact that the vitamin, mineral or their derivative is selected from the group that includes vitamin A, vitamin BZ, vitamin C, vitamin OZ, vitamin K, retinoic acid, Zo nicotinamide, zinc or zinc compounds and calcium or calcium compounds. 21. Application according to claim 16, which is characterized by the fact that the promoter of differentiation is a natural or synthetic hormone. 22. Application according to claim 21, which differs in that the natural or synthetic hormone is hydrocortisone or dexamethasone. 23. Application according to claim 16, which is characterized by the fact that the promoter of differentiation is an amino acid or its derivative. 24. Application according to claim 23, which is characterized by the fact that the amino acid or its derivative is selected from the group that includes I-glutamine (I--diy), ergothioneine (EST), proline and substituted amino acids (MEAA). 25. Application according to claim 16, which differs in that the promoter of differentiation is a chemical compound or its derivative. 26. Application according to claim 25, which is characterized by the fact that the chemical compound or its derivative is selected from the group that includes p-mercaptoethyl, cyclic adenosine dibutyl monophosphate (aBbbB-CAMP), monothioglycerol (MTS), putrescine, dimethyl sulfoxide (OM5O), hypoxanthine, adenine, forskolin, cilostamide and 3-isobutyl-1-methylxanthine. 27. Application according to claim 16, which differs in that the promoter of differentiation is a nucleoside or its analogue. 28. Application according to item 27, which differs in that the nucleoside or its analogue is 5-azacytidine. 29. Application according to claim 16, which is characterized by the fact that the promoter of differentiation is acids or their salts. 30. Application according to claim 29, which is characterized by the fact that the acid or its salt is selected from the group that includes ascorbic acid, pyruvate, okadaic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), disodium salt of EOTA, ethylene glycol tetraacetic acid (ESTA), anticoagulant dextrose citrate of formula A (ASOBA), sodium butyrate and glycerophosphate. 31. Application according to claim 16, which is characterized by the fact that the promoter of differentiation is an antibiotic or a drug. 32. Use according to claim 31, which is characterized in that the antibiotic or drug is selected from the group that includes (418, gentamicin, pentoxifylline (1-(5-oxohexyl)-3,7-60 dimethylxanthine) and indomethacin. 33. Application according to claim 16, which differs in that the promoter of differentiation is a protein. 34. Application according to claim 33, which differs in that the protein is tissue plasminogen activator (TPA). 35. Application according to claim 16, which is characterized by the fact that the tissue culture medium contains autologous plasma; platelets; serum or mammalian serum. 36. Application according to claim 16, which is characterized by the fact that the cells are cultured in blood bags, substrates, tissue culture bags or plastic vessels for tissue culture. 37. Use according to claim 36, characterized in that the tissue culture vessels are attached or non-attached tissue culture vessels. 38. Application according to claim 36, which is characterized by the fact that the tissue culture vessels are closed or open. 39. Use according to claim 38, characterized in that the tissue culture vessels are coated with an agent selected from the group consisting of gelatin, collagen, matrigel or extracellular matrix. 40. Application according to claim 16, which is characterized by the fact that the cells are cultured at temperatures from about 18 to about 40 "C. 41. The use of claim 16, wherein the cells are cultured at a carbon dioxide level of from about 4 to about 10 95. 42. The use of claim 16, wherein the cells are cultured at an oxygen level of from about 10 to about 35 95. 43. Pharmaceutical composition containing reprogrammed target cells defined in any of claims 1-42. 44. A method of obtaining reprogrammed neural stem cells for administration to a patient in need, which includes (u) obtaining committed cells of the first cell line; (ii) reprogramming the committed cells to obtain reprogrammed neural stem cells, the reprogramming comprising contacting the committed cells with antibodies that bind to a receptor that mediates antigen capture, recognition, or display on the surface of the committed cells. Zo 45. A pharmaceutical composition containing reprogrammed neural stem cells obtained by the method of claim 44 and at least one pharmaceutically acceptable excipient. 46. The method of obtaining a medication or a pharmaceutical composition for administration to a patient who needs it, which involves: (i) obtaining committed cells of the first cell line; (ii) reprogramming the committed cells, by contacting an antibody that binds a receptor that mediates antigen recognition and presentation on the surface of the committed cells to obtain reprogrammed target cells, and these reprogrammed target cells are neural stem cells; and (iii), if necessary, combining the reprogrammed neural stem cells with one or more pharmaceutical excipients. 47. pharmaceutical composition obtained by the method according to claim 46. br rt ad bu bu , | | I am 15 | | oh | that oh them | and This vky | in And ! in c, in | | yin : | and what's more difficult to read: ia E rt V v g Hn 0zvki Ez TEG te E SHY y v Ge I s u V, Ya she she Control MRI 0045 RGS svi ke SIBIKRIE Control kr 0 SOZ OK Fig. 1 Day 1 v y EL ovi rt p pen she i i i ih Kiya veto Day 14 a zhi 00 dae M EKA shchi nm y g: 8 V even Beshev Yeyunya tia i SHU sha SHE t Ku piy still i. y is WITH I - M TU ROKY in the study of Я y s y. 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