JP6073135B2 - Treatment with reprogrammed mature adult cells - Google Patents

Description

<Incorporation by reference>
Manufacturer's instructions, instructions for all documents cited or referenced in documents cited herein, as well as products referred to in this specification or in documents incorporated herein by reference, Product specifications and product sheets are incorporated herein by reference and can be used in the practice of the present invention.

  A method of treating various diseases, disorders or illnesses in a patient using reprogrammed cells such as reverse differentiated, transdifferentiated or redifferentiated cells. The method includes obtaining committed cells from the patient, dedifferentiating the committed cells to obtain reverse differentiated target cells, and administering the reverse differentiated cells to the patient. In certain embodiments, the method includes obtaining committed cells from a patient, transdifferentiating committed cells to obtain transdifferentiation target cells, and administering transdifferentiation target cells to a patient. Reverse differentiated or transdifferentiated target cells repair or recruit tissue or cells in the patient.

  Stem cells are characterized by the ability to regenerate themselves by somatic cell division and differentiate into various specialized cell types. Two broad types of mammalian stem cells are embryonic stem cells isolated from placental vesicle inner cell mass and adult stem cells found in adult tissue. In developing embryos, stem cells can differentiate into all specialized embryonic tissue. In adult organisms, stem and progenitor cells replenish specialized cells and maintain normal turnover of regenerating organs such as blood, skin, or intestinal tissue.

  Although stem cells are abundant in developing embryos, the amount of stem cells decreases as development progresses. In contrast, adult organisms contain a limited number of stem cells that are limited to specific body compartments.

  Stem cell therapeutic applications have the potential to change the therapy of a number of diseases or disorders. Some adult stem cell therapies such as bone marrow transplantation already exist, but medical researchers are more interested in cancer, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis, multiple sclerosis and muscle injury, among others. We expect to use stem cells to treat a variety of diseases. Such therapies can take advantage of the ability of the stem cells to differentiate into the cell types necessary to treat the disease.

  However, there are concerns regarding the uncertainty regarding the successful treatment of such diseases using stem cells and the ease with which stem cells can be obtained. For example, hematopoietic stem cells are traditionally extracted by isolation from bone marrow, growth factor mobilized peripheral blood or umbilical cord blood (placenta). Hematopoietic stem cells can also be prepared from embryonic stem (ES) cells extracted from embryos obtained using in vitro fertilization methods. However, extraction from these resources is cumbersome, sometimes dangerous, and can face ethical issues. Furthermore, the number of stem cells obtained from these resources is limited. In addition, stem cells may experience difficulty in differentiation into the cells necessary to treat the disease.

U.S. Application No. 08 / 594,164 U.S. Patent No. 6,090,625 US Application No. 09 / 742,520 U.S. Patent No. 7,112,440 U.S. Application No. 10 / 140,978 U.S. Patent No. 7,220,412 U.S. Application No. 10 / 150,789 U.S. Patent No. 7,410,773 US Application No. 09 / 853,188

Vettese-Dadey, The Scientist, 1999, 13, 21 Nisitani et al., Int Immuno, 1994, 6, 909-916 Le Page, New Scientist, December 16, 2000 Bischoff, Dev Biol, 1986, 115, 129-39 Nakafuku and Nakamura, J Neurosci Res, 1995, 41, 153-168 Metcalf, Nature, 1989, 339, 27-30 Potocnik et al., EMBO J, 1994, 13, 5274-83 Andrews et al., Hybridoma, 1984, 3, 347-361 Kannagi et al., EMBO J, 1983, Vol. 2, pp. 2355-2361 Fox et al., Dev Biol, 1984, 103, 263-266 Ozawa et al., Cell Differ, 1985, 16, 169-173 J Neurosci, 985, 3310 Eaves et al., J Tiss Cult Meth, 1991, 13, 55-62 Kreisseg et al., J Hematother, 1994, 3, 263-89 Korbling et al., Bone Marrow Transplant, 1994, 13, 649-54 Visser et al., Blood Cells, 1980, 6, 391-407 Grogan et al., Blood Cells, 1980, 6, 625-44 Urbankova et al., J Chromatogr B Biomed Appl, 1996, 687, 449-52. Deryugina et al., Crit Rev Immunology, 1993, 13, 115-150 Dexter et al., J Cell Physiol, 1977, 91, 335 Dexter et al. Acta Haematol, 1979, 62, 299 Lebkowski et al., Transplantation, 1992, 53, 1011-9 Lebkowski et al., J Hematother, 1993, 2, 339-42 Haylock et al., Immunomethods, 1994, 5, 217-25 Young et al., Blood, 2006, 108, 2509-2519 Bacigalupo et al., Hematology, 2007, 23-28 Locasciulli et al., Haematologica, 2007, 92, 11-18 Gottdiener et al., Arch Intern Med, 1981, 141, 758-763 Sanders et al., Semin Hematol, 1991, 28, 244-249 Elhasid et al., Leukemia et al., 2000, 14, 931-934 Locatelli et al., Bone Marrow Transplant, 1996, 18: 1095-101

  The present invention relates to the use of reprogrammed cells to repair tissue in a patient or to replenish tissue or cells. For example, the present invention relates to the use of reverse differentiated cells obtained by reverse differentiation of differentiated or committed cells to repair tissue or replenish tissue or cells in a patient. The invention also relates to the use of transdifferentiated cells obtained by transdifferentiation of differentiated or committed cells to repair tissue in a patient or to supplement tissue or cells.

  This application allows commits or somatic cells obtained from a patient to be reprogrammed to result in cells of different lineages, and these reprogrammed cells are administered to the patient to repair or supplement tissue or cells. Based in part on applicants' discovery that they can. Examples of reprogramming methods include reverse differentiation and transdifferentiation.

  Applicants have therefore discovered that committed cells can be reprogrammed to result in cells of different lineages. For example, committed cells can undergo reverse differentiation, resulting in reverse differentiated cells, such as pluripotent stem cells, which are less differentiated cells, and these reverse differentiated cells can be administered to a patient to produce tissue. Alternatively, the cells can be repaired or supplemented. As another example, committed cells obtained from a patient can undergo transdifferentiation to result in transdifferentiation cells, for example, cells of a different lineage than the commit cells, and these transdifferentiation cells are administered to the patient. Tissue or cells can be repaired or supplemented.

  The invention includes a method of repairing or recruiting cells of a tissue or cell lineage in a patient by administering the reprogrammed cells to the patient. In particular, the present invention comprises (i) obtaining committed cells, (ii) dedifferentiating committed cells to obtain reverse differentiated target cells, and (iii) administering the reverse differentiated target cells to a patient, The method includes redifferentiating a reverse differentiation target cell into a cell of the cell lineage to repair or replenish a tissue or cell lineage cell in the patient. These redifferentiated cells can be of the same cell lineage as the committed cells or of a different cell lineage.

  The present invention also includes (i) obtaining committed cells, (ii) transdifferentiating committed cells to obtain transdifferentiation target cells, and (iii) administering the transdifferentiation target cells to a patient. A method of repairing or replenishing cells of a tissue or cell lineage.

  In some embodiments, the patient has bone marrow failure, hematological disorder, aplastic anemia, beta Mediterranean anemia, diabetes, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney disease, multiple sclerosis, congestion Congenital heart failure, hepatitis C virus, human immunodeficiency virus, head injury, spinal cord injury, lung disease, depression, non-obstructive azoospermia, male menopause, menopause and infertility, rejuvenation, scleroderma ulcer, psoriasis, It may be suffering from a disease, disorder or condition including but not limited to wrinkles, cirrhosis, autoimmune disease, hair loss, retinitis pigmentosa, crystalline dystrophy / blindness, diabetes, and infertility. Thus, in some embodiments, the reprogrammed cells can be bone marrow cells that treat aplastic anemia, leukemia, lymphoma or human immunodeficiency virus in a patient.

  In some embodiments, the reprogrammed target cells, such as reverse differentiated cells, transdifferentiated cells or redifferentiated cells, are pluripotent stem cells, pluripotent germ cells, hematopoietic stem cells, neural stem cells, epithelial stem cells, mesenchymal stem cells. , Endoderm and neuroectodermal 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 / bone cells, hair / dermal papilla cells, cartilage / chondrocytes, fat cells / adipocytes, skeletal muscle cells, endothelial cells, cardiac muscle / It can include but is not limited to cardiomyocytes and tropoblasts.

  In certain embodiments, committed cells are obtained from related tissues including blood or bone marrow. Committed cells can be obtained from whole blood and / or by apheresis. The blood may be mobilized or non-mobilized blood. Such committed cells include T cells, B cells, eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, lymphocytes, leukocytes, platelets and erythrocytes. However, it is not limited to these. Alternatively, committed cells can be obtained from neuronal tissue, muscle tissue, or skin epidermis and / or dermal tissue from the central or peripheral nervous system.

  In certain embodiments, committed cells are obtained from a patient's blood or tissue. In some embodiments, the patient from whom committed cells are obtained and the patient to whom a reprogrammed target cell, such as a reverse differentiation target cell or transdifferentiation target cell is administered, are the same patient.

  In some embodiments, the committed cells are reverse differentiated by contacting the committed cells with an agent. For example, committed cells can be incubated with the agent. In certain embodiments, the agent engages with a receptor that mediates capture, recognition or presentation of the antigen on the surface of committed cells. The receptor can be an MHC class I antigen or an MHC class II antigen. In some embodiments, the class I antigen is HLA-A receptor, HLA-B receptor, HLA-C receptor, HLA-E receptor, HLA-F receptor or HLA-G receptor; The class II antigen is HLA-DM receptor, HLA-DP receptor, HLA-DQ receptor or HLA-DR receptor.

In certain embodiments, the agent can be an antibody to the receptor, such as a monoclonal antibody to the receptor. In some embodiments, the antibody is monoclonal antibody CR3 / 43 or monoclonal antibody TAL 1B5. In a further embodiment, the agent modulates MHC gene expression, such as MHC class I ⁺ and / or MHC class II ⁺ expression.

  In some embodiments, the reverse differentiated cells can undergo redifferentiation in discrete steps. For example, reverse-differentiated cells can be expressed as 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 Reverse differentiated cells can be redifferentiated by contacting with growth factors including, but not limited to, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, stem cell factor and bone morphogenetic protein. The resulting redifferentiated target cells can then be administered to a patient.

  In an embodiment of the present invention, committed cells can be transdifferentiated by culturing committed cells under specific culture conditions. For example, committed cells can be cultured with reverse differentiation agents in certain types of culture media. Examples of these culture media include Dulbecco's modified Eagle's medium (DMEM) and Iskov's modified Dulbecco's medium (IMDM). The tissue culture medium may also contain differentiation promoting agents such as vitamin and / or mineral supplements, hydrocortisone, dexamethasone, β-mercaptoethanol. Further culture conditions include using chelating agents or antibiotics, culturing at specific temperatures or carbon dioxide or oxygen levels, and culturing in specific vessels. The culture conditions can determine the type of transdifferentiation target cell that results.

  One embodiment of the present invention is therefore a method of obtaining target cells. The method can include obtaining a committed cell and then reprogramming the committed cell. These methods have been described herein. In some embodiments, the method can include dedifferentiating committed cells. In other embodiments, the method can include transdifferentiating committed cells. In other embodiments, the method may include dedifferentiating committed cells and then redifferentiating the reverse differentiated cells.

  Another aspect of the present invention is the use of one or more reprogrammed target cells in the preparation of a medicament for repairing or supplementing cells of a tissue or cell lineage in a patient or for treating a disease or tissue injury It is.

  Yet another aspect of the invention is a method of treating disease or tissue damage in a patient in need thereof. In certain embodiments, the method includes obtaining a committed cell, reprogramming the committed cell to obtain a reprogrammed target cell, and administering the reprogrammed target cell to a patient. In some embodiments, the target cells can be reprogrammed by reverse differentiation, transdifferentiation and / or redifferentiation. In certain embodiments, the target cells are reverse differentiation target cells, transdifferentiation target cells and / or redifferentiation target cells. The process of obtaining committed cells and reprogramming committed cells is as described herein.

  In some embodiments, reprogrammed target cells such as reverse differentiation target cells, transdifferentiation target cells or redifferentiation target cells can be administered by injection, transplantation or infusion. These cells can be administered parenterally, intramuscularly, intravenously, subcutaneously, intraocularly, orally, transdermally, or injected into the spinal fluid. In certain embodiments, reverse differentiation target cells or transdifferentiation target cells are administered in the form of a pharmaceutical composition. The pharmaceutical composition may comprise a reverse differentiation target cell or transdifferentiation target cell and at least one pharmaceutically acceptable excipient.

  One aspect of the present invention is a pharmaceutical composition for administering a reprogrammed target cell such as a reverse differentiation target cell or a transdifferentiation target cell. The pharmaceutical composition may comprise one or more types of target cells and at least one pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may include adjuvants and / or other excipients suitable for administration to a patient.

  Other aspects of the invention include (i) obtaining committed cells; (ii) reprogramming committed cells to obtain reprogrammed target cells; and (iii) optionally reprogramming target cells. A method of preparing a pharmaceutical composition or medicament comprising the step of combining 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 for obtaining committed cells and reprogramming committed cells are as described herein.

  In this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like are For example, they may have the meanings of `` includes '', `` included '', `` including '' and the like, `` substantially Terms such as `` consisting essentially of '' and `` consists essentially of '' have the meaning assigned to them in U.S. Patent Law, e.g., they are clearly It is noted that elements not listed are taken into account, but excluding those elements found in the prior art or affecting the basic or novel features of the present invention.

  These and other embodiments are disclosed or are obvious from and are encompassed by the following detailed description.

  The following detailed description, which is given by way of example and is not intended to merely limit the invention to the specific embodiments described, may be best understood in conjunction with the accompanying drawings.

FIG. 2 shows immunophenotypic examination of mononuclear cells obtained by apheresis before (upper panel) and after (lower panel) induction of reprogramming. Cells were labeled with an immunoglobulin G1 (IgG1) isotype control and a monoclonal antibody conjugated to R-phycoerythrin (RPE) Cy-5 or phycoerythrin (PE) (vertical axis symbol) for CD34 or CD19, respectively. Cells were also stained with CD45, CD38, CD61 and IgG1 isotype control monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) (horizontal axis symbol). The lower panel shows an increase in hematopoietic stem cells as indicated by an increase in the relative number of CD34 and CD34CD38 cells with a decrease in leukocyte maturation markers such as CD45 and CD19. It is a figure which shows the continuous immunophenotype test | inspection of the peripheral blood sample of a patient with severe aplastic anemia after injection | pouring of autologous HRSC (1 day, 2, 3, 6, and 14 days later). Cells were labeled with monoclonal antibodies against CD34 and CD45 (second horizontal panel) and CD34 and CD38 (third horizontal panel). The top horizontal panel shows forward and side scatter and is autologous HRSC flow cytometry, bone marrow smear and tehphin sections. Days 1-14 show an increase in cells with large forward and side scatter suggesting granulocytes, and days 1-3 show an increase in the relative number of circulating CD34 hematopoietic stem cells. FIG. 3 shows serial immunophenotyping of peripheral blood samples from patients with severe aplastic anemia after injection of autologous human reprogrammed stem cells (HRSC) (1, 2, 3, 6, and 14 days later). Cells were labeled with monoclonal antibodies against CD34 and CD61 (first horizontal panel), CD19 and CD3 (second horizontal panel) and CD33 & 13 and CD7 (third horizontal panel). The FACScan plot shows an increase in the number of myeloid cells as indicated by the gradual increase in cells expressing CD33 & 13, including progenitors, as indicated by an increase in the relative number of cells co-expressing CD33 & 13 and CD7. There was also a gradual increase in the relative number of lymphocytes, as shown by the increase in the relative number of CD19 and CD3 lymphocytes. It is a figure which shows the bone marrow analysis of the severe aplastic anemia patient before and after the injection of autologous HRSC. Bone marrow smears (a and b) before and after treatment show an increase in red blood cells, trephine sections (c and d) before and after treatment show an increase in myeloid cellularity and infusion of HRSC Subsequent clonal analysis of bone marrow includes colony forming unit megakaryocytes (e), colony forming monocytes (f), colony forming granulocytes and macrophages (g), and colony forming myelocytes and red blood cells (h), and burst forming red blood cells. (i) shows increased proliferation. It is a figure which shows the karyotype analysis of the peripheral blood sample of a patient with severe aplastic anemia 4 years after injection | pouring of the autologous HRSC which does not show a genetic abnormality, and the g-staining method. FIG. 5 shows an increase in absolute mean fetal hemoglobin levels in Mediterranean anemia patients after treatment with self-reprogrammed cells. FIG. 5 shows the increase in mean red blood cell volume showing the mean size of red blood cells expressed in femtoliters in a poor Mediterranean patient after treatment with self-reprogrammed cells. FIG. 6 shows an increase in mean erythrocyte hemoglobin, which is the weight of hemoglobin per erythrocyte expressed in picograms in a Mediterranean anemia patient after treatment with self-reprogrammed cells. FIG. 5 shows the reduction in serum ferritin levels expressed in nanograms per milliliter in Mediterranean anemia patients after treatment with self-reprogrammed cells. FIG. 5 shows an increase in C-peptide levels on a fasting and mixed food intake in patients with diabetes after treatment with self-reprogrammed cells. FIG. 3 shows a decrease in glycosylated hemoglobin (HbA1C) levels in diabetic patients after treatment with self-reprogrammed cells. FIG. 5 shows a decrease in creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) levels in muscular dystrophy patients after treatment with self-reprogrammed cells. FIG. 5 shows a decrease in liver enzyme alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in muscular dystrophy patients after treatment with self-reprogrammed cells. FIG. 6 shows the reduction in microalbuminuria levels in 12 patients with renal disease after treatment with self-reprogrammed cells. FIG. 6 shows the reduction of glycosylated hemoglobin (HbA1C) levels in 12 patients with renal disease resulting from diabetes after treatment with self-reprogrammed cells. Magnetic resonance imaging (MRI) of the brain of a patient with multiple sclerosis before treatment with self-reprogrammed cells (left scan) and after 3 months (right scan), showing reduced lesion enhancement after stem cell therapy It is a figure which shows a scan. FIG. 6 shows MRI scans of different parts of the patient's brain before treatment with self-reprogrammed cells (left scan) and after 3 months (right scan). In the scan indicating the pretreatment, the arrow indicates a lesion in the brain, but in the scan indicating the posttreatment, the arrow indicates an improvement in the lesion. FIG. 5 shows a magnetic resonance imaging (MRI) scan of the brain of a patient with multiple sclerosis before (upper scan) and after 6 months (lower scan) treatment with self-reprogrammed cells. FIG. 6 shows additional MRI scans of the brain of a patient with multiple sclerosis before (upper scan) and after 6 months (lower scan) before treatment with self-reprogrammed cells. In the scan showing before treatment, the arrow indicates a lesion in the brain, but in the scan showing after treatment, the arrow is accompanied by a decrease in brain atrophy, as indicated by a decrease in ventricular and groove enlargement. Shows improvement in lesions. FIG. 2 shows a sagittal section magnetic resonance imaging (MRI) scan of a patient with multiple sclerosis before (left scan) and after 6 months (right scan) treatment with self-reprogrammed cells. FIG. 2 shows a cross-sectional MRI scan of the spinal cord of a patient before treatment with self-reprogrammed cells (left scan) and after 6 months (right scan). In the scan indicating before treatment, the arrow indicates a lesion on the spinal cord, while in the scan indicating after treatment, the arrow indicates improvement of the lesion. FIG. 5 shows a decrease in liver enzyme alanine aminotransferase (ALT) levels in patients infected with hepatitis C after treatment with self-reprogrammed cells. FIG. 5 shows a decrease in aspartate aminotransferase (AST) levels in patients infected with hepatitis C after treatment with self-reprogrammed cells. FIG. 2 shows a magnetic resonance imaging (MRI) scan of the brain of a patient having a head injury due to a car accident. Prior to treatment (upper scan), the ventricles show enlargement and misalignment due to extensive hematoma. After treatment with self-reprogrammed cells (lower scan), the ventricles show a decrease in brain atrophy parameters, such as a decrease in ventricular and sulcus expansion due to improved hematoma. FIG. 6 shows additional MRI scans of the patient's brain before treatment (upper scan) and after (lower scan). FIG. 2 is a chest radiograph of a patient with lung disease before (left x-ray) and after (right x-ray) treatment with self-reprogrammed cells. After treatment, the patient has shown improved lung volume and reduced lesion size as indicated by a decrease in the low-absorption zone. Diagram showing sex hormone levels of follicle stimulating hormone (fsh), luteinizing hormone (lh), progesterone (pro) and testosterone (test) in patients with non-occlusive azoospermia and treated with self-reprogrammed cells It is. Levels show a significant increase in free testosterone with increasing testicular size as measured by ultrasound (data not shown). FIG. 5 shows retinal sensitivity and visual impairment in patients suffering from visual impairment before (upper panel) and after (lower panel) treatment with self-reprogrammed cells. In the retinal sensitivity result, the orange portion indicates a decrease in retinal sensitivity. In the visual impairment results, the white part shows normal vision, and the pink, orange and black parts show increased visual impairment. After treatment, the patient experienced improved vision because the orange part in the pre-treatment retinal sensitivity results became green and white after treatment, and the black part in the pre-treatment visual impairment results turned white after treatment did.

<Definition>
As used herein, a “commit cell” is a cell that exhibits differentiation characteristics. These cells are often considered mature and specialized. Examples include leukocytes, erythrocytes, epithelial cells, neurons and chondrocytes.

  As used herein, “uncommitted cells” are cells that do not exhibit mature differentiation characteristics. These cells are often considered immature and are not specialized. An example of an uncommitted cell is a stem cell that is an immature cell capable of self-renewal (unlimited division) and differentiation (specialization).

  As used herein, “reprogramming” refers to the process by which committed cells of the initial cell lineage are changed to cells of different cell types. This different cell type can be of a different cell lineage. Reprogramming can occur in processes such as reverse differentiation, transdifferentiation, or redifferentiation.

  As used herein, a “reprogrammed cell” is a cell that has undergone reprogramming of committed cells. Reprogrammed cells can include reverse differentiated cells, transdifferentiated cells, and / or redifferentiated cells.

  As used herein, “reverse differentiation” is the process by which committed cells, ie mature specialized cells, return to a more primitive cell stage. A “reversely differentiated cell” is a cell that results from the reverse differentiation of a committed cell.

  As used herein, “transdifferentiation” is the process by which committed cells of the initial cell lineage are changed to other cells of different cell types. In some embodiments, transdifferentiation can be a combination of reverse differentiation and redifferentiation. “Transdifferentiated cells” are cells produced by transdifferentiation of committed cells. For example, committed cells such as whole blood cells can be transdifferentiated into neurons.

  As used herein, “redifferentiation” refers to the process by which uncommitted or reverse differentiated cells differentiate into more mature specialized cells. “Redifferentiated cells” are cells that result from the redifferentiation of uncommitted cells or reverse differentiated cells. When redifferentiated cells are obtained by redifferentiation of reverse differentiated cells, the redifferentiated cells can be of the same or different lineage as the committed cells that have undergone reverse differentiation. For example, committed cells such as leukocytes can be reverse differentiated to give rise to reverse differentiated cells such as pluripotent stem cells, and then the reverse differentiated cells can be redifferentiated to produce lymphoid cells of the same lineage as leukocytes (commit cells). Spheres can be generated or redifferentiated to give rise to neurons of a different lineage than leukocytes (commit cells).

  As used herein, a “target cell” is a cell obtained for administration to a patient to repair or supplement tissue or cells. For example, the target cell may be a reprogrammed target cell, such as a reverse differentiation target cell or a transdifferentiation target cell, whereby the reverse differentiation or transdifferentiation target cell is administered to the patient.

<Commit cell>
As described above, the committed cell of the present invention is a cell exhibiting differentiation characteristics. Commit cells can include components related to antigen presentation, capture or recognition. For example, committed cells can be MHC class I ⁺ and / or MHC class II ⁺ cells.

  The committed cell may be a cell derived from or derived from an undifferentiated cell. Thus, in one embodiment, committed cells are also undifferentiated cells. Thus, by way of example, committed cells can be lymphoid or myeloid stem cells that are differentiated compared to pluripotent stem cells.

  Commit cells are living organisms such as blood or related tissue, including bone marrow or umbilical cord blood, neuronal tissue from the central or peripheral nervous system, muscle tissue, or epidermal and / or dermal tissue of the skin (i.e., by mouth sweeping) May be derived from biological materials. The biological material can be of postnatal origin.

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

  In certain embodiments, committed cells are derived from whole blood or its processed products, such as plasma or buffy coat, because their removal from a subject can be performed with minimal medical supervision. is there. Blood samples are generally treated with an anticoagulant such as heparin or citrate. Cells in a biological sample can be processed to enrich for specific cell types, remove specific cell types, or dissociate cells from a tissue mass. Useful methods for purifying and separating cells include centrifugation (density gradient centrifugation), flow cytometry and affinity chromatography (such as the use or panning of magnetic beads containing monoclonal antibodies to cell surface markers) (Vettese- Dadey, The Scientist, 1999, Vol. 13, p. 21). As an example, Ficoll-Hypaque separation is useful for removing red blood cells and granulocytes to leave mononuclear cells such as lymphocytes and monocytes.

  Examples of committed cells that can be obtained from blood are CFC-T cells, CFC-B cells, CFC-Eosin cells, CFC-Bas cells, CFC-Bas cells, CFC-GM cells, CFC-M, CFC-MEG cells , BFC-E cells, CFC-E cells, T cells, B cells, eosinophils, basophils, neutrophils, monocytes, megakaryocytes and erythrocytes.

Commit cells derived from blood can be identified by their expression of specific antigens. For example, B cells are CD19 ⁺ , CD21 ⁺ , CD22 ⁺ and DR ⁺ cells. T cells are CD2 ⁺ , CD3 ⁺ , and CD4 ⁺ or CD8 ⁺ cells. Immature lymphocytes are CD4 ⁺ and CD8 ⁺ cells. Activated T cells are DR ⁺ cells. Natural killer cells (NK) are CD56 ⁺ and CD16 ⁺ . T lymphocytes are CD7 ⁺ cells. White blood cells are CD45 ⁺ cells. Granulocytes are CD13 ⁺ and CD33 ⁺ cells. Monocyte macrophage cells are CD14 ⁺ and DR ⁺ cells.

  In certain embodiments, committed cells express B lymphocytes (activated or deactivated), T lymphocytes (activated or deactivated), cells of the macrophage monocyte lineage, class I or class II antigens. It can be a competent nucleated cell, a cell that can be induced to express class I or class II antigens, or an enucleated cell (ie, a cell without nuclei—red blood cells, etc.).

  In an alternative embodiment, the committed cells may be selected from any one of the group of cells including large granular lymphocytes, null lymphocytes and natural killer cells, each expressing CD56 and / or CD16 cell surface receptors. it can.

  Since committed cells are essentially primary cultures, it may be necessary to supplement the population of cells with appropriate nutrients to maintain survival. Appropriate culture conditions are known to those skilled in the art. Nonetheless, treatment of the cell population is preferably initiated as soon as possible after removal of the biological sample from the patient, generally within 12 hours, preferably within 2-4 hours. Cell survival can be confirmed using well-known techniques such as trypan blue exclusion or propidium iodide.

<Reverse differentiated cells>
Reverse differentiation is a type of reprogramming process whereby cell structure and function are gradually changed to yield less specialized cells. Reverse differentiation can occur naturally and cells can undergo limited reverse differentiation in vivo in response to tissue damage. Alternatively, reverse differentiation is incorporated herein by reference in its entirety, U.S. Application No. 08 / 594,164, currently U.S. Patent No. 6,090,625; U.S. Application No. 09 / 742,520, currently U.S. Patent No. 7,112,440; Application No. 10 / 140,978, currently U.S. Pat.No. 7,220,412; U.S. Application No. 10 / 150,789, currently U.S. Pat.No. 7,410,773; U.S. Application No. 09 / 853,188. .

  The reverse differentiated cells of the present invention 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, endoderm stem cells, mesenchymal stem cells and embryonic stem cells. Not.

  In certain embodiments, committed cells are blood derived and reverse differentiated to yield reverse differentiated cells of the hematopoietic cell lineage. Examples of these reverse differentiated cells include, but are not limited to, pluripotent stem cells, lymphoid stem cells, and myeloid stem cells.

  Committed cells can be reverse differentiated by contacting the cells with an agent that is operably linked to the cells. The cells are incubated to allow cells operably linked by the agent to evolve through a reverse differentiation process and eventually become undifferentiated.

  The contacting step can include linking the agent to a surface antigen on the committed cell. The agent may act in a direct or indirect connection with committed cells. An example of direct ligation is that the committed cell is at least on its cell surface, such as a β chain, having a homologous region (a region generally found to have the same or similar sequence) as can be found on B cells. In the case of having one cell surface receptor, the agent is directly linked to the cell surface receptor. Another example is when a committed cell has a cell surface receptor on its cell surface, such as an alpha chain with a homologous region that can be found on T cells, and the agent is bound to the cell surface receptor. Connect directly.

  An example of indirect ligation is that a committed cell has at least two cell surface receptors on its cell surface, and the linkage of an agent with one of the receptors affects the other receptor, which in turn commits This is a case of inducing cell reverse differentiation.

  The agent for reverse differentiation of committed cells can be a compound or a composition. For example, the agent may have the ability to bind to cell surface receptors on the surface of committed cells. In certain embodiments, the agent is operably linked to a receptor present on the surface of the commit cell, and the receptor, such as a receptor that can be expressed by the commit cell, is expressed by the commit cell. Can be expressed.

  For example, the agent may be one or more cyclic adenosine monophosphate (cAMP), CD4 molecule, CD8 molecule, part or all of a T cell receptor, ligand (fixed or free), peptide, T cell receptor (TCR), antibodies, cross-reactive antibodies, monoclonal antibodies or polyclonal antibodies may be included, but are not limited to these. Hematopoietic growth factors such as growth factors such as erythropoietin and granulocyte monocyte colony stimulating factor (GM-CSF) can also be used.

  When the agent is an antibody, a cross-reactive antibody, a monoclonal antibody or a polyclonal antibody, the agent is an antibody against any one or more of the following, a cross-reactive antibody, a monoclonal antibody or a polyclonal antibody May be one or more of: MHC class II antigen beta chain, MHC HLA-DR antigen beta chain, MHC class I or class II antigen alpha chain, HLA-DR antigen alpha chain, MHC class II antigen Or α and β chains of MHC class I antigens. An example of an antibody is CR3 / 43 (supplied by Dako).

The term `` antibody '' refers to various fragments (whether obtained by proteolytic cleavage or recombinant techniques) and derivatives that retain binding activity, such as Fab, F (ab ′) ₂ and scFv antibodies, As well as mimetics or biological equivalents thereof. In addition, what is included as an antibody is that the part of the amino acid sequence is modified by, for example, substitution of an amino acid residue to enhance binding, or the cell is used to reduce the possibility of an adverse immune reaction. Antibodies to organisms desired to be treated by the inventive method are prepared in different species (an example of this is a “humanized mouse monoclonal antibody”), a genetically modified variant.

  The agent used to effect the conversion of committed cells into reverse differentiated cells can preferably act outside the committed cells. For example, a commit cell can include a receptor that can be operably linked by an agent, and the agent is operably linked to the receptor.

  For example, the receptor can be a cell surface receptor. Specific examples of cell surface receptors include, but are not limited to, MHC class I and class II receptors. The receptor may comprise an alpha component and / or a beta component, as in the case of MHC class I and class II receptors.

  The receptor may comprise a β chain having a homologous region, for example, a homologous region of at least the HLA-DR β chain.

  Alternatively, or in addition, the receptor may comprise an α chain having a homologous region, eg, at least a homologous region of the α chain of HLA-DR. The receptor may be a major histocompatibility complex (MHC) class I or class II antigen. In certain embodiments, the cell surface receptor is HLA-DR receptor, DM receptor, DP receptor, DQ receptor, HLA-A receptor, HLA-B receptor, HLA-C receptor, HLA- It may include, but is not limited to, E receptor, HLA-F receptor or HLA-G receptor. In some embodiments, the cell surface receptor can be an HLA-DR receptor.

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

Examples of agents may be those that modulate MHC gene expression, such as MHC class I ⁺ and / or MHC class II ⁺ expression.

  In certain embodiments, the agent can be used with a biological response modifier. Examples of biological response modifiers include, but are not limited to, alkylating agents, immunomodulators, growth factors, cytokines, cell surface receptors, hormones, nucleic acids, nucleotide sequences, antigens or peptides. For example, the alkylating agent may be or include cyclophosphamide.

  Other biological response modifiers can upregulate the expression of MHC class I and / or MHC class II antigens, and in some embodiments, agents that bind to MHC receptors act more efficiently. It may contain compounds that can make this possible.

  Since any cell type can be made to express MHC class I and / or MHC class II antigens, this is whether they constitutively express MHC class I and / or MHC class II antigens. Regardless, it can provide a method of dedifferentiating various cell types.

  Commit cells are typically incubated with the agent for at least 2 hours, generally 2 to 24 hours, preferably 2 to 12 hours. Incubations are generally performed at about room temperature or a temperature from about 22 ° C. to 33 ° C. to about 37 ° C. The progress of the reverse differentiation treatment can be confirmed periodically by removing an aliquot of the sample and examining the cells using a microscope and / or flow cytometry. Alternatively, the device may include a tracking means that monitors the progress of the reverse differentiation procedure online.

In addition to the use of reverse differentiation agents, committed cells can be cultured in autologous plasma or serum, or in fetal serum or horse serum. In some cases, committed cells can be cultured with anticoagulants, chelators or antibiotics. The temperature range at which the cells are incubated can be expanded to 18-40 ° C. and can also include 4-10% CO ₂ and / or 10-35% O ₂ . Furthermore, the incubation can be carried out in a coated or uncoated blood bag, tissue culture bag or plastic container.

  Certain types of reverse differentiated cells can be obtained by reverse differentiation using specific culture conditions. For example, by culturing committed cells with a reverse differentiation agent in Dulbecco's modified Eagle's medium (DMEM), non-essential amino acids (NEAA), L-glutamine (L-glu) and β-mercaptoethanol (2βME) It can be differentiated into pluripotent cells. Committed cells can also be first exposed to a chelator.

  As another example, to obtain mesenchymal cells, commit cells are cultured with reverse differentiation agents using DMEM (low glucose) and L-glu or DMEM (low glucose), L-glu, 2βME and NEAA. Can do. In addition, the antibiotic gentamicin can be used for cell culture.

<Transformed cells>
Transdifferentiated cells can be obtained by culturing committed cells together with a reverse differentiation agent using a tissue culture medium. Commit cells are thereby transdifferentiated, the committed cells are converted to cells of other cell types, and in some embodiments the committed cells are converted to cells of different lineages.

  The type of target cell obtained by transdifferentiation depends on the culture conditions. These conditions include the type of tissue culture medium, presence / absence of various differentiation promoters, presence / absence of various sera, incubation temperature, presence / absence of oxygen or carbon dioxide, and type of container or vessel used for incubation. It depends on.

  Examples of tissue culture media used for transdifferentiation include Iskov modified Dulbecco medium (IMDM), Dulbecco modified Eagle medium (DMEM), Eagle minimum essential (EME) medium, alpha-minimal essential medium (α-MEM), Roswell Park Memorial Institute (RPMI; where the medium was developed) 1640, Ham-F-12, E199, MCDB, Leibovitz L-15, William E medium or commercially formulated tissue culture medium, including but not limited to.

Differentiation promoters include anticoagulants, chelating agents and antibiotics. Examples of such agents may be one or more of the following: A (retinol), B ₃ , C (ascorbic acid), ascorbic acid 2-phosphate, D ₂ , D ₃ , K, Vitamins and minerals such as retinoic acid, nicotinamide, zinc or zinc compounds, and calcium or calcium compounds or derivatives thereof; natural or synthetic hormones such as hydrocortisone and dexamethasone; L-glutamine (L-glu), ethylene glycol tetraacetic acid ( EGTA), amino acids such as proline and non-essential amino acids (NEAA) or derivatives thereof; β-mercaptoethanol, dibutyl cyclic adenosine monophosphate (db-cAMP), monothioglycerol (MTG), putrescine, dimethyl sulfoxide (DMSO) , Hypoxanthine, adenine, forskolin, cilostamide and 3-isobutyl-1-methylxanthine Derivatives; nucleosides such as 5-azacytidine and analogs thereof; ascorbic acid, pyruvate, okadaic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose citrate formulation A (ACDA), disodium EDTA, sodium butyrate and Acids or salts thereof such as glycerophosphate; antibiotics or drugs such as G418, gentamicin, pentoxyphyllin (1- (5-oxohexyl) -3,7-dimethylxanthine) and indomethacin; and tissue plasminogen activator Proteins such as (TPA).

These differentiation promoting agents can be used to obtain specific types of target cells. For example, vitamin B ₃ may be used to obtain the acinar cells, or hydrocortisone, such as islet cells, dexamethasone, mesenchymal origin or epithelial origin (e.g., renal epithelial cells, associated structures such as skin and dermal papilla cells ) And β-mercaptoethanol can be used to obtain ectoderm cells such as neuronal cells, including CNS accessory cells.

  The culture medium may contain autologous plasma; platelets; serum such as fetal blood draw; or serum of mammalian origin such as horse serum. Furthermore, the conversion process can occur in blood bags, scaffolds, tissue culture bags or plastic tissue culture containers. The tissue culture vessel may be an adherent or non-adherent tissue culture vessel, or gelatin, collagen, matrigel or extracellular matrix that promotes attachment or levitation depending on the required type of tissue or specialized cells to be prepared. Or may be uncoated.

Further culture conditions are temperatures that can be about 10 to about 60 ° C. or about 18 to about 40 ° C .; levels of carbon dioxide (CO ₂ ) that can be about 0 to about 20%, or about 4 to about 10%; and Oxygen (O ₂ ), which can be about 0 to about 50%, or about 10 to about 35%.

  Examples of methods used to obtain target cells and used in conjunction with reverse differentiation agents are shown in Table 1.

  In particular, with regard to the culture conditions described for each cell type in Table 1, further transdifferentiation results from cessation of use of the reverse differentiation agent by subsequent dilution of the culture conditions in the corresponding culture medium.

  During the culture, the culture medium optionally containing various differentiation promoting agents can be diluted by adding more medium without the reverse differentiation agent. Without being bound by theory, it is likely that dilution will increase differentiation because cells will be less dense and growth stimulators will be less concentrated. Thus, the addition of media may further promote transdifferentiation, affecting what type of cells are obtained within the cell lineage. For example, if the target cell is a neuron, the addition of culture medium results in a developmental change to more mature neurons (both in the same lineage) rather than neuronal progenitor cells. As another example, anterior differentiated skeletal muscle progenitor cells differentiate only by serial dilution of the culture medium achieved by gradually decreasing the serum concentration.

<Redifferentiated cells>
Reverse differentiated cells can be used to obtain target cells by recommitting or redifferentiating reverse differentiated cells into a target cell type. This can be done by contacting reverse differentiated cells with growth factors. For example, retinoic acid has been used to differentiate stem cells into neurons. Co-culture of methylcellulose with subsequent bone marrow stromal system and IL-7 has been used to differentiate stem cells into lymphocyte progenitors (Nisitani et al., Int Immuno, 1994, 6, 909-916) . Le Page (New Scientist, December 16, 2000) teaches that stem cells can be differentiated into lung epithelial cells. Bischoff (Dev Biol, 1986, 115, 129-39) teaches how muscle satellite cells are differentiated into mature muscle fibers. Neural progenitor cells can be expanded using basic fibroblast growth factor and epidermal growth factor (Nakafuku and Nakamura, J Neurosci Res, 1995, 41, 153-168). Hematopoietic stem cells can be expanded using a number of growth factors including GM-CSF, erythropoietin, stem cell factor and interleukins (IL-1, IL-3, IL-6). See Metcalf (Nature, 1989, 339, 27-30) for a review of these various factors.

  Potocnik et al. (EMBO J, 1994, 13, 5274-83) even showed the differentiation of stem cells into hematopoietic cells using hypoxia (5%) conditions.

The redifferentiated cell can be of the same lineage as the committed cell from which the reverse differentiated cell was obtained. Alternatively, the redifferentiated cell may be of a different lineage from the committed cell from which the reverse differentiated cell was obtained. For example, B lymphocytes can be reverse differentiated into CD34 ⁺ CD38 ⁻ HLA-DR ⁻ stem cells. The stem cells can then be redifferentiated or recommitted along the B cell lineage (same lineage) or lymphocyte lineage (different lineage).

<Target cell>
The target cell of the present invention is a reprogrammed cell that can be obtained by reverse differentiation, transdifferentiation, or redifferentiation as described above. According to the present invention, the target cells are pluripotent stem cells, lymphoid stem cells, myeloid stem cells, neural stem cells, skeletal muscle satellite cells, epithelial stem cells, endoderm and neuroectodermal stem cells, germ cells, non-embryonic and embryos Sex stem cells, mesenchymal stem cells, kidney cells, alveolar epithelial cells, endoderm cells, neurons, ectoderm cells, islet cells, acinar cells, oocytes, sperm, hematopoietic cells, hepatocytes, skin / keratinocytes, melanocytes, May include bone / bone cells, hair / dermal papilla cells, cartilage / chondrocytes, fat cells / adipocytes, skeletal muscle cells, endothelial cells, cardiomyocytes / cardiomyocytes and tropoblasts However, it is not limited to these.

  As described above, committed cells and / or reverse differentiated cells are cultured under specific conditions to induce reverse differentiation and / or transdifferentiation and / or redifferentiation to obtain target cells. The period of culturing committed cells and / or reversely differentiated cells is adjusted by determining that the target cells have been generated without adjusting by a specific time.

Determining the generation or number change of reverse differentiation, transdifferentiation or redifferentiation target cells can be done by changing the relative number of committed cells that down-regulate the expression of lineage-related markers or transcription factors, and / or the cell surface specific to the target cell This can be done by monitoring the change in the relative number of cells with the marker. Alternatively, or in addition, a decrease in the number of cells with cell surface markers characteristic of committed cells (not target cells) can be monitored. For example, target cells include many stage-specific markers such as POU5F1 (OCT-4), TERT, KLF4, UTF1, SOX2, Nanog or stage-specific embryonic markers 3 and 4 (SSEA-3 and SSEA-4), May be embryonic stem cells characterized by high molecular weight glycoproteins TRA-1-60 and TRA-1-81 and alkaline phosphatase (Andrews et al., Hybridoma, 1984, 3, 347-361; Kannagi et al., EMBO J 1983, 2, 2355-2361; Fox et al., Dev Biol, 1984, 103, 263-266; Ozawa et al., Cell Differ, 1985, 16, 169-173). They also do not express SSEA-1, whose presence is an indicator of differentiation. Other markers such as Nesteine of neuroepithelial stem cells are known for other types of stem cells (J Neurosci, 985, 3310). Mesenchymal stem cells are, for example, positive for SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a and CD124 and negative for CD34, CD45 and CD14. Pluripotent stem cells are CD34 ⁺ DR ⁻ TdT ⁻ cells (other useful markers are CD38 ⁻ and CD36 ⁺ ). Lymphoid stem cells are DR ⁺ , CD34 ⁺ and TdT ⁺ cells (also CD38 ⁺ ). Myeloid stem cells are CD34 ⁺ , DR ⁺ , CD13 ⁺ , CD33 ⁺ , CD7 ⁺ and TdT ⁺ cells.

  Additional cellular markers for target cells can be discovered by microarray analysis. The analysis involves isolating RNA from reverse differentiated and / or transdifferentiated and / or redifferentiated target cells, labeling the isolated RNA with a dye, and hybridizing the isolated RNA to a microarray. Including making soybeans. The microarray can include genes or oligonucleotides that represent the entire genome, or can include genes or oligonucleotides that are specific for a particular organ system, tissue system, disease, condition, etc. Cell markers can be identified in which genes / oligonucleotides exhibit high signal intensity and thereby are up-regulated or down-regulated in target cells. This information can be applied to determine target cells based on the presence of markers identified by microarray analysis, or marker groups or marker patterns.

  Target cell confirmation can also be performed using a number of in vitro assays, such as the CFC assay (see also the Examples). Very primitive hematopoietic stem cells are often measured using the long-term culture initiating cell (LTC-IC) assay (Eaves et al., J Tiss Cult Meth, 1991, 13, 55-62). LTC-IC maintains hematopoiesis for 5-12 weeks.

  For other cell types such as cells of the central nervous system, pancreas, liver, kidney, skin etc., immunohistochemistry, flow cytometry, microarray or reverse transcription polymerase chain reaction (RT Cell culture can be continued until target cells as characterized by -PCR) appear. This can also include functional assays such as transplantation into an immunodeficient host or correction or improvement of the underlying clinical condition as observed herein.

  Target cells can be identified by microarray or RT-PCR showing the acquisition of new lineage specific transcription factors, proteins and signals in the target cells. For example, reverse-differentiated stem cells that have been transformed into target cells of the ectoderm lineage may express genes such as Nestin, Criptol, isI1, LHX1 and / or EN1, and when further differentiated into neurons, neurofilaments (NF ) Is expressed. On the other hand, cells that have been transformed into target cells of the endoderm lineage may express genes such as sox7, sox17, Nodal, PDX1 and / or FOXA2, but target cells that have further differentiated into islet cells are insulin Genes such as (INS) and neurog3 (NGN3) can be expressed. In particular, conversion to the desired target cell may involve down-regulation of mature transcription factors associated with the initial starting population that has undergone conversion.

  Furthermore, confirmation of target cell production can be found by recognizing specific structural and / or morphological characteristics of the target cell, such as cell shape, size, and the like. These properties are known in the art for the target cells of the present invention.

  Once the relative number of desired cell types has increased to an appropriate level, which can be, for example, as low as 0.1% or as high as 5%, the resulting modified cell population can be used in a number of ways. It is important to recognize the proliferative capacity of stem cells with respect to the number of target cells generated, eg, pluripotent stem cells. Under certain circumstances, it may appear that the number of stem cells or other reverse differentiated cells generated is low, but only 50 pluripotent hematopoietic stem cells can reconstitute the entire hematopoietic system in donor mice It was shown in the test. Thus, therapeutic utility does not require the formation of a large number of cells.

  Reverse differentiation, transdifferentiation, or redifferentiation target cell transformation of committed cells can also be performed in vivo by administering an agent mixed with a pharmaceutically acceptable carrier or diluent to a patient. However, in many cases, it is preferable to perform reverse differentiation, transdifferentiation, or redifferentiation in vitro / ex vivo.

  The treated population of cells obtained in vitro can then be used with minimal treatment. For example, they can simply be mixed 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 cell population of reverse differentiation, transdifferentiation or redifferentiation target cells or to purify cells from the cell population. This can conveniently be done using a number of methods (see Vettese-Dadey--The Scientist, 1999, volume 13). For example, cells can be purified based on cell surface markers using chromatography and / or flow cytometry. Nonetheless, other cells (e.g., stromal cells) that are present in the population may maintain stem cell survival and function, thus broadening the scope of reverse differentiation, transdifferentiation, or redifferentiation target cells from the cell population. Purification is often not necessary or desirable.

  Flow cytometry is a well-established, reliable and powerful technique for characterizing cells within a mixed population as well as sorting cells. Thus, the purification or isolation means can include a flow cytometer. Flow cytometry operates on the physical properties of particles in suspension that can be identified when interrogated with light. Such particles can of course be cells. Physical properties include cell size and structure or cell surface markers bound by monoclonal antibodies conjugated to fluorescent molecules that have become very common in recent years.

Kreisseg et al. (J Hematother, 1994, Volume 3, pp. 263-89) stated that “the availability of anti-CD34 monoclonal antibodies makes multi-parameter flow cytometry an optimal tool for measuring hematopoietic stem and progenitor cells. " Kreisseg further describes a general approach for quantification and characterization of CD34 expressing cells by flow cytometry. In addition, Korbling et al. (Bone Marrow Transplant, 1994, 13, 649-54) teaches the purification of CD34 ⁺ cells by flow cytometry based on immunoadsorption followed by HLA-DR expression. As mentioned above, CD34 ⁺ is a useful marker associated with stem / progenitor cells. Flow cytometry techniques for sorting stem cells based on other physical characteristics can also be used. For example, Visser et al. (Blood Cells, 1980, 6, 391-407) teach that stem cells can be isolated based on their size and degree of structuredness. Grogan et al. (Blood Cells, 1980, 6, 625-44) also teaches that “viable stem cells can be selected from simple hematopoietic tissues with high and verifiable purity”.

  Similar to selecting cells based on the presence of cell surface markers or other physical properties (positive selection), cell populations can be enriched and purified using negative criteria. For example, cells with lineage specific markers such as CD4, CD8, CD42 and CD3 can be removed from the cell population by flow cytometry or affinity chromatography.

  A very useful technique for purifying cells involves the use of antibodies or other affinity ligands conjugated to magnetic beads. The beads are incubated with the cell population to capture cells with cell surface markers such as CD34 to which the affinity ligand binds. The sample tube containing the cells is placed in a magnetic sample concentrator where the beads are attracted to the side of the tube. After one or more washing steps, the cells of interest are partially or substantially completely purified from other cells. When used in a negative selection mode, instead of washing the cells bound to the beads by discarding the liquid phase, the liquid phase is retained and then the cells bound to the beads are efficiently removed from the cell population.

  These affinity ligand-based purification methods can be used for cell types where an appropriate marker has been characterized or can be characterized. Urbankova et al. (J Chromatogr B Biomed Appl, 1996, 687, 449-52) teaches a micro-preparation of hematopoietic stem cells from mouse bone marrow suspension by gravity field flow fractionation. Urbankova et al. Further commented that this method was used to characterize stem cells from mouse bone marrow because stem cells are larger than other cells in the bone marrow and therefore can be separated from the mixture. Yes. Thus, physical parameters other than cell surface markers can be used to purify / enrich stem cells.

  A cell population comprising a reprogrammed target cell such as a reverse differentiation, transdifferentiation or redifferentiation target cell and / or a purified reprogrammed target cell such as a reverse differentiation, transdifferentiation or redifferentiation target cell generated by the method of the present invention, Can be maintained in vitro using known techniques. Generally, minimal such as Hanks, RPMI 1640, Dulbecco's minimal essential medium (DMEM) or Iskov modified Dulbecco medium supplemented with mammalian serum, such as FBS, and optionally autologous plasma to give the cells a suitable growth environment Use growth medium. Stem cells can be cultured on a feeder layer, such as a layer of stromal cells (see Deryugina et al., Crit Rev Immunology, 1993, 13, 115-150). Stromal cells are thought to secrete factors that maintain progenitor cells in an undifferentiated state. Long-term culture systems for stem cells are described by Dexter et al. (J Cell Physiol, 1977, 91, 335) and Dexter et al. (Acta Haematol, 1979, 62, 299).

For example, Lebkowski et al. (Transplantation, 1992, 53, 1011-9) purify human CD34 ⁺ hematopoietic cells using a technique based on the use of monoclonal antibodies covalently immobilized on polystyrene surfaces. And that CD34 ⁺ cells purified by this method can be maintained with a viability of greater than 85%. Lebkowski et al. (J Hematother, 1993, 2, 339-42) also teach how to isolate and culture human CD34 ⁺ cells. See also Haylock et al. (Immunomethods, 1994, Volume 5, pp. 217-25) for a review of the various methods.

  Cell populations containing stem cells and purified preparations containing stem cells can be frozen / preserved for future use. Suitable techniques for freezing cells and then restoring them are known in the art.

  In one embodiment, reverse differentiation, transdifferentiation or redifferentiation occurs in cells from or in a buffy coat blood sample. The term “buffy coat” refers to a layer of white blood cells that forms between a layer of red blood cells and a layer of plasma when uncoagulated blood is centrifuged or left to stand.

<Method of treatment>
The reprogrammed target cells of the present invention, such as reverse differentiation target cells, transdifferentiation target cells, or redifferentiation target cells, can be mixed with various components to produce the compositions of the present invention. The composition can be mixed with one or more pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition (which can be for use in humans or animals). Suitable carriers and diluents include, but are not limited to, isotonic saline solutions, such as phosphate buffered saline. The composition of the present invention can be administered by direct injection. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, transdermal administration, or intrathecal injection.

  A composition comprising target cells can be delivered by injection or transplantation. Cells can be delivered in suspension or embedded in a support matrix such as a natural and / or synthetic biodegradable matrix. Natural matrices include, but are not limited to, collagen matrices. Synthetic biodegradable matrices include, but are not limited to, polyanhydrides and polylactic acid. These matrices can provide support for fragile cells in vivo.

  The composition may also comprise a reverse differentiation, transdifferentiation or redifferentiation target cell of the invention and at least one pharmaceutically acceptable excipient, carrier or vehicle.

  Delivery may be by controlled delivery, ie, delivered over a period of time that may be from minutes to hours or days. Delivery may be systemic (eg, by intravenous injection) or directed to a specific site in the subject. Cells can be introduced in vivo using liposome delivery.

Target cells can be administered at a dose of 1 × 10 ^{5 to} 1 × 10 ⁷ cells per kg. For example, a 70 kg patient can be administered 14 × 10 ⁶ CD34 ⁺ cells for tissue reconstitution. The dose can be any combination of target cells as indicated herein.

  The methods of the invention can be used to treat various diseases, conditions or disorders. Such conditions include bone marrow failure, hematological disease, aplastic anemia, beta Mediterranean anemia, diabetes, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney disease, liver disease, multiple sclerosis, congestive disease Heart failure, hepatitis C virus, human immunodeficiency virus, head injury, lung disease, depression, non-obstructive azoospermia, menopause, menopause and infertility, rejuvenation, scleroderma ulcer, psoriasis, wrinkles, cirrhosis, Including but not limited to disorders associated with autoimmune diseases, hair loss, retinitis pigmentosa, crystalline dystrophy / blindness or tissue degeneration.

  Aplastic anemia is a rare but fatal bone marrow disorder characterized by pancytopenia and myelocytopenia (Young et al., Blood, 2006, 108, 2509-2519). This disorder can be caused by immune-mediated pathophysiology by activated type I cytotoxic T cells that express Th1 cytokines, particularly γ-interferons that target hematopoietic stem cell compartments, leading to bone marrow failure and thus hematopoietic dysfunction (Bacigalupo et al., Hematology, 2007, 23-28). Extending this approach to older patients or patients lacking family donors remains a challenge, but the majority of aplastic anemia patients can be treated by transplanting stem cells from HLA-matched siblings (Locasciulli Et al., Haematologica, 2007, 92, 11-18). Despite reasonable survival after HLA-matched allogeneic stem cell transplantation, this treatment has several potential risks due to immunosuppressive therapy used to prevent graft-versus-host disease (GVDH). For example, high-dose cyclophosphamide with or without anti-thymocyte globulin (ATG) provides long-term immunosuppression and increases the likelihood of opportunistic infections in patients. Another potential risk is transplant failure that can last weeks or months after stem cell transplant (Gottdiener et al., Arch Intern Med, 1981, 141, 758-763; Sanders et al., Semin Hematol, 1991 Year, 28, 244-249). Furthermore, the risk of transplant failure increases with the number of transfusions received prior to stem cell transplant.

  Mediterranean anemia is an inherited autosomal recessive blood disease characterized by a reduced synthesis rate of one of the globulin chains that make up hemoglobin. Thus, there is a lack of production of normal globulin proteins often resulting from regulatory gene mutations, which results in the formation of abnormal hemoglobin molecules and causes anemia. Different types of Mediterranean anemia include alpha Mediterranean anemia, beta Mediterranean anemia and Delta Mediterranean anemia, which affect the production of alpha globulin, beta globulin and delta globulin, respectively. Treatment methods include chronic blood transfusion, iron chelation, splenectomy, and allogeneic hematopoietic cell transplantation. However, chronic blood transfusions are not available to most patients due to the lack of HLA-compatible bone marrow donors, while allogeneic hematopoietic cell transplantation is associated with many potential complications such as infection and graft-versus-host disease .

  Diabetes is a syndrome that results in abnormally high blood sugar levels (hyperglycemia). Diabetes refers to a group of diseases that result in hyperglycemia resulting from a deficiency of insulin secretion or insulin action in the body. Diabetes is generally divided into two types, type 1 diabetes characterized by a decrease in the production of insulin or type 2 diabetes characterized by resistance to the action of insulin. Both types of symptoms commonly associated with diabetes, such as excessive urine production, resulting compensatory thirst and increased fluid intake, visual impairment, unexplained weight loss, lethargy and energy metabolism It leads to hyperglycemia that mainly causes change. Diabetes is considered a chronic disease without therapy. Treatment options include insulin injection, exercise, proper diet, or for some patients with type 2 diabetes, some medications, such as promoting insulin secretion by the pancreas, reducing glucose produced by the liver, insulin It is limited to drug therapy such as increasing the sensitivity of cells to

  Motor neuron disease refers to a group of neurological disorders that affect motor neurons. Such diseases include amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS) and progressive myopathy (PMA). ALS is characterized by degeneration of both upper and lower motor neurons, which stops transmission to the muscle, resulting in their weakness and eventual atrophy. PLS is a rare motor neuron disease involving only upper motor neurons that causes difficulty in balancing, leg weakness and stiffness, spasm, and speech disturbance. PMA is a subtype of ALS that affects only lower motor neurons that can cause muscle atrophy, fiber bundle contraction and weakness. There is no known therapy for motor neuron disease. Riluzole has been approved as a drug for ALS, which is thought to slow progression rather than ameliorate the effects of ALS but reduce motor neuron damage. For PLS, only therapies such as baclofen, which can reduce spasm, or quinine, which can reduce spasm, can address the symptoms.

  Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of nigrostriatal tract due to degeneration of dopaminergic neurons within the substantia nigra. The cause of PD is unknown but is associated with progressive death of dopaminergic (tyrosine hydroxylase (TH) positive) midbrain neurons, including movement disorders. Thus, PD is characterized by muscle stiffness, tremors, slow movements and potentially immobility. Therefore, there is currently no sufficient therapy for Parkinson's disease or treatment to prevent or treat Parkinson's disease or its symptoms. Symptomatic treatment of disease-related movement disorders includes oral administration of dihydroxyphenylalanine (L-DOPA), which can result in substantial improvement in motor function, but the effect diminishes as degeneration of dopaminergic neurons progresses. An alternative strategy is to introduce neurotransplantation based on the idea that dopamine supplied from cells transplanted into the striatum can replace nigrostriatal cells that have been lost, and an enzyme responsible for L-DOPA Or by dopamine synthesis, such as by introducing potential neuroprotective molecules that may prevent the death of TH-positive neurons or stimulate regeneration and functional recovery in the damaged nigrostriatal system. Includes gene therapy that can be used to replace dopamine in the striatum.

  Spinal cord injury is characterized by damage to the spinal cord and in particular nerve fibers resulting in damage to some or all muscles or nerves below the site of injury. Such damage may occur due to traumatic injury of the spine that fractures, dislocations, fractures, or compresses one or more vertebrae, or due to atraumatic damage caused by arthritis, cancer, inflammation or disc degeneration. Therapies after spinal cord injury include drug therapy such as methylprednisolone, a corticosteroid that reduces nerve cell damage and reduces inflammation at the site of injury, or drug therapy that controls pain and muscle spasm, and spinal immobilization or Although surgery may be included to remove prolapsed discs or objects that may damage the spine, there are no known means to reverse spinal cord injury.

  Muscular dystrophy (MD) is a series of hereditary muscle diseases that weaken skeletal muscle. MD may be characterized by progressive muscle weakness, muscle protein deficiency, myocyte apoptosis and tissue atrophy. Nine diseases in particular, Duchen, Becker, Limb, Congenital, Facial Scapulohumeral, Myotonic, Oropharyngeal, Distal, and Emerydrefus are classified as MD, There are more than 100 diseases that exhibit MD characteristics. There is no known therapy for MD and no specific therapy. Physical therapy can maintain muscle tone, and surgery can be used to improve quality of life. Furthermore, symptoms such as muscle tonicity can be treated with drugs, but there is no long-term therapy.

  Kidney disease damages the kidneys and reduces the ability of the kidneys to function, including removal of waste and excess water from the blood, regulation of electrolytes, blood pressure, acid-base balance, and glucose and amino acid reabsorption Refers to the state. Other causes include glomerulonephritis, lupus and malformations and obstructions in the kidney, but the two main causes of kidney disease are diabetes and hypertension. There is no therapy for kidney disease, and thus therapy slows disease progression and treats the cause of the disease, such as by controlling blood glucose and hypertension, monitoring the diet, etc .; for example, fluid retention, anemia Focus on addressing bone disease, treating complications of the disease; compensating for lost renal function, such as by dialysis or transplantation.

  Multiple sclerosis is an autoimmune condition in which the immune system attacks the central nervous system, resulting in demyelination. MS compromises the ability of nerve cells in the brain and spinal cord to communicate with each other because the body's own immune system attacks and damages the myelin sheath that envelops the neuronal axons. When myelin is lost, axons can no longer transmit signals effectively. This can lead to various neurological symptoms that usually progress to physical and cognitive impairment. There are no known therapies for MS, and treatment attempts to restore function after a seizure (sudden onset or worsening of MS symptoms), prevent new seizures, and prevent disability. For example, treatment with corticosteroids may help to end a seizure, but treatment with interferon during the first seizure has been shown to reduce the likelihood of developing clinical MS.

Human immunodeficiency virus (HIV) is a lentivirus that can lead to acquired immune deficiency syndrome (AIDS), a condition in which the immune system begins to fail. HIV primarily infects vital cells in the human immune system such as T helper cells, macrophages and dendritic cells. HIV infection is caused by direct viral killing of infected cells, by increasing the rate of apoptosis of infected cells, or by killing infected CD4 ⁺ T cells by CD8 cytotoxic lymphocytes that recognize infected cells. ⁺ Results in low levels of T cells. Currently there are no vaccines or therapies for HIV or AIDS. Treatment of HIV infection consists of highly active antiretroviral therapy, ie HAART. The current HAART option is a combination (or “cocktail”) consisting of at least three anti-retroviral drugs. In general, these classes are two nucleoside analog reverse transcriptase inhibitors (NARTI or NRTI) plus protease inhibitors or non-nucleoside reverse transcriptase inhibitors (NNRTI).

  Congestive heart failure refers to a condition in which the heart cannot pump enough blood to other organs of the body. This condition can result from coronary artery disease, scar tissue on the heart caused by myocardial infarction, hypertension, valvular heart disease, heart defects and heart valve infection. Treatment programs generally consist of drugs such as rest, proper diet, changes in daily activities, and angiotensin converting enzyme (ACE) inhibitors, beta blockers, digitalis, diuretics, vasodilators. However, treatment programs do not reverse heart damage or conditions.

  Hepatitis C is an infection in the liver caused by the hepatitis C virus. Hepatitis C can progress to scarring (fibrosis) and severe scarring (cirrhosis). Cirrhosis can lead to other complications such as liver failure and liver cancer. Current therapies include the use of a combination of pegylated interferon alpha and the antiviral drug ribavirin. The success rate can vary from 50-80% depending on the genotype of the virus.

  Head trauma refers to head trauma that may or may not cause brain damage. Common causes of head injuries include traffic accidents, household and work accidents, falls and attacks. Skull fractures, scalp tears, subdural hematoma (bleeding under the dura), epidural hematoma (bleeding between the dura and skull), brain contusion (brain of the brain), concussion (function of trauma) Various types of problems such as temporary loss), coma or death can be caused by head trauma. Treatment of head trauma varies with the type of trauma. If the brain is damaged, there is no immediate means of repairing it, and often the damage can be irreversible with available treatment means.

  Lung disease is a broad term for diseases of the respiratory system, including the lungs, thoracic cavity, bronchi, trachea, upper respiratory tract and nerves and muscles for breathing. Examples of pulmonary disease include obstructive pulmonary disease with narrowed bronchi; restrictive or fibrotic lung disease that causes loss of compliance, incomplete lung dilation and increased lung stiffness; common cold or pneumonia Respiratory tract infections such as those caused by cancer; chest cavity diseases; and pulmonary vascular diseases that affect the pulmonary circulation. Treatment of pulmonary disease depends on the type of disease, but can include medications such as corticosteroids and antibiotics, oxygen, mechanical ventilation, radiation therapy and surgery.

  Depression is a mental disorder characterized by a low pride and a low mood state with a loss of interest and joy in normal enjoyable activities. Biologically, depression is the raphe nucleus, a group of small nuclei in the upper brainstem that is the source of serotonin; the supraoptic nucleus that controls biological rhythms such as sleep / wake cycles; against various stressors Hypothalamic-pituitary-adrenal axis, a chain of structures activated during the body's reaction; ventral tegment area considered to be involved in the brain's "reward" circuit; reward, laughter, joy, jealousy and fear Accompanied by changes in activity in multiple parts of the brain, including the nucleus accumbens, which is thought to play a role; and the anterior cingulate cortex activated by negative experience. Depression therapy is an antidepressant, exercise and psychotherapy that increases the amount of extracellular serotonin in the brain. However, the effectiveness of these therapies continues to be questioned.

  Non-occlusive azoospermia is a morbidity in men that does not have measurable levels of sperm in semen due to problems associated with spermatogenesis. This is often caused by hormonal imbalances and can be treated with drugs that restore the imbalances.

  Male menopause is a menopause-like condition experienced in middle-aged men associated with decreased production of testosterone and dehydroepiandrosterone hormones. Therapies include hormone replacement therapy and exercise.

  Scleroderma is a chronic autoimmune disease that affects connective tissue. Although skin hardening can affect connective tissue throughout the body, it is the most obvious sign of the disease. There is no known therapy for scleroderma.

  Psoriasis is a chronic autoimmune disease that causes red desquamation spots that appear on the skin. The cause is psoriasis and is associated with excessive proliferation of skin cells. One hypothesis suggests that it is associated with T cells that migrate to the dermis and trigger the release of cytokines that induce rapid production of skin cells. Psoriasis therapy includes drugs that target T cells.

  Retinitis pigmentosa is a type of progressive retinal dystrophy in which photoreceptors or retinal pigment epithelium are abnormal and result in blindness. There are limited therapies for treating retinitis pigmentosa.

  The conditions described herein can be treated using a specific type or combination of types of target cells. In a preferred embodiment, the diseases described herein can be treated by infusion of cell types as outlined in Table 2.

As an example, a patient can be treated for a condition as described above by the following steps.
1) insert a fistula cannula into the patient's arm;
2) Collecting white blood cells by apheresis using an automated system such as COBE® Spectra Device (Gambro PCT);
3) generating autologous reverse differentiated stem cells from the patient's leukocytes;
4) Wash autologous reverse differentiated stem cells and then infuse intravenously into the patient;
5) Monitor the patient's progress, including performing blood tests and assessing the site of injury.

  The present invention will be further illustrated by the following non-limiting examples that further illustrate the invention and are not intended to limit or limit the scope of the invention.

<Example>
<Example 1>
<Materials and methods>
This clinical trial evaluated the safety of a single infusion of autologous 3-hour reprogrammed cells after exposure to hematopoietic induction culture conditions in 4 patients with aplastic anemia.

The clinical trial was approved by the King Edward Memorial (KEM) Hospital Ethics Committee and was conducted in collaboration with the Institute of Immunohematology (IIH). Patients were required to meet the criteria outlined in Table 3. As a result, 4 patients with severe (3 males) and hypoplastic (1 female) anemia were enrolled in the study. These four patients were selected and monitored by IIH / KEM staff. The clinical and treatment history of the patients is listed in Table 4, while their CD34 ⁺ cell injection volume is shown in Table 5.

  The patient was infused with 2 units of irradiated concentrated red blood cells and 4 units of platelet count to maintain hemoglobin levels greater than 8 g / dl and platelet counts greater than 50000. Patients were apheresised by processing 2-3 times the patient's total blood volume using a Cobe Spectra apheresis device and a leukocyte separation kit (both from Gambro BCT). Apheresis required cervical and antecubital vein catheter insertion using a single lumen catheter for venous access.

An aliquot of cells was aseptically collected for analysis of CD34 after collection of 150-200 ml buffy coat. Thereafter, the buffy coat was subjected to reprogramming under hematopoietic induction culture conditions. Briefly, the reprogramming procedure requires aseptic addition of 1000 μg of purified CR3 / 43 (specially prepared by DakoCytomation for TriStem Corp.) into a leukocyte bag diluted in 30 ml of Iskov modified medium. It was. The bags were then incubated for 3 hours in a sterile tissue culture incubator maintained at 37 ° C. and 5% CO ₂ . After completion of the reprogramming process, the transformed cells were analyzed for CD34 + cell content. Thereafter, the cells were washed twice with a physiological saline solution using a cobe cell processor 2991. After agitation and resuspension in saline solution, the cell suspension was injected into the patient via the jugular vein under gravity using an infusion set. Vital signs including patient CBC count were continuously monitored before and after injection of self-reprogrammed cells.

  All clinical monitoring was performed by IIH / KEM staff. The need for infusion was judged before and after infusion from infusion records obtained after administration of any unit of blood product. Infusion units are used only after obtaining consent from the patient and relatives as witnesses. All blood products were irradiated at Tata Memorial Hospital prior to infusion to the patient. Patients were also asked about their health before and after reprogrammed cell injection. All patients had a copy or ledger of the patient's condition and all laboratory tests and clinical follow-up. The monitoring period for these patients was initially set at 2 years but was extended. During the first month after infusion, the patient stayed in a sterile positive pressure chamber in the hospital.

One million cells were stained with the following panel of monoclonal antibodies (all from DakoCytomation) according to the manufacturer's instructions.
Panel 1 consisting of isotype negative controls IgG1-FITC, IgG1-PE-Cy5 and IgG1-RPE conjugate
Panel 2 consisting of anti-human CD45-FITC and CD34-RPE-Cy5
Panel 3 consisting of anti-human CD38-FITC and CD34-RPE-Cy5
Panel 4 consisting of CD61-FITC and CD34-RPE-Cy5
Panel 5 consisting of CD33 / 13RPE and CD7-FITC
Panel 6 consisting of CD45 and glycophorin-A-RPE
Panel 7 consisting of CD3-FITC and CD19-RPE

  Cell analysis was performed on a FACSCaliber system (BD bioscience) using BD cell Quest software.

  For clonal assays, patient bone marrow mononuclear cells (MNC) before and after reprogrammed cell injection were seeded in methocult GFH4434 supplemented with recombinant growth factors according to manufacturer's instructions (Stem Cell Technologies). Differentiation into hematopoietic cell colonies was evaluated over time using phase contrast inverted microscopy and scored.

  Patients' CBC, liver enzymes and hemoglobin variants were continuously monitored before and after treatment. After discharge from the hospital, patients, CBC, liver enzymes and hemoglobin variants and peripheral blood karyotype analysis and G-staining were monitored by an independent facility for reconfirmation purposes. These tests were frequently performed after injection of self-reprogrammed cells.

  Peripheral blood samples and bone marrow cells were analyzed before and after injection of self-reprogrammed cells. This trial was repeated every 6 months in the first year and annually after 2 years after the start of self-reprogrammed stem cell therapy. In addition, reprogrammed cells were analyzed to examine cell stability prior to injection, which was also performed after a 3-hour conversion step, as well as after establishment of long-term culture for up to 1 month of the converted cells. . Karyotype analysis and G-staining were monitored by a third independent facility.

  Bone marrow smear and trephine sections were performed before and after injection of self-reprogrammed cells. This study was performed 14-20 days after injection of self-reprogrammed cells and thereafter annually.

  All smear and trephine sections were scanned using a microscope connected to a camcorder before and after reprogrammed cell injection to assess and maintain engraftment records.

<result>
All patients withstood apheresis and single reprogrammed cell infusion treatment and had no adverse events. Patient A and patient D became transfusion-independent after a single infusion of reprogrammed hematopoietic stem cells (RHSC) (see Tables 6 and 7). Platelet, neutrophil and erythrocyte engraftment occurred in patients A and D, respectively, 3 and 6 days after infusion. Fetal hemoglobin switching was observed in patients A and D (see Tables 4 and 5), but not in patients B and C (data not shown). Prior to infusion, liver enzymes were elevated in patient A and patient D despite being HCV negative (as measured by ELISA). Liver enzymes began to normalize after RHSC injection and reached normal levels 4 years after RHSC injection. Patient B and patient C died 2 years after injection and 6 months, respectively. Fetal Hb switching was observed in patients 001 and 004 (see Tables 6 and 7). These two patients showed long-term engraftment after a single infusion of autologous HRSC.

  Flow cytometry of mononuclear cells obtained by apheresis before and 3 hours after induction of hematopoietic reprogramming is shown in FIG. The number of CD34 positive cells generated after hematopoietic reprogramming is shown in Table 4. Representative flow cytometry of mononuclear cells obtained by apheresis before and after hematopoietic reprogramming shows a significant increase in the number of CD34 positive cells with and without CD45, CD38 and CD7 expression (Fig. 1). Following injection, CD34 cells circulated in peripheral blood for 3-6 days, after which a significant increase in cells expressing CD33 & 13 with or without CD7 with high forward and lateral scatter was shown (Figure 2). As differentiated into myelocytes at a sustainable level. This pattern of reprogramming was observed in all patients.

  Little colonies formed from patient bone marrow aspirate before infusion of autologous HRSC after seeding into methylcellulose cell culture (see FIG. 3). Only the clonal assay in patient B who suffered from hypoplastic anemia showed suppression of hematopoiesis. However, all bone marrow aspirates obtained from patients 14-20 days after infusion produced various hematopoietic colonies in the normal range with a slight increase in the number of red blood cell burst-forming cells (BFU-red blood cells). Bone marrow smears and trephine sections 14-20 days after autologous HRSC infusion (see Figure 3) show significant numbers of myeloid cells at various stages of differentiation, including mature and immature megakaryocytes, in all patients compared to baseline. Showed an increase. Hyperplasia of erythrocytes at various stages of differentiation was also observed in all samples.

  There was no change in karyotyping and G-staining patterns in peripheral blood or bone marrow samples obtained before and after autologous HRSC infusion in all patients (patient A and patient D over 4 years maximum) ( (See Figure 4).

  Following infusion of autologous HRSCs into patients with aplastic anemia, primitive (CD38 negative) and committed (CD38 positive) CD34 cells circulated in the peripheral circulation for 3 days prior to reprogramming to myeloid cells (FIG. 2). Myeloid cell engraftment occurred 3 days after infusion of HRSC in patient A, patient B and patient D. On the other hand, engraftment of myeloid cells occurred on day 20 in patient 003 when analyzed by flow cytometry. A single infusion of HRSC without preconditioning treatment results in long-term engraftment of 2 out of 4 patients with aplastic anemia. Patient A and patient B showed long-term engraftment without blood transfusion after infusion of HRSC. Engraftment of neutrophils, erythrocytes and platelets in such patients was accompanied by increased HbF switching or HbF hemoglobin levels (see Tables 6 and 7). This was not observed in the other two patients who died. HbF switching in these two patients is the ability of infused HRSC to reprogram to the juvenile Hb phenotype and thus engraftment and reconstitution as observed for cord blood stem cell transplantation (Elhasid et al., Leukemia et al., 2000, 14, 931-934; Locatelli et al., Bone Marrow Transplant, 1996, 18, 1095-101).

  Long-term engraftment with preservation of chromosome number and staining clearly reflects the safety of HRSC injection in hematological disorders where clonal development is not a rare event with conventional therapies.

  Importantly, autologous HRSC was capable of long-term engraftment and survival in some patients with severe aplastic anemia without the use of immunosuppressive therapy, similar to that observed for syngeneic stem cells.

  In summary, after 14 days of infusion, analysis of the bone marrow of the infused patient revealed that myeloid and predominant occupants of myeloid cells at various stages of differentiation, red blood cell and megakaryocyte lineage and severe aplastic anemia. A decrease in cells and stromal cells was shown. There was a significant increase in the amount of red blood cells in the bone marrow and a corresponding increase in hemoglobin levels and reticulocyte count. There was also a steady increase in fetal hemoglobin—an important component in the improvement of sickle cell anemia and beta Mediterranean anemia—and fetal hemoglobin-expressing red blood cells after injection. Furthermore, there was a significant improvement in red blood cell index as determined by red blood cell size, hemoglobin content and concentration. Reprogrammed cells showed normal karyotype and genetic stability after injection. Finally, engraftment and long-term regrowth were observed in 3 additional patients with aplastic anemia.

<Example 2>
<Materials and methods>
Self-reprogrammed hematopoietic cells (target cells) were tested in 21 patients with beta Mediterranean anemia. Nineteen patients had severe beta-Mediterranean anemia and two had moderate beta-Mediterranean anemia. One patient with moderate beta Mediterranean anemia had a Mediterranean anemia / HbE variant (common in Far Eastern and Indian patients) and the other had Mediterranean anemia / sickle cell anemia.

  The patient was apheresised by processing 2-3 times the patient's total blood volume. Self-reprogrammed cells were obtained by reprogramming leukocytes until target cells as indicated by their discriminating properties were obtained as described above. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
Toxicity after reprogrammed cell infusion in patients with beta-Mediterranean anemia as measured by liver and kidney enzymes including vital sign monitoring, echocardiography, bone density, karyotype analysis and G-staining when compared to baseline There were no adverse side effects. When compared to baseline, there was a statistically significant mean reduction (50%) in the need for blood transfusion in patients with severe beta-Mediterranean anemia approximately 9 months now after reprogrammed cell infusion. Two Mediterranean anemia patients with moderate beta Mediterranean anemia (one with Mediterranean anemia / Hb E and the other with Mediterranean anemia / sickle cell anemia) are now transfused almost 9 months after reprogrammed cell infusion. It is independent.

  Mean body weight and height after injection of reprogrammed cells were significantly greater when compared to baseline, and organ sizes in patients with spleen and / or liver swelling were normalized. Absolute mean fetal hemoglobin concentration was significantly increased in patients with severe and moderate Mediterranean anemia after infusion of reprogrammed cells when compared to baseline (Figure 5). Also, the mean red blood cell index, which reflects improvements in red blood cell size, hemoglobin content (FIG. 6) and concentration (FIG. 7), was significantly improved compared to baseline.

  Finally, mean serum ferritin (iron overload biomarker) was significantly reduced in Mediterranean anemia patients after reprogrammed cell infusion (FIG. 8). Iron overload is a leading cause of death and morbidity in patients with Mediterranean anemia, sickle cell anemia, and transplanted iron overload-induced disorders.

<Example 3>
<Materials and methods>
Two patients with diabetes were apheresised by treating 2-3 times the patient's total blood volume. Self-reprogrammed mesenchymal stem cells, pluripotent stem cells and islet cells (target cells) by reprogramming the apheresis leukocytes until target cells as indicated by their discriminating properties occur as described above Got. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
Following infusion of self-reprogrammed cells, patients were synthesizing normal levels of insulin as measured by fasting and 90 minutes food intake stimulating C peptide. This normal level of C-peptide is maintained for up to 3 months after reprogrammed cell injection (Figure 9). Furthermore, HbA1C levels indicating glycemic control were normalized after reprogrammed cell injection (FIG. 10). For example, patients who had more than 10% HbA1C prior to infusion now have 5.8% HbA1C after receiving reprogrammed cells. Moreover, the blood glucose levels in these patients appear to have reached normal levels after infusion when compared to baseline. In addition, a dramatic reduction in insulin intake / injection by diabetics was observed after reprogrammed cell infusion.

<Example 4>
<Materials and methods>
Four patients with amyotrophic lateral sclerosis (ALS) received self-reprogrammed cells. Diagnosis of this disease does not include specific biomarkers. This disease is diagnosed by clinical exclusion of other similar disorders.

  The patient was apheresised by processing 2-3 times the patient's total blood volume. Self-reprogramming pluripotent stem cells, alveolar epithelial cells and neurons (target cells) by reprogramming the apheresis leukocytes until target cells as indicated by their distinguishing properties are generated as described above Got. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
There was a significant improvement in pulmonary function testing (PFT) in patients with ALS who received self-reprogrammed cells. The dysfunction in this pulmonary function test is one of the causes leading to premature death. Most patients experienced less stiffness in the limbs and neck, and some reported improved speech. Other patients showed improved walking ability as well as head lifting.

<Example 5>
<Materials and methods>
Apheresis was performed on 4 patients with Parkinson's disease by processing 2-3 times the patient's total blood volume. Self-reprogrammed pluripotent stem cells and neurons (target cells) were obtained by reprogramming the apheresis leukocytes until target cells were generated as indicated by their discriminating properties as described above. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
It is notable that patients with significant tremor effects of the disease experienced tremors and a significant reduction in conventional drugs used to manage this disorder. The first patient was taking 4 tablets of Sinemet (dopamine modulating agent) daily, but only 1 tablet a day after 4 months of infusion.

<Example 6>
<Materials and methods>
Apheresis was performed on two patients with spinal cord injury by processing 2-3 times the patient's total blood volume. Autoreverse differentiated pluripotent stem cells and neurons (target cells) were obtained by reprogramming the apheresis leukocytes until target cells as indicated by their discriminating properties were generated as described above. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

result
One patient was unable to follow up. The other patient had limb paralysis due to C5-C6 spinal cord injury. The patient was unable to get up or rotate the torso in bed. After treatment, he was able to sit with his back extended for a very long time, and was able to rotate his body in bed. He could stand alone after pressing his body against the wall. In addition, he can move the toes in small increments and reports bladder sensations.

  MRI analysis before and after reprogrammed cell injection showed a slight decrease in lesion size after reprogrammed cell injection. He began to actively receive physical therapy and felt that it was generally much better than before.

<Example 7>
<Materials and methods>
Two patients with muscular dystrophy (MD) were obtained by reprogramming the apheresis leukocytes until the target cells as indicated by their discriminating characteristics were generated as described above. Treated with potent stem cells, mesenchymal stem cells and skeletal muscle cells (target cells). The first patient suffers from a limb-type MD, which is a type of MD where the most heavily affected muscle is typically the hip and shoulder muscles, and the second patient is a nemelin MD I was suffering.

  The patient was apheresised by processing 2-3 times the patient's total blood volume. Self-reprogramming cells were obtained by reprogramming according to the present invention. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

result
Muscle atrophy associated with MD can be measured by monitoring the level of the muscle enzyme creatine phosphokinase (CPK). This enzyme decreased in response to infusion of reverse differentiated stem cells (FIG. 11). Lactate dehydrogenase, an enzyme that rises during tissue degradation, also decreased (FIG. 11). The patient also experienced a decrease in the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both associated with inflammation and damage to liver cells and skeletal muscle.

  In addition, patients improved in pulmonary function tests after infusion of reprogrammed cells as compared to baseline and patient mobility determined by patient camera recordings before and after infusion of reprogrammed cells. showed that.

<Example 8>
Self-reprogrammed pluripotent stem cells obtained by reprogramming patients with renal disease by reprogramming the apheresis leukocytes until target cells as indicated by their discriminating properties occur as described above Treated with leaf stem cells and kidney cells (target cells). The patient was apheresised by processing 2-3 times the patient's total blood volume. Self-reprogramming cells were obtained by reprogramming according to the present invention. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
Patients who received infusions of self-reprogrammed cells experienced improvements in urine volume, serum creatinine and various fluid marker levels that suggested healthier kidney function such as BUN or UEA. For example, a 75-year-old diabetic male patient showed improved renal function after 24 months of treatment with self-reprogrammed cells (see Table 8). Patients showed increased levels of hemoglobin, a protein molecule in red blood cells that carry oxygen, and insulin-like growth factor 1 (IGF-1), a growth factor. Patients also showed a decrease in urea, an organic compound, creatinine, a degradation product of creatine phosphate in muscle, uric acid, an organic compound excreted in urine, and phosphorus, a mineral found in bone. High amounts of these markers indicate poor renal function. In addition, patients showed a reduction in glycosylated hemoglobin (HbA1C), a form of hemoglobin, commonly used as an indicator of plasma glucose levels in people with diabetes.

  Similarly, a 51 year old diabetic man showed similar improvement after 12 months of treatment (see Table 8). This patient showed increased levels of hemoglobin and decreased levels of blood urea nitrogen (BUN), a measure of the amount of nitrogen in the blood in the form of creatinine, HbA1C, and urea.

  Analysis of 12 patients with type II diabetes and treated with self-reprogrammed cells, is associated with leakage of albumin into the urine and is an indicator of renal disease and vascular endothelial dysfunction and cardiovascular disease, A decrease in the level of microalbumin was revealed (Figure 13). Twelve patients also experienced decreased levels of HbA1C (FIG. 14).

  In addition, a 45-year-old woman suffering from autoimmune glomerulonephritis showed improved renal function within 18 months after treatment with self-reprogrammed cells (see Table 9).

  In addition, a 59-year-old female patient with end-stage renal disease showed improvement in creatinine levels, urea, hemoglobin levels, and parathyroid hormone (PHT) after treatment with reprogrammed cells (see Table 10). Renal function marker levels in a 45 year old female patient suffering from autoimmunity. The patient also reduced the number of hemodialysis sessions she attended per month from 12 to about 8.

  A 46 year old patient suffering from chronic renal failure due to diabetes also showed improvement in creatinine levels, urea and hemoglobin levels after treatment with reprogrammed cells (see Table 11). The patient reduced the number of hemodialysis sessions he participated per month from 12 sessions to approximately 5 sessions and received subcutaneously delivered epoetin alfa (EPREX®) twice a week for 4000 units to 2 weeks. Every time it was reduced to 4000 units.

<Example 9>
<Materials and methods>
Patients with multiple sclerosis were treated with self-reprogrammed pluripotent stem cells, mesenchymal stem cells and neurons (target cells). Target cells were obtained by reprogramming the apheresis leukocytes until target cells were generated as indicated by their distinguishing properties as described above. The patient was apheresised by processing 2-3 times the patient's total blood volume. Self-reprogramming cells were obtained by reprogramming according to the present invention. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
Patients treated with self-reprogrammed cells showed reduced lesions in the brain and spinal cord. Lesion reduction can occur within 3 months of receiving treatment (FIGS. 15a-b). Reduction in brain tissue damage occurred within 6 months (FIGS. 16a-b).

  Patients treated with self-reprogrammed cells also showed removal of spinal cord lesions (FIGS. 17a-b). In addition, these patients showed improved Kurtzke Overall Disability Rating Scale (EDSS) scores, showed remission, and did not participate in conventional therapy until 4 years after receiving a single infusion.

<Example 10>
<Materials and methods>
Female patients with HIV were treated with self-reprogrammed hematopoietic stem cells. The patient was apheresised by processing 2-3 times her total blood volume. Target cells were obtained by reprogramming the apheresis leukocytes until target cells were generated as indicated by their distinguishing properties as described above. Patients were administered self-reprogrammed cells by intravenous infusion into the carotid artery or arm or femoral vein.

<result>
Prior to treatment, the screening test for HIV-1 and HIV-2 antibodies showed a test value of 3.68. A value of 1.0 or higher is considered positive.

  Two months after treatment with self-reprogrammed cells, the test value for HIV-1 and HIV-2 antibody screening was 0.46, indicating that the patient did not show positive results for HIV-1 and HIV-2. Was shown. Six months after treatment, the test value for HIV-1 and HIV-2 antibody screening was 0.48, further indicating that the patient did not show positive results for HIV.

<Example 11>
The effect of treatment with self-reprogrammed cells has been shown in patients suffering from other conditions and diseases. The target cells shown herein were obtained by reprogramming the apheresis leukocytes until target cells were generated as indicated by their discriminating properties as described above.

Congestive heart failure A 61-year-old male patient suffering from congestive heart failure was injected with self-reprogrammed cardiomyocytes, pluripotent stem cells, mesenchymal stem cells and endothelial cells. Treatment resulted in an improvement in ejection fraction (EF); brain natriuretic peptide precursor levels (Pro BNP), a predictor associated with coronary artery disease; fasting blood glucose; and HbA1C levels). In addition, previously dilated hearts have a reduced left ventricular end-diastolic diameter (LVID / D), left ventricular end-systolic diameter (LVID / S) and end-diastolic ventricular septal thickness (IVSD) as measured by echocardiography. As shown in Table 12, it returned to normal.

<Hepatitis C>
Thirteen patients infected with hepatitis C virus (HCV) and having beta Mediterranean anemia were injected with self-reprogrammed hematopoietic cells, pluripotent stem cells, mesenchymal stem cells and hepatocytes. The effect of treatment showed a reduction or clearance of HCV load (see Table 1) and improvement of blood markers such as liver enzymes, bilirubin, albumin, prothrombin time, international standard rate (for blood clotting) (see Table 14) . In particular, patients experienced normalization of the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both of which are associated with liver cell inflammation and damage (FIGS. 18a-b).

  For example, a 44-year-old male patient suffering from cirrhosis caused by hepatitis C virus infection also showed improvement in liver enzymes alanine aminotransferase and aspartate aminotransferase, international standard rates (for blood coagulation), bilirubin and albumin and random blood glucose (See Table 15). In fact, this patient had received albumin therapy prior to treatment with reprogrammed cells, but did not receive albumin after treatment.

<Head injury>
A patient suffering from a head injury due to a car accident was treated by injection of self-reprogrammed pluripotent stem cells and neurons. Treatment includes repair of damaged brain tissue (Figures 19a-b), ejection fraction (EF); brain natriuretic peptide precursor levels (Pro BNP) that are predictors associated with coronary artery disease; fasting blood glucose levels; and It resulted in improved HbA1C levels (see Table 10).

<Lung disease>
Patients suffering from restrictive lung disease associated with motor neuron disease were treated by injection of self-reprogrammed pluripotent stem cells, mesenchymal stem cells, alveolar epithelial cells and endothelial cells. Six months after treatment, forced inspiratory vital capacity (FVC), the volume of air that can be forcibly expelled after sufficient inspiration, increases from 50% to 71%, while forcibly exhaling in one second The one-second forced expiratory volume (FEV ₁ ), the volume of air that can be increased, increased from 64% to 68%. Furthermore, the ratio of FEV ₁ to FVC, which is about 75-80% in healthy adults, decreased from 100% to 82%. A lung X-ray scan shows lower opacity in the lung cavity after treatment (FIG. 20).

<menopause>
Self-reprogrammed pluripotent stem cells, pluripotent germ cells and oocytes were administered by injection to a 51 year old patient in menopause. After treatment, patients experienced an increase in various hormone and protein levels including insulin-like growth factor 1 (IGF-1), estradiol and low density lipoprotein (LDL) (see Table 16).

<depression>
Patients suffering from depression were treated by injection of self-reprogrammed pluripotent stem cells and neurons. After treatment, patients experienced an increase in various hormone and protein levels including insulin-like growth factor 1 (IGF-1), cortisol and testosterone (see Table 17).

<Non-obstructive azoospermia>
Patients suffering from non-occlusive azoospermia were treated by infusion of self-reprogrammed pluripotent stem cells, pluripotent germ cells and sperm. After treatment, the patient experienced an increase in testosterone over a period of 8 months (FIG. 21).

Visual Impairment Patients suffering from visual impairment due to removed benign tumors were treated by injection of self-reprogrammed pluripotent stem cells and neurons. After treatment, the patient experienced increased retinal sensitivity and improved vision (Figure 22).

  Although the preferred embodiment of the present invention has been described in detail, the present invention, specified by the above paragraph, is described above, since many obvious variations thereof are possible without departing from the spirit or scope of the invention. It should be understood that the invention is not limited to the specific details set forth in.

Claims (35)

Tissue in a patient suffering from a neurological disease, disorder or condition selected from the group consisting of Parkinson's disease, motor neuron disease, amyotrophic lateral sclerosis, spinal cord injury, multiple sclerosis, depression, and visual impairment Or the use of a population of reprogrammed cells comprising neurons in the preparation of a drug or pharmaceutical composition for repairing or replenishing cells, comprising
The reprogrammed cells are obtained by reprogramming committed cells from the patient to obtain reprogrammed cells;
The reprogramming is
(i) contacting the committed cell with one or more reverse differentiation agents linked to a receptor that mediates capture, recognition or presentation of an antigen on the surface of the committed cell, thereby allowing the A reverse differentiation step to obtain a population of cells comprising cells, and
a population of cells comprising (ii) was reverse differentiated cells, were cultured in tissue culture medium containing one or more differentiation promoting agent, whereby the regeneration step of a population of reprogrammed cells of the patient is obtained
Including the door,
The differentiation enhancer is another reverse differentiation agents used.   The use according to claim 1, wherein the committed cells are obtained from whole blood.   Use according to claim 2, wherein the committed cells are obtained by apheresis.   Use according to claim 2, wherein the committed cells are obtained from mobilized or non-mobilized blood.   The commit cell is selected from the group consisting of T cells, B cells, eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, lymphocytes, leukocytes, platelets and erythrocytes. The use according to claim 2.   The use according to claim 1, wherein the committed cells are incubated with one or more of the reverse differentiation agents.   The use according to claim 1, wherein the receptor is MHC class I antigen or MHC class II antigen.   The use according to claim 1, wherein one or more of the reverse differentiation agents is an antibody against the receptor.   9. Use according to claim 8, wherein one or more of the reverse differentiation agents are monoclonal antibodies against the receptor.   Use according to claim 9, wherein the antibody is selected from the group consisting of monoclonal antibody CR3 / 43 and monoclonal antibody TAL 1B5.   The use according to claim 1, wherein the medicament or pharmaceutical composition is suitable for administration by injection or transplantation.   12. Use according to claim 11, wherein the medicament or pharmaceutical composition is suitable for administration by parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, transdermal or spinal fluid injection.   The tissue culture medium is Iskov modified Dulbecco medium (IMDM), Dulbecco modified Eagle medium (DMEM), Eagle minimum essential (EME) medium, alpha-minimal essential medium (α-MEM), Roswell Park Memorial Institute (RPMI; medium developed) 2) Use according to claim 1 selected from the group consisting of 1640, Ham-F-12, E199, MCDB, Leibovitz L-15, William E medium or commercially available culture medium.   The use according to claim 1, wherein the one or more differentiation promoting agents are anticoagulants or chelating agents.   The use according to claim 1, wherein the one or more differentiation promoting agents are vitamins, minerals or derivatives thereof. 16. Use according to claim 15 , wherein the vitamin, mineral or derivative thereof is retinoic acid.   The use according to claim 1, wherein the one or more differentiation promoting agents are natural or synthetic hormones. 18. Use according to claim 17 , wherein the natural or synthetic hormone is hydrocortisone.   The use according to claim 1, wherein the one or more differentiation promoting agents are amino acids or derivatives thereof. 20. The use according to claim 19 , wherein the amino acid or derivative thereof is selected from the group consisting of L-glutamine (L-glu) and a nonessential amino acid (NEAA).   The use according to claim 1, wherein the one or more differentiation promoting agents is a compound or a derivative thereof. The use according to claim 21 , wherein the compound or derivative thereof is selected from the group consisting of β-mercaptoethanol and putrescine.   The use according to claim 1, wherein the one or more differentiation promoting agents is an acid or a salt thereof. The acid or salt thereof is selected from the group consisting of ascorbic acid, ethylenediaminetetraacetic acid (EDTA), disodium EDTA, ethylene glycol tetraacetic acid (EGTA), and anticoagulant citrate dextrose formulation A (ACDA). Use as described in 23 .   Use according to claim 1, wherein the tissue culture medium comprises autologous plasma, platelets, serum or serum of mammalian origin. The use according to claim 1, wherein the population of reverse differentiated cells is cultured in a blood bag, scaffold, tissue culture bag or plastic tissue culture vessel. 27. Use according to claim 26 , wherein the tissue culture container is an adherent or non-adherent tissue culture container. 27. Use according to claim 26 , wherein the tissue culture vessel is coated or uncoated. 29. Use according to claim 28 , wherein the tissue culture vessel is coated with a substance selected from the group consisting of gelatin, collagen, matrigel or extracellular matrix. Use according to claim 1, wherein the population of reverse differentiated cells is cultured at a temperature of 18-40 ° C. Use according to claim 1, wherein the population of reverse differentiated cells is cultured at a carbon dioxide level of 4-10%. Use according to claim 1, wherein the population of reverse differentiated cells is cultured at an oxygen level of 10-35%. As defined in any one of claims 1 to 37, a pharmaceutical composition comprising a population of reprogrammed cells, including neurons. (i) and obtaining a commit cells to reprogram (ii) the committed cells, and a to obtain the reprogrammed cell, Parkinson's disease, motor neuron disease, amyotrophic lateral sclerosis, spinal cord A method of obtaining a population of reprogrammed cells comprising neurons for administration to a patient suffering from a neurological disease, disorder or condition selected from the group consisting of injury, multiple sclerosis, depression, and vision impairment There,
The reprogramming is
(i) contacting the committed cell with one or more reverse differentiation agents linked to a receptor that mediates capture, recognition or presentation of an antigen on the surface of the committed cell, thereby allowing the A reverse differentiation step to obtain a population of cells comprising cells, and
(ii) a redifferentiation step of culturing a population of cells comprising reverse differentiated cells in a tissue culture medium comprising one or more differentiation promoting agents, thereby obtaining a population of reprogrammed cells of the patient ;
It includes,
A method wherein the differentiation promoting agent is separate from a reverse differentiation agent. 35. A pharmaceutical composition comprising reprogrammed cells obtained by the method of claim 34 and at least one pharmaceutically acceptable excipient.