KR101766203B1 - Treatment using reprogrammed mature adult cells - Google Patents

Abstract

A method of treating a variety of diseases, disorders or conditions in a patient using reprogrammed cells such as de-differentiated, transduced, or regenerated cells. The method includes obtaining a population of cells from a patient, de-differentiating the population of cells to obtain a de-differentiated target cell, and administering the de-differentiated cells to the patient. In certain embodiments, the method comprises obtaining a population of cells from a patient, transforming the population of cells to obtain a transformed target cell, and administering the transformed target cell to the patient. Degenerate or transient differentiation target cells restore or replenish the tissues or cells of the patient.

Description

[0001] TREATMENT USING REPROGRAMMED MATURE ADULT CELLS [0002]

Citation by reference

All documents cited or referenced herein are incorporated by reference herein in their entirety, together with any manufacturer's instructions, manuals, product specifications, product descriptions mentioned herein or in any document, which can be used to practice the present invention.

Field of invention

A method of treating a variety of diseases, disorders or conditions in a patient using reprogrammed cells such as de-differentiated, transduced, or regenerated cells. The method includes obtaining a population of cells from a patient, de-differentiating the population of cells to obtain a de-differentiated target cell, and administering the de-differentiated cells to the patient. In certain embodiments, the method comprises obtaining a population of cells from a patient, transforming the population of cells to obtain a transformed target cell, and administering the transformed target cell to the patient. Degenerate or transient differentiation target cells restore or replenish the tissues or cells of the patient.

Stem cells are characterized by their ability to self-renew through cell mitosis and differentiate into a wide range of specialized cell types. Two types of mammalian stem cells are adult stem cells found in embryonic stem cells and mature tissues isolated from the inner cell mass of blastocysts. In developing embryos, stem cells can differentiate into all specialized embryonic tissues. In mature organisms, stem cells and progenitor cells supplement specialized cells and maintain normal replacement of regenerative organs such as blood, skin or intestinal tissue.

Stem cells decrease in amount of stem cells as development progresses, but they are abundant in developing embryos. In contrast, adult organisms contain a limited number of stem cells confined to specific body regions.

Therapeutic applications of stem cells have the potential to alter multiple disease or disorder treatments. While some adult stem cell therapies (eg, bone marrow transplant) have already been carried out, medical researchers have used stem cells to treat more than any other cancer, including Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis, multiple sclerosis and muscle damage We expect to be able to treat a wide variety of diseases. Such therapies can take advantage of the ability of stem cells to differentiate into the cell types needed to treat the disease.

However, it is uncertain whether stem cells can successfully treat such diseases, and there is concern about the ease with which stem cells can be obtained. For example, hematopoietic stem cells have traditionally been isolated from bone marrow, growth factor mobilized peripheral blood, or umbilical cord blood (placenta). In addition, hematopoietic stem cells can be prepared from embryonic stem (ES) cells that are extracted from embryos obtained using in vitro fertilization techniques. However, the extraction process from these sources is complex, sometimes dangerous, and can lead to ethical concerns. In addition, the number of stem cells that can be obtained from their source is limited. In addition, stem cells may face difficulty in differentiating into cells necessary for treating the disease.

The present invention relates to the use of reprogrammed cells to restore tissues or replenish tissues or cells of a patient. For example, the present invention relates to reconstituting a patient's tissue or replenishing a patient's tissue or cells using degenerated cells obtained from the differentiation of the differentiated or committed cells. The present invention also relates to the use of transformed differentiated cells obtained from the differentiation of differentiated or committed cells to restore the tissue of the patient or supplement the tissue or cells of the patient.

The present application is based on the applicant's finding that reprogramming of somatic cells or somatic cells obtained from a patient can produce other lines of cells and that these reprogrammed cells can be administered to the patient to restore or supplement tissue or cells I have left. Examples of reprogramming procedures include de-differentiation and transitional differentiation.

Thus, applicants have found that a feeder cell can undergo reprogramming to produce other lines of cells. For example, a feeder cell can undergo regeneration to produce undifferentiated cells, such as multipotent stem cells, such as pluripotent stem cells, which are administered to a patient to restore or supplement tissue or cells . As another example, a feeder cell obtained from a patient can undergo a transduction differentiation to produce a cell different from a transduced differentiated cell, for example, a feeder cell, and these transduced differentiated cells are administered to a patient to restore Can be supplemented.

The present invention includes a method of restoring or replenishing a tissue or cell of a cell line of a patient by administering the reprogrammed cell to the patient. In particular, the present invention relates to a method for the treatment of cancer, comprising: (i) obtaining a population of cells, (ii) de-differentiating the population of cells to obtain a de-differentiated target cell, and (iii) The target cell comprises a method of regenerating or replenishing the tissue or cells of the cell line of the patient, wherein the target cell is regenerated into cells of one cell line of the patient. These regenerated cells may be the same cell lineage as the recipient cells or different cell lineages.

The present invention also provides a method of treating a subject suffering from (i) obtaining a population of cells, (ii) transforming the population of cells to obtain a transformed differentiated target cell, and (iii) Lt; RTI ID = 0.0 > cell < / RTI >

In some embodiments, the subject is selected from the group consisting of, but not limited to, myeloid disorders, hematological conditions, aplastic anemia, beta-Mediterranean anemia, diabetes mellitus, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, A human immunodeficiency virus, a head trauma, a spinal cord injury, a lung disease, a depression, an obesity-prone disease, male menopause, menopause and infertility, rejuvenation, a peptic ulcer, psoriasis, wrinkle, liver cirrhosis, autoimmune disease , Hair loss, retinitis pigmentosa, crystalline dystrophy / blindness, diabetes and infertility. Thus, in certain 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, reprogrammed target cells (e. G., Degenerated cells, transformed target cells, or regenerated cells) include, but are not limited to, pluripotent stem cells, pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, Cells, mesenchymal stem cells, endoderm and neural ectodermal stem cells, germ cells, outermost stem cells, embryonic stem cells, kidney cells, alveolar epithelial cells, endodermal cells, neurons, ectodermal cells, islet cells, progenitor cells, gonads, sperm, hematopoiesis And may include cells, hepatocytes, skin / keratinocytes, melanocytes, bone / bone cells, hair / dermal papilla cells, cartilage / cartilage cells, adipocytes, skeletal muscle cells, endothelial cells, myocardial / myocardial cells and nutrient cells.

In certain embodiments, the committed cells are obtained from blood or related tissues (including bone marrow). Growth cells can be obtained from whole blood and / or can be obtained through component harvesting. Blood can be mobilized or non-fermented blood. Such cells include, but are not limited to, T cells, B cells, eosinophils, basophils, neutrophils, guk cells, mononuclear cells, red blood cells, granulocytes, mast cells, lymphocytes, leukocytes, platelets and red blood cells. Alternatively, the gauge cells can be obtained from neuronal tissue of the central nervous system or peripheral nervous system, muscle tissue or epidermis and / or dermal tissue of the skin.

In certain embodiments, the challenge cells are obtained from the patient ' s blood or tissue. In some embodiments, the patient receiving the reprogrammed target cell, such as a reprogramming target cell or a transduced differentiation target cell, is the same patient.

In some embodiments, the feeder cells are de-differentiated by contacting the feeder cells with the agent. For example, a feeder cell can be cultured with the agent. In certain embodiments, the agent grafts a receptor that mediates capture, recognition, or presentation of an antigen at the surface of the population of cells. The receptor may be an MHC class I antigen or an MHC class II antigen. In some embodiments, the class I antigen is an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E receptor, an HLA-F receptor or an HLA- -DP receptor, an HLA-DQ receptor, or an HLA-DR receptor.

In certain embodiments, the agent may be an antibody to a receptor, e. G., A monoclonal antibody to a receptor. In some embodiments, the antibody is a monoclonal antibody CR3 / 43 or a monoclonal antibody TAL1B5. In a further embodiment, the agent modulates MHC gene expression, e. G., MHC class I ^< ⁺ & gt ^; and / or MHC class II ⁺ expression.

In some embodiments, the dedifferentiated cells may undergo regeneration at a separate step. For example, degenerated cells may be treated with growth factors (including, but not limited to, basic fibroblast growth factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, stem cell factor, interleukin- -7, basic fibroblast growth factor, epidermal growth factor, granulocyte-macrophage colony stimulating factor, granulocyte-colony stimulating factor, erythropoietin, stem cell factor and osteogenic protein) . The resulting regenerated target cells can then be administered to the patient.

In certain embodiments of the invention, the feeder cells can be transformed by culturing the feeder cells under specific culture conditions. For example, feed cells can be combined with an inverse agent to be cultured in a particular type of culture medium. Examples of these culture media may include Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM), and the like. In addition, the tissue culture medium may comprise a differentiation promoting agent such as vitamin and / or mineral supplements, hydrocortisone, dexamethasone,? -Mercaptoethanol, and the like. In addition, further incubation conditions include the use of a chelating agent or antibiotic, incubation at a certain temperature or carbon dioxide or oxygen level, and incubation in a particular vessel. The culture conditions can determine the type of transformed target cell to be produced.

Accordingly, one aspect of the present invention is a method for obtaining target cells. The method can include obtaining a population of cells and then reprogramming the population of cells. These processes are as described herein. In some embodiments, the methods of the invention can include de-differentiating the feed cells. In another embodiment, the method of the invention may comprise converting a population of cells. In another embodiment, the methods of the invention can include de-differentiating the feed cells and then regenerating the de-differentiated cells.

Another aspect of the invention is the use of one or more reprogrammed target cells in the manufacture of a medicament for restoring or supplementing tissue or cells of a cell line of a patient or for treating a disease or tissue injury.

Another aspect of the invention is a method of treating such disease or tissue damage in a patient in need thereof. In certain embodiments, the methods of the invention include obtaining a population of cells, reprogramming the population of cells to obtain a reprogrammed target cell, and administering the reprogrammed target cells to the patient. In some embodiments, the target cells can be reprogrammed through de-differentiation, transfection, and / or regeneration. In certain embodiments, the target cell is a degenerated target cell, a transformed target cell, and / or a regenerated target cell. Methods for obtaining feeder cells and reprogramming feeder cells are as described herein.

In some embodiments, reprogrammed target cells, such as a degenerated target cell, a transduced differentiated target cell, or a regenerated target cell, can be administered via injection, implantation or infusion. These cells may be administered by parenteral, intramuscular, intravenous, subcutaneous, intradermal, oral, transdermal, or injection into the spinal fluid. In certain embodiments, the dedifferentiated target cell or the transduced target cell is administered in a pharmaceutical composition. The pharmaceutical composition may include a degenerated target cell or a transduced differentiated target cell and one or more pharmaceutically acceptable excipients.

One aspect of the invention is a pharmaceutical composition for administering a reprogrammed target cell (e.g., a degenerated target cell or a transduced target cell). The pharmaceutical composition may comprise one or more types of target cells and one or more pharmaceutically acceptable carriers. Optionally, the pharmaceutical composition may comprise adjuvants and / or other excipients suitable for administration to a patient.

Another aspect of the present invention is a method of producing a recombinant vector comprising (i) obtaining a population of cells, (ii) reprogramming the population of cells to obtain a reprogrammed target cell, and (iii) optionally reprogramming the target cells with one or more pharmaceutical excipients ≪ / RTI > or a pharmaceutically acceptable salt thereof. In some embodiments, the reprogrammed target cells are mixed with one or more pharmaceutical excipients. In another embodiment, the reprogrammed target cells are not mixed with one or more pharmaceutical excipients. Methods for obtaining feeder cells and reprogramming feeder cells are as described herein.

The terms "including", "including", "including", and the like used in the specification and especially in the claims and / or paragraphs may have the meanings in accordance with the United States Patent Act; By way of example, and not limitation, such terms are intended to include both the objects of reference and others. In addition, terms such as "consisting essentially of" also have meanings in accordance with the United States Patent Act. For example, this term includes elements that are not explicitly mentioned, but are meant to exclude elements found in the prior art or that affect the elementary or novel features of the invention.

These and other aspects are described or implied in the following detailed description.

The following detailed description sets forth examples of the present invention, but it is not intended to limit the invention to the specific embodiments described, but may be best understood by reference to the accompanying drawings.
Figure 1 shows the results of immunophenotyping of collected mononuclear cells before induction (top panel) and after induction (bottom panel) of reprogramming. Cells were labeled with monoclonal antibodies conjugated to immunoglobulin G1 (IgG1) isotype control and to picoerithrine (PE) against R-phycoerythrin (RPE) Cy-5 or CD19 against CD34 (vertical legend) . In addition, the cells were stained for CD45, CD38, CD61 and IgG1 isotype control monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) (horizontal legend). The lower panel shows an increase in hematopoietic stem cells as the relative number of CD34 and CD34CD38- cells increases and leukocyte maturation markers such as CD45 and CD19 decrease.
FIG. 2a sequentially shows the results of immunophenotyping of peripheral blood samples of patients with severe Aplastic anemia after injection of autologous HRSC (day 1, day 2, day 3, day 6, and day 14). Cells were labeled with monoclonal antibodies against CD34 and CD45 (second horizontal panel) and CD34 and CD38 (third horizontal panel). The upper horizontal panel shows that forward scattering and lateral scattering are the flow cytometry analyzes of HRSC, bone marrow smear and terpin flake. On day 1 through day 14, there is an increase in cells with giant anterior and lateral scattering, which is an indicator of granulocyte and shows an increase in the relative number of circulating CD34 hematopoietic stem cells from day 1 to day 3. FIG. 2B shows the results of immunophenotyping of peripheral blood samples of patients with aplastic anemia after injection of severe autologous human reprogrammed stem cells (HRSC) (day 1, day 2, day 3, day 6 and day 14) Show. 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 a numerical increase in myeloid cells, which is manifested by a gradual increase in CD33 & 13 expressing cells, including progenitor cells, which is manifested by an increase in the relative number of cells co-expressing CD33 & In addition, the relative number of lymphocytes increased gradually, as shown by the relative increase in the number of CD19 and CD3 lymphocytes.
Figure 3 shows the results of bone marrow analysis in patients with severe aplastic anemia before and after injection of autologous HRSC. Bone marrow smears before (a) and after (b) showed an increase in red blood cells; The terpin flakes before (c) and after (d) showed an increase in bone marrow cell integrity; Clonal analysis of the bone marrow after injection of HRSC showed that the colony forming unit guk cells (e), colony forming mononuclear cells (f), colony forming granulocytes and macrophages (g), and colony forming bone marrow cells and red blood cells (h) 0.0 > (i). ≪ / RTI >
FIG. 4 shows karyotype analysis and G-fractionation of peripheral blood samples from patients with severe aplastic anemia after 4 years of autologous HRSC injection, showing no genetic abnormality.
Figure 5 shows that absolute mean fetal hemoglobin levels were increased in patients with Mediterranean anemia after treatment with self reprogrammed cells.
Figure 6 shows the mean size of red blood cells in patients with Mediterranean anemia after self-reprogrammed cells and shows an increase in mean hematocrit expressed in femtoliter.
Figure 7 shows the weight of hemoglobin per cell in patients with Mediterranean anemia after self-reprogrammed cells and an increase in mean cellular hemoglobin, expressed in picograms.
Figure 8 shows a decrease in blood ferritin levels, expressed in nanograms per milliliter, of patients with Mediterranean anemia after treatment with self reprogrammed cells.
Figure 9 shows the increase in C-peptide levels after fasting and after mixed feeding in diabetic patients after treatment with self-reprogrammed cells.
Figure 10 shows a decrease in glycated hemoglobin (HbA1C) levels in diabetic patients after treatment with self-reprogrammed cells.
Figure 11 shows the reduction of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) levels after treating muscular atrophy with self-reprogrammed cells.
Figure 12 shows a decrease in liver enzyme alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in patients with muscular atrophy after treatment with self reprogrammed cells.
Figure 13 shows a decrease in urinary microalbumin levels in 12 kidney disease patients after treatment with self reprogrammed cells.
Figure 14 shows a reduction in glycated hemoglobin (HbA1C) levels after 12 patients with renal disease due to diabetes were treated with self reprogrammed cells.
15A shows a magnetic resonance imaging (MRI) scan of the brain of a multiple sclerosis patient before (left scan) self-reprogrammed cells and three months after the treatment (right scan) Indicating a decrease in lesion growth. Figure 15B shows MIR scans of different parts of the patient's brain before self-reprogrammed cells (left scan) and 3 months after treatment (right scan). In the pre-treatment scan, the arrows indicate the brain's lesion, while the post-treatment scan indicates an improvement in the lesion.
16A shows a magnetic resonance imaging (MRI) scan of the brain of a multiple sclerosis patient before (top scan) self-reprogrammed cells and after six months (bottom scan) after treatment. Figure 16b shows additional MRI scans before self-reprogrammed cells (upper scan) and 6 months after treatment (lower scan). In the pre-treatment scan, arrows point to the brain's lesion, whereas in the post-treatment scan the arrows indicate an improvement in the lesion.
Figure 17a shows a sagittal MRI scan of a patient with multiple sclerosis before treatment (left scan) with self reprogrammed cells and 6 months (right scan) after treatment. FIG. 17B shows a cross-sectional MRI scan of the spine before self-reprogrammed cells (left scan) and 6 months (right scan) after treatment. In the scans showing d before treatment, the arrows indicate the lesion of the brain, while the arrows after the treatment indicate the improvement of the lesion.
Figure 18a shows a decrease in liver enzyme alanine aminotransferase (ATL) levels after treatment with self-reprogrammed cells in patients infected with hepatitis C, Figure 18b shows a decrease in the level of aspartate aminotransferase (AST) Show.
19A shows a magnetic resonance imaging (MRI) scan of a patient's brain suffering head trauma from a car accident. Before treatment (upper scan), the ventricles are enlarged and show extensive hematoma scattering. After treatment with autologous reprogrammed cells (lower scan), the ventricles show a decrease in brain atrophy, such as a decrease in the ventricle and cerebral cortex, as well as improvement in the hematoma. Figure 19b shows an additional MRI scan of the patient's brain before (upper scan) and after (lower scan) the patient's treatment.
Figure 20 shows the chest X-ray of patients with pulmonary disease before treatment with the self reprogrammed cells (left X-ray) and after treatment (right X-ray). After treatment, the patient exhibited improved lung volumes and reduced lesion size as a reduction of the low density area.
FIG. 21 shows sex hormone levels of follicle-stimulating hormone (fsh), luteinizing hormone (lh), progesterone (pro) and testosterone (test) after treating non-obstructive azoospermia patients with self reprogrammed cells. These concentrations show a significant increase in free testosterone with an increase in ultrasound-determined testicular size (data not shown).
Figure 22 shows the patient's retinal sensitivity and visual impairment before (top panel) and after treatment (bottom panel) treatment of a patient suffering from impaired vision with self reprogrammed cells. In retinal sensitivity results, the orange area indicates reduced retinal sensitivity. In the visual impairment results, the white areas indicate normal vision, and the pink, orange and black areas indicate increased visual impairment. In the retinal sensitivities before treatment, the orange area was converted to green and white after treatment, and the black area in the pre-treatment visual impairment was converted to white after treatment, and the patient's vision improved after treatment.

Justice

As used herein, "population cells" are cells that exhibit differentiated characteristics. These cells are often considered mature and specialized. Examples include leukocytes, erythrocytes, epithelial cells, neurons and chondrocytes.

As used herein, "non-aqueous cells" are cells that do not exhibit matured differentiation characteristics. These cells are often considered immature and unspecialized. One example of non-aqueous cells is stem cells, which are immature cells capable of self-renewal (infinite cleavage) and differentiation (specialization).

As used herein, "reprogramming" refers to the process by which the cells of the first cell lineage are transformed into cells of another cell type. These other cell types may be of other cell lines. Reprogramming can occur through processes such as de-differentiation, transitional differentiation, or regeneration.

As used herein, "reprogrammed cells" are cells that have undergone reprogramming of feed cells. The reprogrammed cell can include a degenerated cell, a transformed differentiated cell, and / or a regenerated cell.

As used herein, "dedifferentiation" is the process by which a number of cells, mature specialized cells, return to the more primitive cellular stage. "Regenerated cell" is a cell generated by the reprogramming of a number cell.

As used herein, "transduction differentiation" is the process by which the first cell lineage cell transforms into another cell of another cell type. In some embodiments, the conversion differentiation may be a combination of de-differentiation and regeneration. "Converted differentiation cell" is a cell generated by the conversion differentiation of the feeder cell. For example, a number of cells, such as whole blood cells, can be differentiated into neurons.

As used herein, "regeneration" refers to the process by which non-aqueous or degenerated cells differentiate into more mature and specialized cells. "Regenerative cells" are cells that are produced by regeneration of non-aqueous cells or degenerated cells. When the regenerated cells are obtained through regeneration of the de-differentiated cells, the regenerated cells may be the same or different strain than the recipient cells that have undergone de-differentiation. For example, a number of cells such as white blood cells can be differentiated to form degenerated cells such as pluripotent stem cells, and then the regenerated cells are regenerated to form lymphocytes which are the same lineage as the white blood cells (feed cells) It can form neurons that are different from white blood cells (feed cells).

As used herein, a "target cell" is a cell that is obtained to administer to a patient to restore or supplement tissue or cells. For example, the target cell may be a reprogrammed target cell, such as a degenerated target cell or a transduced differentiated target cell, whereby the degenerated or transformed target cell is administered to the patient.

Water cell

As described above, the committed cells of the present invention are cells exhibiting differentiated characteristics. A feeder cell can include all members involved in antigen presentation, capture or recognition. For example, the feeder cells may be MHC class I ⁺ and / or MHC class II ⁺ cells.

In addition, the feeder cell may be any cell derived or inducible from undifferentiated cells. Thus, in one embodiment, the committed cells are also undifferentiated cells. Thus, for example, the feeder cells may be lymphoid stem cells or myeloid stem cells differentiated in association with pluripotent stem cells.

The feeder cell may be an organism such as blood or related tissue including bone marrow or umbilical cord blood, neuronal tissue from the central nervous system or peripheral nervous system, epidermis from muscular tissue or skin and / or dermal tissue (i. E., Via oral scraping) Material. ≪ / RTI > The biological material may be of postnatal origin.

The biological material may be obtained using methods known in the art as being suitable for the tissue type. Examples include, but are not limited to, incision, syringe extraction, and swabbing component collection.

In certain embodiments, the feeder cells are derived from whole blood or processed products thereof (e.g., plasma or smoke), since they can be removed from the subject under minimal medical supervision. Blood samples are typically treated with an anticoagulant such as heparin or citrate. Cells in a biological sample can be treated to proliferate a particular cell type, to remove a particular cell type, or to separate cells from a tissue ingrowth. Methods useful for purifying and separating cells include centrifugation (e.g., density gradient centrifugation), flow cytometry and affinity chromatography (e.g., the use of magnetic beads containing monoclonal antibodies to cell surface markers or panning) See: Vettese-Dadey, The Scientist 1999, 13:21). For example, Ficoll-Hypaque separation is useful for removing monocytes such as lymphocytes and monocytes by removing red blood cells and granulocytes.

Examples of feeder cells that can be derived from blood include but are not limited to CFC-T cells, CFC-B cells, CFC-eosin cells, CFC-Bas cells, CFC-Bas cells, CFC- , CFC-MEG cells, BFC-E cells, CFC-E cells, T cells, B cells, eosinophils, basophils, neutrophils, monocytes, megakaryocytes and red blood cells.

Blood-derived feeder cells can be identified by 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 ⁺ cells. T lymphocytes are CD7 ⁺ cells. Leukocytes are CD45 ⁺ cells. Granulocytes are CD13 ⁺ and CD33 ⁺ cells. Monocyte macrophages are CD14 ⁺ and DR ⁺ cells.

In certain embodiments, the committed cells are selected from the group consisting of B lymphocytes (activated or inactivated), T lymphocytes (activated or inactivated), cells from the macrophage mononuclear system, nucleated cells capable of expressing Class I or Class II antigens, Class I or Class II A cell or a nucleus cell that can be induced to express an antigen (i.e., a cell that does not contain a nucleus such as a red blood cell).

In other embodiments, the committed cells can be selected from any one of the group of cells, including large granulocytes, balloons lymphocytes, and natural killer cells, each expressing CD56 and / or CD16 cell surface receptors.

Because feed cells are essentially primary cultures, it may be necessary to supplement the nutrients to maintain viability in the cell population. Suitable culture conditions are well known to those skilled in the art. However, it is preferable that the treatment of the cell cluster is started as soon as possible after the removal of the biological sample from the patient, typically within 12 hours, preferably within 2 to 4 hours. Cell viability can be checked using well-known techniques, such as Trypan blue stain exclusion or propidium iodide.

De-differentiation  cell

Degeneration is a type of reprogramming process that progressively changes the structure and function of cells to produce less specialized cells. De-differentiation can occur naturally, in which the cells can undergo a limited de-differentiation in vivo in response to tissue damage. As an alternative, de-differentiation may be accomplished using the techniques described in U.S. Patent Application No. 08 / 594,164 (U.S. Patent No. 6,090,625), U.S. Patent Application No. 09 / 742,520 (U.S. Patent No. 7,112,440), U.S. Patent Application No. 10 / 140,978 No. 7,220,412), U.S. Patent Application No. 10 / 150,789 (U.S. Patent No. 7,410,773), and U.S. Patent Application Serial No. 09 / 853,188. All of these patents are incorporated herein by reference.

The dedifferentiated cells of the present invention include, but are not limited to, pluripotent stem cells, lymphoid stem cells, myeloid stem cells, neuronal stem cells, skeletal muscle subcutaneous cells, epithelial stem cells, endodermal stem cells, mesenchymal stem cells and embryonic stem cells . ≪ / RTI >

In certain embodiments, the feeder cells are derived from the blood and de-differentiated to form hematopoietic lineage differentiated cells. Examples of these degenerated cells include, but are not limited to, pluripotent stem cells, lymphoid stem cells, and myeloid stem cells.

Feeding cells can be de-differentiated by contacting these cells with agents that are operatively grafted to the cells. Subsequently, the cells are cultured so that the cells, which are operatively grafted by the agent, progress through the de-differentiation process and ultimately undifferentiated.

The contacting step can include an agent that grafts with surface antigens on a population of cells. The agent may act directly or indirectly with graft cells. An example of direct grafting is when a feeder cell has one or more cell surface receptors on its cell surface, for example, a homologous region (a region commonly found to have the same or similar sequence) as can be found in B cells , Wherein the agent is directly conjugated to a cell surface receptor. Another example is when a feeder cell has a cell surface receptor on its cell surface, for example, an a-chain having a homologous region, such as may be found in a T cell, wherein the agent is directly associated with a cell surface receptor Graft.

An example of indirect grafting is where the recipient cell has at least two cell surface receptors on its cell surface and the grafting of one of the receptors with the agent affects the other receptor and then leads to the de-differentiation of the recipient cells.

Agents for the de-differentiation of population cells may be chemical compounds or compositions. For example, the agent can be combined with a cell surface receptor at the surface of the feeder cell. In certain embodiments, the agent is operably associated with a receptor present on the surface of the population of cells (e.g., a receptor that can be expressed by a feeder cell).

For example, the agent can be, but is not limited to, cyclic adenosine monophosphate (cAMP), CD4 molecule, CD8 molecule, some or all of the T-cell receptor, ligands (immobilized or free), peptides, T- TCR), antibodies, cross-reactive antibodies, monoclonal antibodies or polyclonal antibodies. In addition, growth factors such as hematopoietic growth factors (e.g., erythropoietin and granulocyte-monocyte colony stimulating factor (GM-CSF)) may be used.

If the agent is an antibody, a cross-reactive antibody, a monoclonal antibody or a polyclonal antibody, the agent may be a beta-chain of the MHC class II antigen, a beta chain of the MHC HLA-DR antigen, a MHC class I or a class II antigen an antibody against any one or more of the alpha and beta chains of the alpha chain, the alpha chain of the HLA-DR antigen, the MHC class II antigen or the MHC class I antigen, a cross-reactive antibody, a monoclonal antibody, May be any one or more of the antibodies. An example of an antibody is CR3 / 43 (supplier Dako).

The term "antibody" is intended to encompass a variety of fragments (derived by proteolytic cleavage or recombinant techniques) and derivatives (such as Fab, F (ab ') ₂ and scFv antibodies) . ≪ / RTI > It is also possible, for example, to modify the amino acid sequence by rearrangement of amino acid residues to improve binding, or to alter the amino acid sequence of the organism and other species that are the source of the cells necessary for the treatment of the antibody according to the method of the invention to reduce the potential for immune side- (E.g., humanized murine monoclonal antibodies).

The agent used to convert the feeder cells into the de-differentiated cells can preferably act extracellularly of the feeder cells. For example, the feeder cell may comprise a receptor operably grafted by the agent, and the agent is operatively associated with the receptor.

For example, the receptor may be a cell surface receptor. 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, such as in the case of MHC class I and class II receptors.

The receptor may comprise a homologous region of the? -Chain having a homologous region, such as at least the? -Chain of HLA-DR.

Alternatively or additionally, the receptor may comprise a homologous region of the a-chain of a homologous region, such as at least the a-chain of HLA-DR. The receptor may be a Class I or Class II antigen of the main histocompatibility complex (MHC). In certain embodiments, cell surface receptors include but are not limited to HLA-DR, DM, DP, DQ, HLA-A, HLA-B, HLA- F receptor or an HLA-G receptor. In some embodiments, the cell surface receptor may be an HLA-DR receptor.

The agent may be an antibody to a receptor, for example a monoclonal antibody to a receptor.

An example of a preparation may be one that modulates MHC gene expression, such as MHC class I ⁺ and / or MHC class II ⁺ expression.

In certain embodiments, the preparation can be used in combination with a bioreaction regulator. Examples of bioreactor regulatory substances 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 or may not be a cyclophosphamide.

Other biologic response modulators may include compounds that are capable of up-regulating MHC class I and / or class II antigen expression, and in some embodiments, allowing agents that bind to MHC receptors to work more effectively.

As any cell type can be made to express MHC class I and / or class II antigens, it is possible that the cell will be capable of differentiation into a wide variety of cell types, whether or not essentially expressing or expressing class I and / or class II MHC antigens May be provided.

The feed cells are generally cultured with the agent for at least 2 hours, typically 2 to 24 hours, preferably 2 to 12 hours. The incubation is typically conducted at a temperature of about room temperature or for example from about 22 캜 to about 37 캜 (including 33 캜). Progression of the de-differentiation process can be monitored periodically by aliquotting a small sample and examining the cells using a microscope and / or flow cytometry. Alternatively, the instrument may include tracking means for online monitoring of the progress of the de-differentiation process.

In addition to the use of reversing agents, feed cells can be cultured in autologous plasma or serum or in fetal serum or horse serum. Optionally, the feed cells can be incubated with an anticoagulant, a chelating agent or an antibiotic. The temperature range for cell culture can range from 18 to 40 DEG C and can also include 4 to 10% CO ₂ and / or 10 to 35% O ₂ . In addition, the culture can be carried out in coated or uncoated blood bags, tissue culture bags or plastic containers.

Certain types of dedifferentiated cells can be obtained by de-differentiation using specific culture conditions. For example, supernatant cells can be obtained by mixing Dulbecco's modified Eagle's medium (DMEM), non-essential amino acid (NEAA), L-glutamine (L-glu) Lt; RTI ID = 0.0 > pluripotent < / RTI > cells. In addition, the feeder cells may be exposed to the chelating agent first.

As another example, to obtain mesenchymal cells, feed cells are combined with the reverse-repellant (s) using DMEM (low glucose) and L-glu or DMEM (low glucose), L-glu, 2? ME and NEAA, can do. In addition, the antibiotic gentamicin can be used for cell culture.

Transformed differentiation cell

The transformed differentiated cells are obtained by culturing the feed cells in a tissue culture medium blended with a reverse-blending agent. Whereby the feeder cells are transformed into cells of another cell type undergoing conversion differentiation; In some embodiments, the committed cells are transformed into other lines of cells.

The type of target cells obtained through the transduction differentiation depends on the culture conditions. These conditions may vary depending on the type of tissue culture medium used in culture, the presence / absence of various differentiation promoters, the presence / absence of different sera, the incubation temperature, the presence / absence of oxygen or carbon dioxide, and the type of container or container .

Examples of tissue culture media used for conversion differentiation include, but are not limited to, Iscove transformed Dulbecco's medium (IMDM), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential (EME) medium, (? -MEM), Roswell Park Memorial Institute (RPMI) 1640, Ham-F-12, E199, MCDB, LABORBIES L-15, Williamsburg medium E or formulated tissue culture medium commercial products.

Differentiation promoters include anticoagulants, chelating agents and antibiotics. Examples of such preparations may be one or more of the following: vitamins and minerals or derivatives thereof [for example, A (retinol), B _3, C (ascorbyl bait), ascorbyl 2-phosphate bait, D _2, D _3, K, Retinoic acid, nicotinamide, zinc or zinc compounds and calcium or calcium compounds; Natural or synthetic hormones (e.g., hydrocortisone and dexamethasone); Amino acid or a derivative thereof such as L-glutamine (L-glu), ethylene glycol tetrasaccharic acid (EGTA), proline and nonessential amino acid (NEAA);? -Mercaptoethanol, dibutylcyclic adenosine monophosphate -cAMP), monothioglycerol (MTG), putrescine, dimethylsulfoxide (DMSO), hypoxanthine, adenine, phocolin, cilostamide and 3-isobutyl-1-methylxanthine or derivatives thereof ; Nucleosides such as 5-azacytidine and analogs thereof; ascorbic acid, pyruvate, okadinic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), anticoagulant Citrate dextrose A (ACDA), disodium EDTA, sodium (E.g., G418, gentamycin, pentoxypyrin (1- (5-oxohexyl) -3, 7-dimethylxanthine) and indomethacin); acids or salts thereof such as butyrate and glycerophosphate; And tissue plasminogen activator ( TPA).

These differentiation promoters can be used to obtain specific types of target cells. For example, it is possible to generate the acinar cells or islet hydrocortisone such as by using the vitamin B _3; Dexamethasone can be used to produce mesenchymal or epithelial cells (e.g., kidney epithelial cells, skin and related structures (e.g., dermal papilla cells)); β-mercaptoethanol can be used to generate ectodermal cells, including neuronal cells, including the CNS subculture.

The culture medium was autogenous plasma; Platelets; Serum such as fetal blood; Or serum of mammalian origin such as horse serum. In addition, the conversion process may proceed in a blood bag, a scaffold, a tissue culture bag, or a plastic tissue culture vessel. The tissue culture vessel may be an adsorptive or non-adsorbent tissue culture vessel or may be coated with a formulation such as gelatin, collagen, matrigel or extracellular matrix which promotes adsorption or floatation separation depending on the type of tissue or specialized cell to be produced, Or may not be coated.

Further incubation conditions include a temperature that may be from about 10 to about 60 DEG C, or from about 18 to about 40 DEG C; A carbon dioxide (CO ₂ ) level that can be from about 0 to about 20% or from about 4 to about 10%; And an oxygen (O _2), which may be from about 0 to about 50%, or about 10 to about 35%.

An example of the method of using a reversed-parting agent to obtain a target cell is described in Table 1. < tb > < TABLE >

The methods of the present invention combine with a reverse-repellant to obtain various types of target cells. Target cell type The culture conditions used in combination with the inverse agent Additional culture conditions
Pluripotent stem cells
or
Of universal precursor cells
Heterogeneous colony
DMEM, NEAA, L-glu, 2? ME Pre-exposure of cells confined to chelating agents or anticoagulants
In the case of precursor cells, recovery of the reverse-repellent agent by dilution with only the culture medium
Mesenchymal stem cells ㆍ DMEM (low glucose), L-glu; or
DMEM (low glucose), L-glu, 2? ME, NEAA ㆍ Gentamicin









ㆍ universal germ cell
ㆍ oocytes
ㆍ Sperm


RPMI 1640, optionally NEAA, L-glu,
2? ME (in the case of oocytes); or

EME medium, retinol, L-glu, sodium pyruvate, sodium lactate, NEAA (in the case of oocytes); or

DMEM, Hams F12, Vitamin C, Vitamin E, Retinoic acid, Retinol, Pyruvate, optionally Pentoxyfilin (for sperm); or

The culture conditions for the above-mentioned pluripotent stem cells and the culture conditions (in the case of sperm or oocyte)
GAIC
At about 30 to 32 占 폚 (sperm) and about 38 to 39 占 폚 (oocyte).
Cells may be exposed to chelating agents such as EDTA and EGT prior to de-differentiation and regeneration.
In the case of sperm, it may include the addition of okadinic acid, DMSO and zinc or zinc compounds; Gamete 100 can be used as a primary medium instead.
In the case of oocytes, addition of dp-cAMP, disodium DETA, phoscholine, cilostamide and hypoxanthine may be included.
In the case of oocytes, Medium 199 can be used as a primary medium instead.

kidney DMEM, Hams F12 and hydrocortisone, optionally vitamin K; or
The culture conditions for the pluripotent stem cells listed above and the culture conditions for hydrocortisone

Alveolar epithelium
ㆍ Endoderm DMEM, NEAA, L-glu, optionally 2? ME, nicotinamide; or
Culturing conditions and nicotinamide for the pluripotent stem cells listed above, and optionally dexamethasone, retinoic acid, db-cAMP; or
ㆍ IMDM, L-glu, ascorbic acid, MTG Antibiotic G418 and Matrigel-coated tissue culture vessels

ㆍ Neuron
ㆍ Ectoderm DMEM and Hams F12, NEAA, 2? ME, and optionally, putrescine, retinoic acid, L-glu, hydrocortisone, ascorbate; or
Culturing conditions for the pluripotent pluripotent stem cells listed above and optionally supplemented with pu trocin, retinoic acid, hydrocortisone, ascorbate

ㆍ island cell
Proliferation cell ㆍ DMEM, Hams F12, Vitamin B ₃ ; or
RPMI 1640 and vitamin B ₃ ; or
Culture conditions for the pluripotent stem cells listed above and vitamin B ₃ (nicotinamide), and optionally dexamethasone





Hematopoietic cell





IMDM and optionally hydrocortisone; or
IMDM, L glutamine and MTG; or
The culture conditions for the pluripotent stem cells listed above and the MTG used in place of 2? ME and optionally the vitamins The incubation temperature may be 33 ° C.
The incubation temperature may be room temperature to amplify megakaryocytes in the culture.
Differentiated cells may be exposed to chelating agents prior to conversion for amplification of erythroid precursor cells in culture.
RPMI 1640 can be used as the primary medium for the proliferation of lymphocyte precursor cells.
Sodium butyrate and / or 5-azacytidine may be added to the culture for the promotion of protic erythropoiesis.


Hepatocyte DMEM or IMDM or? -Min essential medium, L-glu and optionally dexamethasone, L-ascorbic acid-2-phosphate and nicotinamide; or
Williams < RTI ID = 0.0 > badges E, < / RTI > sodium pyruvate, dexamethasone; or
The culture conditions for the pluripotent stem cells listed above and the dexamethasone


Skin / keratinocyte Hams F12, DMEM (medium ratio 3: 1), hydrocortisone, L-glu and optionally adenine; or
E199 or DMEM and L-glu, and optionally hydrocortisone and adenine; or
The culture conditions and hydrocortisone for the pluripotent stem cells listed above, and optionally the calcium or calcium compound or ascorbate
Zetamycin may be added as an antibiotic.

ㆍ It can be cultured at 36.4 ℃.
Melanocytes MCDB and Leibovitz L-15, TPA and optionally 3-isobutyl-1-methylxanthine; or
Medium 199 and hydrocortisone

Bone cell / bone DMEM,? -Glycerophosphate, dexamethasone, ascorbate and L-glu and optionally vitamin D ₃ ; or
Culturing conditions for the pluripotent stem cells listed above and additionally glycerophosphate, dexamethasone and ascorbate and optionally vitamin D ₃




Dermal papilla cells / hair William E medium, L-glu, hydrocortisone and / or vitamin D ₂ , adenine and linoleic acid; or
DMEM, Hams F12 (as a basal medium), L-glu, hydrocortisone and / or vitamin D ₂ , adenine and linoleic acid; or
The culturing conditions for the pluripotent stem cells listed above, hydrocortisone, vitamin D ₂ , adenine and linoleic acid

Cartilage cell / cartilage DMEM, pyruvate, ascorbate 2-phosphate, dexamethasone and proline; or
The culture conditions for the pluripotent stem cells listed above, ascorbate 2-phosphate, dexamethasone and proline
Fat cell DMEM, dexamethasone and indomethacin; or
ㆍ dexamethasone and indomethacin



Skeletal muscle
DMEM, low glucose and optionally hydrocortisone, dexamethasone, L-glutamine and sodium pyruvate; or
DMEM and Ham F12 or F10 (as base medium) or DMEM and medium 199; or
The culture conditions for the pluripotent stem cells listed above and the culture conditions for glucose, gentamicin and low serum Gentamicin can be added as an antibiotic.
ㆍ Low serum concentration can be added.
ㆍ Gelatin-coated culture containers can be used.
ㆍ It can be cultured at a temperature raised to 39 ℃.
≪ / RTI > 5-azacytidine.
Blood vessels (endothelium) DMEM, NEAA and 2? ME; or
ㆍ IMDM and dexamethasone


Myocardium / myocardial cell
ㆍ 4: 1 DMEM and M199 medium or DMEM (low glucose) and additionally ascorbic acid and / or DMSO; or
The culture conditions for the pluripotent stem cells listed above and, furthermore, the anti-gravity culture or vibration Environment For complete differentiation, gelatin-coated culture vessels and autologous or platelet-depleted plasma
Complete differentiation can be observed on the cover slip slide.

Nutrient cells
Continuous dilution to the same culture medium except for DMEM, L-glu, 2? ME, NEAA, and reverse osmotic agent; or
≪ RTI ID = 0.0 > RPMI 1640, 2? ME, sodium pyruvate and L-glutamine Can be used to produce the self-growth factors and hormones necessary to differentiate mesenchymal and ectodermal cells (including reproductive stem cells and progenitor cells).

In particular, in the case of the culture conditions described for each cell type in Table 1, the continuous dilution of the culture conditions with the corresponding culture medium to remove the reverse agent results in an increased conversion differentiation.

During the incubation, the culture medium optionally containing various differentiation promoting agents may be diluted by further adding the medium from which the de-differentiation agent is excluded. While not intending to be bound by theory, dilution appears to increase differentiation because the cells are less dense and the proliferating factor is less concentrated. Thus, the addition of medium can further increase the differentiation of the differentiation and affect which cell types can be obtained in the cell lineage. For example, if the target cell is a neuron, the addition of the culture medium can lead to an expression shift towards more mature neurons (both of which are in the same line) than neuronal precursor cells. As another example, pro-differentiated skeletal muscle progenitor cells will differentiate only by serial dilution of the culture medium achieved by progressively decreasing the serum concentration.

Regrowth cell

Degenerated cells can be used to acquire target cells by rejuvenating or regenerating these degenerated cells as target cell types. This can be accomplished by contacting the degenerated cells with a growth factor. For example, retinoic acid has been used to differentiate stem cells into neuronal cells. Co-cultures of bone marrow stromal cell line and IL-7 with methylcellulose have been used to differentiate stem cells into lymphocyte precursors (Nisitani et al., Int Immuno 1994, 6: 909-916). Le Page (New Scientist Dec. 16, 2000) teaches that stem cells can be differentiated into lung epithelial cells. Bischoff (Dev Biol 1986, 115: 129-39) teaches how to differentiate muscle-associated cells into mature muscle fibers. Neuronal progenitor cells can be proliferated by basic fibroblast growth factor and epidermal growth factor (Nakafuku and Nakamura, J Neurosci Res 1995, 41: 153-168). Hematopoietic stem cells can be proliferated using a number of growth factors including GM-CSF, erythropoietin, stem cell factors and interleukins (IL-1, IL-3, IL-6) For review, see Matcalf (Nature 1989, 339: 27-30).

Potocnik et al. (EMBO J 1994, 13: 5274-83) have demonstrated that even low oxygen (5%) conditions are used to differentiate stem cells into hematopoietic cells.

Regenerative cells may be the same lineage as the recipient cells that are the origin of the degenerated cells. Alternatively, the regenerated cells may be in a different lineage from the recipient cells that are the origin of the de-differentiated cells. For example, B lymphocytes can be differentiated into CD34 ⁺ CD38 ^- HLA - DR ^- stem cells. These stem cells can subsequently be regenerated or re-seeded along the B-cell lineage (the same lineage) or the lymphatic lineage (the different lineage).

Target cell

The target cells of the present invention are reprogrammed cells that can be obtained by de-differentiation, transfection or regeneration as described above. According to the present invention, target cells include, but are not limited to, pluripotent stem cells, lymphoid stem cells, myeloid stem cells, neuronal stem cells, skeletal muscle subcutaneous cells, epithelial stem cells, endodermal and neuroectodermal stem cells, And embryonic stem cells, mesenchymal stem cells, kidney cells, alveolar epithelial cells, endoderm cells, neurons, ectodermal cells, islets, progenitor cells, oocytes, sperm, hematopoietic cells, hepatocytes, melanocytes, Cells, hair / dermal papilla cells, cartilage / chondrocytes, adipocytes, skeletal muscle cells, endothelial cells, myocardial / myocardial cells and nutrient cells.

As described above, the feeder cells and / or the degenerated cells are cultured under specific conditions to induce de-differentiation and / or transduction and / or regeneration, and obtain target cells. The time period for culturing the feeder cells and / or the regenerated cells is not controlled by the specific time but is controlled by the determination that the target cells have been produced.

Regeneration, differentiation, or regeneration The determination of the production or the change in the number of target cells to produce a change in the number of cells that down regulate the expression of the lineage-associated marker or transcription factor and / or the cell surface markers characteristic of the target cell Can be accomplished by monitoring the relative number of < / RTI > Alternatively or additionally, a decrease in the number of cells that do not exist in the target cell and have typical cell surface markers in the feeder cell can be monitored. For example, the target cells may be selected from the group consisting of POU5F1 (OCT-4), TERT, KLF4, UTF1, SOX2, Nanog or step-specific embryo markers 3 and 4 (SSEA-3 and SSEA- -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 the presence of SSEA-1, an indicator of differentiation. Other markers for different types of stem cells are known, for example nestin for neural epithelial stem cells (J Neurosci 985, 5: 3310). Mesenchymal stem cells are positive for, for example, SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a and CD124 and negative for CD34, CD45 and CD14. The 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 cell markers for target cells can be developed through microarray analysis. This assay can include separating RNA from degenerated and / or transfected and / or regenerated target cells, labeling the isolated RNA with a dye, and hybridizing the isolated RNA to the microarray. The microarray may comprise a gene or oligonucleotide representing the complete genome or may comprise a gene or oligonucleotide directed at a particular organ system, tissue system, disease, pathology, and the like. Cell markers can be identified by which genes / oligonucleotides exhibit high signal intensity and are therefore up-regulated or down-regulated in target cells. This information can then be applied to determine the presence of a marker identified by microarray analysis or even a target cell based on a group or pattern of markers.

In addition, many in vitro assays such as CFC assays can be used to identify target cells (see Examples). Often, highly primitive hematopoietic stem cells are measured using Long Term Culture Initiation Cell (LTC-IC) assays (Eaves et al., J Tiss Cult Meth. 1991, 13: 55-62). LTC-IC lasts 5-12 weeks for hematopoiesis.

In the case of other cell types such as cells of the central nervous system, pancreas, liver, kidney, skin, etc., the cell culture can be performed by immunohistochemistry, flow cytometry, microarray or reverse transcription polymerase chain reaction -PCR) until the property is confirmed. This may also include functional assays, for example, implantation of an immunodeficient host or modification or mitigation of essential clinical conditions as observed in the present invention.

Target cells can be identified by microarray or RT-PCR, which show the acquisition of new lineage-specific transcription factors, proteins and signals in target cells. For example, degenerative stem cells transformed into ectodermal target cells can express genes such as Nestin, Criptol, isl1, LHX1 and / or EN1 and, when further differentiated into neurons, Lt; / RTI > On the other hand, cells transformed into endoderm lineage target cells can express genes such as sox7, sox17, Nodal, PDX1 and / or FOXA2. In addition, target cells differentiated into pancreatic islet cells are insulin (INS) and neurog3 ). ≪ / RTI > In particular, conversion towards the target cell of interest can be achieved by down-regulation of mature transcription factors associated with the first starting colony undergoing transformation.

In addition, the production of the target cell can be determined by identifying specific structural and / or morphological characteristics of the target cell (e.g., cell shape, size, etc.). Such characteristics are known in the art in connection with the target cells of the present invention.

Once the relative number of target cell types has increased to an appropriate level (eg, greater than or equal to 0.1% or less than 5%), the resulting modified cell population can be used in many ways. Regarding the number of target cells formed (eg, pluripotent stem cells), it is important to evaluate the proliferative capacity of stem cells. Although in some circumstances the number of stem cells or other degenerated cells formed may be small, studies have shown that only 50 universal hematopoietic stem cells can reconstitute the entire hematopoietic system in donor mice. Thus, therapeutic uses do not require the formation of large numbers of cells.

In addition, the de-differentiation, conversion or differentiation of target cells into target cells can be carried out in vivo by administering to the patient an agent mixed with a pharmaceutical carrier or diluent. However, in many cases, it is desirable to perform in vitro / in vivo differentiation, differentiation or regeneration.

The treated cell clones obtained in vitro can be subsequently used through a minimal process. For example, these cells can be administered to patients in need of stem cells simply by combining with a pharmaceutically acceptable carrier or diluent.

However, it may be desirable to proliferate cell populations or purify cells from cell populations for de-differentiation, transduction or subdivision target cells. This can be done easily using many methods (Vattese-Dadey-The Scientist 1999, 13). For example, cells can be purified based on cell surface markers using chromatography and / or flow cytometry. Nonetheless, it is not necessary or desirable to broadly refine, differentiate, or differentiate target cells from cell populations because other cells present in the cell population (such as stromal cells) can sustain stem cell viability and function I can not.

Flow cytometry is a reliable and robust established technique for identifying cells and classifying cells in a mixed cell population. Thus, the purification or separation means may comprise a flow cytometer. Flow cytometry operates on the basis of the physical characteristics of the particles in the suspension, and particles can be detected and distinguished by light rays. Of course, these particles may be cells. Physical features include cell surface markers that contain cell size and structure, or that have been highly expressed in recent years and that are bound by monoclonal antibodies conjugated to fluorescent molecules.

Kreisseg et al. (J Hematother 1994, 3: 263-89) states that "the availability of anti-CD34 monoclonal antibodies has led to the selection of multivariate flow cytometry as a tool for the determination of hematopoietic stem and progenitor cells" have. Kreisseg also describes a general technique 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 following immune adsorption based on HLA-DR expression. As discussed above, CD34 ⁺ is a useful marker in relation to stem / progenitor cells. In addition, flow cytometric analysis techniques are available that classify stem cells based on other physical characteristics. For example, Visser et al. (Blood Cells 1980, 6: 391-407) teaches that stem cells can be isolated based on their size and degree of structurization. In addition, Grogan et al. (Blood Cells 1980, 6: 625-44) teaches that viable stem cells can be classified as high purity as verifiable from simple hematopoietic tissue.

In addition to selecting cells (positive selection) based on the presence of cell surface markers or other physical properties, cell populations can be propagated and purified using negative criteria. For example, cells possessing 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 linked to magnetic beads. The beads are cultured with cell clusters, and cells bearing cell surface markers such as CD34 to which affinity ligands bind are captured. The sample tube containing the cells is placed in a magnetic sample concentrator where the beads are drawn to the side of the tube. After one or more washing steps, the cells are partially or substantially completely purified from other cells. When used in a negative screening method, instead of washing the cells bound to the bead by discarding the liquid phase, the liquid phase is preserved and consequently cells bound to the bead are effectively removed from the cell population.

These affinity ligand-based purification methods can be used with any cell type in which a suitable marker has already been identified or can be identified. Urbankova et al. (J Chromatogr B Biomed Appl 1996, 687: 449-52) teaches the microbial production of hematopoietic stem cells from mouse bone marrow suspension by gravity-flow fractionation. In addition, Urbankova et al. Mentioned that the above method was used for identification of the characteristics of these stem cells since the stem cell derived from mouse bone marrow is larger than other cells in the bone marrow and thus can be separated from the mixture. Thus, stem cells can be purified and propagated using other physical parameters other than cell surface markers.

Cell populations comprising reprogrammed target cells, such as de-differentiation, differentiation or subdivision target cells, and / or purified reprogrammed target cells, such as de-differentiation, differentiation or subdivision target cells prepared by the methods of the present invention, And can be maintained in vitro using known techniques. Typically, minimal growth medium (eg, Hanks, RPMI 1640, Dulbecco's minimal essential medium (DMEM) or Iscove's modified Dulbecco's medium) is used supplemented with mammalian serum (eg, FBS) Thereby providing a suitable growth environment. Stem cells can be cultured on culture-assisted cell layers such as stromal cell layers (Deryugina et al., Crit Rev Immunology 1993, 13: 115-150). Stromal cells are known to secrete factors that maintain precursor cells in undifferentiated state. Long-term culture systems for stem cells are described in 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) can purify human CD34 ⁺ hematopoietic cells using techniques based on the use of monoclonal antibodies covalently immobilized on polystyrene surfaces It is taught that CD34 ⁺ cells purified by this method can be maintained at a survival rate of 85% or more. Lebkowski et al. (J Hematother 1993, 2: 339-42) teach how to isolate and culture human CD34 ⁺ cells. In addition, Haylock et al., Immunomethods 1994, 5: 217-25 can be referred to for review of various methods.

Purified preparations, including stem cells and cell populations and stem cells, can be frozen / cryopreserved for future use. Techniques suitable for freezing and restoring cells in the future are well known in the art.

In one aspect, de-differentiation, transduction, or regeneration occurs or occurs within a cell from a buffy coat blood sample. The term "buffy coat" refers to a white cell layer formed between a red blood cell layer and a plasma layer when centrifuged or allowed to stand for non-coagulated blood.

Treatment method

The reprogrammed target cells of the present invention (e.g., degenerated target cells, transformed target cells, and regenerated target cells) may be combined with various components to produce compositions of the present invention. The composition may be combined with one or more pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include, but are not limited to, isotonic saline solution (e.g., phosphate buffered saline). The composition of the present invention can be administered by direct injection. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intramedullary, oral, transdermal or intraspinal injection.

The composition comprising the target cells may be delivered by injection or implantation. The cells may 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 a support to cells that are susceptible to breakdown in vivo.

In addition, the compositions may comprise the dedifferentiated, transduced or subdivided target cells of the invention and one or more pharmaceutically acceptable excipients, carriers or vehicles.

Also, the delivery can be by controlled delivery, i.e., over a period of time that can be from minutes to hours or even days. Delivery may be systemic (e.g., by intravenous injection) or may be localized to the specific site of interest. Cells can be delivered in vivo via liposome delivery.

The target cells may be administered at a dose of 1 x 10 ⁵ to 1 x 10 ⁷ cells per kg. For example, a 70 kg patient can receive 14 x 10 ⁶ CD34 ⁺ cells for tissue reconstruction. This dose may correspond to any combination of target cells listed herein.

The methods of the invention may be used to treat a variety of diseases, conditions, or disorders. Such conditions include but are not limited to myelosuppression, hematological conditions, aplastic anemia, beta-mediterranean anemia, diabetes mellitus, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, renal disease, liver disease, multiple sclerosis, congestion Atherosclerosis, menopause and infertility, rejuvenation, corneal ulceration, psoriasis, wrinkles, liver cirrhosis, autoimmune diseases, hair loss , Retinitis pigmentosa, and disorders associated with crystalline dystrophy / blindness or other tissue regression.

Aplastic anemia is a rare, but fatal, bone marrow disorder characterized by hematopoietic and hypocellular bone marrow (Young et al., Blood 2006, 108: 2509-2519). This disorder is caused by the immune-mediated pathophysiology of activated Th1 cytokines, particularly activated I-type cytotoxic T cells expressing gamma-interferons that target hematopoietic stem cell fractions, leading to bone marrow failure, (Bacigalupo et al., Hematology 2007, 23-28). Most of the patients with aplastic anemia can be treated with stem cell transplants obtained from HLA-matched siblings (Locasciulli et al., 2007; 92: 11-18) It is facing a considerable challenge. Despite a reasonable survival rate after HLA-matched allogeneic stem cell transplantation, this procedure has some potential risk due to immunosuppressive therapy used to prevent graft-versus-host disease (GVDH) disease. For example, high doses of cyclophosphamide used with or without antithymocyte globulin (ATG) result in prolonged immunosuppression and may be prone to opportunistic infections in patients. Other potential risks are graft failure, which can occur weeks or months after stem cell transplantation (Gottdiener et al., Arch Intern Med 1981, 141: 758-763; Sanders et al., Semin Hematol 1991, 28: 244-249 ). In addition, the risk of graft failure increases by the number of blood transfusions prior to stem cell transplantation.

Mediterranean anemia is a hereditary autosomal recessive hemorrhagic disease characterized by a decrease in the synthesis rate of one of the globin chains that make up hemoglobin. Therefore, the mutation of the regulatory gene is the main cause, abnormal hemoglobin molecule is formed, and normal globin protein is not produced, resulting in anemia. Other types of Mediterranean anemia include alpha anemia, beta anemia and delta anemia, affecting the production of alpha globulin, beta globulin and delta globin, respectively. Treatments include chronic transfusion, iron chelation, splenectomy and allogeneic hematopoietic cell transplantation. However, chronic transfusion has not been applied to most patients because there is no HLA-matched bone marrow donor, while allogeneic hematopoietic cell transplantation is implicated in the possibility of many complications such as infection and graft-versus-host disease.

Diabetes is a syndrome (hyperglycemia) that causes abnormal hyperglycemia. Diabetes refers to a group of diseases in which insulin secretion in the body or defects in insulin action causes hyperglycemia. Diabetes is typically divided into two types: type 1 diabetes mellitus with reduced production of insulin and type 2 diabetes mellitus with resistance to the effects of insulin. Both types cause hyperglycemia and result in symptoms commonly associated with diabetes (eg, excessive urine production, resulting compensatory thirst and increased fluid absorption, blurry vision, unexplained weight loss, changes in lethargy and energy metabolism) . Diabetes is considered a refractory chronic disease. Therapeutic options include treatment with some drugs (eg, promoting insulin secretion by the pancreas, reducing glucose produced by the liver, and increasing the sensitivity of the cells to insulin) in the case of insulin injections, exercise, proper diet or type 2 diabetes Drugs, etc.).

Motor neuron disease refers to a group of neurological disorders that affect motor neurons. Such diseases include amyotrophic lateral sclerosis (ALS), primary colorimetric sclerosis (PLS), and progressive atrophy (PMA). ALS appears to be a regression of both upper and lower motor neurons, and this degeneration stops the message to the muscles, resulting in muscle weakness and ultimately atrophy. PLS is a rare motor neuron disease that affects only the upper motor neurons and causes balance difficulties, angular weakness, weak stiffness, stiffness, or speech impairment. PMA is a subtype of ALS that affects only lower limb motor neurons and leads to atrophy, fiber spasm and muscle weakness. There is no known method for treating motor neuron disease. Riluzole, which is known to reduce motor neuron damage, has been approved as a treatment for ALS, but this drug slows the progression of ALS rather than improving ALS. The treatment of PLS is just to manage symptoms, such as baclofen, which can reduce stiffness, or quinine, which can reduce spasticity.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a loss of the suprabonyloid pathway resulting from degeneration of dopaminergic neurons in the black body. The cause of PD is unknown, but it is associated with progressive death of dopaminergic (tyrosine hydroxylase (TH) positive) middle cerebral neurons, including movement disorders. Thus, PD is characterized by muscle stiffness, tremor, restlessness and latent inability to move. Thus, there is currently no way to satisfactorily prevent or treat Parkinson's disease or its symptoms. A major treatment for Parkinson's disease-related dyskinesia involves oral administration of dihydroxyphenylalanine (L-DOPA), which can induce a substantial improvement in motor function, but as the regression of dopaminergic neurons progresses, . Another strategy is to introduce dopamine in the affected striatum by introducing an enzyme that provides L-DOPA and neurotransplantation based on the idea that dopamine supplied from cells transplanted into the proximal organ can replace the lost acrosarcoma cells Include gene therapies that can be used to replace or replace dopamine synthesis by preventing the TH-positive neurons from killing or by introducing potential neuroprotective molecules that can stimulate regeneration and function restoration of the damaged black line system .

Spinal cord injury is characterized by the disruption of some or all of the muscles or nerves below the injury site due to the damage of the spinal cord and especially the nerve fibers. These injuries can occur through trauma to the spinal cord and can result in fractures, dislocations, cracks or compression of one or more vertebrae, or through non-traumatic injuries caused by arthritis, cancer, inflammation or disc regression. Treatment after spinal cord injury may damage drugs such as methylprednisolone, a corticosteroid that reduces nerve cell damage and reduces inflammation at the site of injury, as well as medications that control pain and atrophy, as well as fixed or escaped discs or spinal cord of the spinal cord It may involve surgery to remove all possible objects, but there is no known means to restore spinal cord injury.

Muscular dystrophy (MD) refers to a group of muscular dystrophies that weaken skeletal muscles. MD is characterized by progressive muscle weakness, muscle protein deficiency, muscle cell apoptosis and tissue atrophy. Although nine diseases (duchenne atrophy, Becker muscle atrophy, limb-related muscle atrophy, congenital atrophy, facial shoulder upper arm muscle atrophy, muscle tension degeneration atrophy, oculopharyngeal atrophy, Muscle atrophy) is classified as MD, but there are more than 100 diseases that show MD characteristics. No specific treatment for MD is known at all. Physical therapy can maintain muscle tone and improve quality of life by using surgery. In addition, symptoms such as myotonia can be treated with drugs, but there is no long-term therapy.

Renal disease refers to conditions that damage the kidneys and reduce renal function (including removal of waste and excess water from the blood, regulation of electrolytes, blood pressure, acid-base balance, and reabsorption of glucose and amino acids). The two main causes of kidney disease are diabetes and hypertension, and other causes include glomerulonephritis, lupus and kidney malformations and disorders. There is no cure for kidney disease, so treatment moderates blood glucose and hypertension and monitors diet to alleviate disease progression and cure the cause of the disease; For example, managing fluid retention, anemia, and bone disease to treat complications of the disease; It is focused on restoring lost kidney function through dialysis or transplantation.

Multiple sclerosis is an autoimmune condition in which the immune system attacks the central nervous system and induces dehydration. MS affects the ability of neurons in the brain and spinal cord to communicate with each other because the body's immune system attacks and damages myelin that encircle neuronal axons. If myelin is lost, the axon can no longer effectively send the signal. This can result in a variety of neurological symptoms and these symptoms usually develop into physical and cognitive disabilities. There is no known treatment for MS; Cure is an attempt to restore function after an attack (sudden onset or worsening of MS symptoms) and prevent new attacks and disorders. For example, treatment with corticosteroids may serve to stop the attack, while treatment with interferon during the first attack has been shown to reduce the likelihood of clinical MS.

Human immunodeficiency virus (HIV) is a lentivirus that can cause acquired immune deficiency syndrome (AIDS), a condition in which the immune system does not work. HIV mainly infects living cells in the human immune system (eg helper T cells, macrophages and dendritic cells). HIV infection results in a low level of CD4 ⁺ T cells due to direct virus killing of infected cells, by an increased percentage of apoptotic cells in infected cells, or by the killing of CD4 ⁺ T cells infected by CD8 cytotoxic lymphocytes that recognize infected cells do. Currently, there is no vaccine or cure for HIV or AIDS. Treatment of HIV infection consists of highly active antiretroviral therapy (HAART). The current HAART option is a combination (or "cocktail") consisting of three or more drugs of at least two types of antiretroviral agents. Typically, these classes are the addition of a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI) to two nucleoside analogue reverse transcriptase inhibitors (NARTI or NRTI).

Congestive heart failure is a condition in which the heart is unable to pump enough blood to other organs in the body. This condition may be caused by coronary artery disease, scar tissue from the heart caused by myocardial infarction, hypertension, heart valve disease, heart disease and heart valve infection. Therapeutic programs typically consist of rest, proper diet, changes in daily activities, and medications (eg, angiotensin-converting enzyme (ACE) inhibitors, beta blockers, digitalis, diuretics, vasodilators). However, the treatment program does not restore the damage or condition of the heart.

Hepatitis C is an infectious disease caused by hepatitis C virus in the liver. Hepatitis C can progress to wounds (fibrosis) and advanced wounds (cirrhosis). Liver cirrhosis can cause liver failure and other complications (eg, liver cancer). Current therapies use a combination of pegylated interferon alpha and the antiviral drug ribavirin. The success rate may vary from 50 to 80% depending on the viral genotype.

Head trauma refers to damage to the head that may or may not cause brain damage. Common causes of head trauma include traffic accidents, home and occupational injuries, falls and assault. (Head trauma), cerebral contusion (temporary impairment due to trauma), coma or even hemorrhage from epidural hematoma (epidural hematoma), epidural hematoma (hemorrhage between epidural and skull) Various types of problems can occur, including death. Treatment of head trauma may vary according to the type of injury. If the brain is damaged, there is no way to quickly heal the brain damage, and brain damage can not be restored by the treatment methods in use.

Pulmonary disease is a generic term for diseases of the respiratory system, including the lungs, thoracic cavity, bronchi, organs, upper respiratory tract and respiratory tract nerves and muscles. Examples of lung diseases include obstructive lung disease caused by bronchial stricture; Inhibitory or fibrotic lung disease caused by loss of lung elasticity resulting in incomplete lung enlargement and increased lung stiffness; Airway infections, which can often be caused by cold air or pneumonia; Respiratory tumors such as those caused by cancer; Thoracic disease; And pulmonary vascular disease that affects pulmonary circulation. Treatment of pulmonary disease varies depending on the type of disease, but may include drugs such as corticosteroids and antibiotics, oxygen, mechanical ventilation, radiation therapy and surgery.

Depression is a mental disorder characterized by low self-esteem and normally delightful activity accompanied by a loss of interest or pleasure, as well as markedly depressed mood. Biologically, depression is the nucleus of the cerebral stem, the source of serotonin, the seam nucleus; A neoclassical nuclear nucleus that regulates bodily rhythms such as sleep / awakening cycles; Hypothalamic-pituitary-adrenal cortex axis, a chain structure that is activated during a physical response to various stresses; Known as the "compensation" circuit of the brain; Chongqing willing nucleus considered to play a role in reward, favor, pleasure, addiction and fear; And altered activity in many parts of the brain, including the frontal cortex that is activated by negative experience. Treatment of depression includes antidepressants, exercise and psychotherapy that increase the amount of extracellular serotonin in the brain. However, the efficacy of these treatments remains questionable.

Obstructive azoospermia is a condition that can not be measured by semen present in semen due to problems with male sperm formation. This is often caused by hormonal imbalance and can be treated with drugs that restore this imbalance.

Male menopause is a menopause-like condition experienced by middle-aged men due to decreased production of hormone testosterone and dehydroepiandrosterone. Treatment includes hormone replacement therapy and exercise.

Cuspidosis is a chronic autoimmune disease that affects connective tissue. Although scleroderma can affect the connective tissue of the whole body, skin hardening is the most visible symptom in this disease. No direct treatment of cicatricialis is known.

Psoriasis is a chronic autoimmune disease in which red scales appear on the skin. The cause of psoriasis is related to the excessive growth of skin cells. One hypothesis suggests that psoriasis is associated with T-cells, where T-cells migrate to the dermis to induce secretion of cytokines and cytokines rapidly produce skin cells. Treatment of psoriasis includes drugs that target T-cells.

Retinal pigment epithelium is an advanced photoreceptor or retinal pigment epithelium with progressive retinal formation that is loss of vision. Treatment of retinitis pigmentosa is limited.

The conditions described herein can be treated with a particular type of target cell or a combination of these types. In a preferred embodiment, the conditions described herein can be treated by the infusion of the cell types listed in Table 2.

Therapeutic treatment of various conditions using reprogrammed (i. E., Reprogramming or transfection or regeneration) target cells Condition Therapeutic cell types (discontinued or combinations thereof) Aplastic anemia Hematopoietic Beta-mediterranean anemia Hematopoietic diabetes Mesenchymal stem cells, pluripotent stem cells and / or islet cells Motor neuron disease Pluripotent stem cells, alveolar epithelial cells, ectodermal cells and / or neurons Parkinson's disease Pluripotent stem cells and / or neurons Spinal cord injury Pluripotent stem cells and / or neurons Obesity Pluripotent stem cells, mesenchymal stem cells and / or skeletal muscle cells Kidney disease Kidney cells, mesenchymal stem cells and / or pluripotent stem cells Multiple sclerosis Pluripotent stem cells, mesenchymal stem cells and / or neurons
Congestive heart failure Myocardial cells, mesenchymal stem cells, pluripotent stem cells and / or endothelial cells Hepatitis C virus Hematopoietic cells, pluripotent stem cells, mesenchymal stem cells and / or hepatocytes Human immunodeficiency virus Hematopoietic Head trauma Pluripotent stem cells and / or neurons
Lung disease Pluripotent stem cells, mesenchymal stem cells, alveolar epithelial cells and / or endothelial cells depression Pluripotent stem cells and / or neurons Non-obstructive azoospermia
Or menopause Universal stem cells, pluripotent germ cells and / or sperm Menopause and infertility Allogeneic stem cells, oocytes, universal germ cells
recovery Pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, keratinocytes, dermal papilla cells, neurons, sperm, oocytes, cartilage cells, myocardial cells, skeletal muscle cells, neurons, endothelial cells and / Arousal ulcer Pluripotent stem cells, endothelial cells, keratinocytes and / or mesenchymal stem cells psoriasis Mesenchymal stem cells or pluripotent stem cells wrinkle Mesenchymal stem cells, keratinocytes and / or pluripotent stem cells Liver cirrhosis Hepatocytes, mesenchymal stem cells, endodermal cells, pluripotent stem cells Autoimmune disease Mesenchymal stem cells and / or pluripotent stem cells hair loss Dermal papilla cells and / or melanocytes Retinitis pigmentosa or crystalline dystrophy / blindness
Neurons and / or pluripotent stem cells

By way of example, a patient in this condition may be treated through the following steps:

1) Insert a Fistula-cannula into the patient's arm;

2) Collect white blood cells using an automated system such as the COBE ^® Spectra device (Gambro PCT);

3) produce self-differentiating stem cells from the patient's leukocytes;

4) Wash autotransplanted stem cells and inject intravenously into the patient;

5) Monitor the progress of the patient, including blood tests and wound site assessment.

The present invention is described in more detail by the following non-limiting examples which further illustrate the invention, but these examples should not be construed or interpreted as limiting the scope of the invention.

Example

Example  One

Materials and methods

This clinical trial evaluated the safety of single-dose infusion of autologous, 3-hour reprogrammed cells following exposure to four patients with Aplastic Anemia.

This clinical study was approved by the Ethics Committee of the King Edward Memorial (KEM) Hospital and conducted in collaboration with the Institute of Immunohematology (IIH). The patient met the criteria listed in Table 3 absolutely. As a result, four patients with severe anemia (three males) and one with hypomemic anemia (one female) participated in the study. These four patients were selected and monitored by IIH / KEM staff. The clinical and therapeutic power of the patients is described in Table 4, and their CD34 ⁺ cell injection doses are listed in Table 5.

Inclusion criteria
Each criterion is required
1 - absolute neutrophil count <0.5x10 ⁹ / L 2 - platelet count <20x10 ⁹ / L  3 - Less than 1% of anemia due to retinal erythrocytes Only one of these criteria
Required  Bone marrow cell integrity less than 4 - 25%  5 - Less than 30% hematopoietic cells and less than 50%  6 - Patient evaluation within 3 months after diagnosis  7 - Patients without prior immunosuppressive therapy

Two units of irradiated erythrocyte packs and four units of platelets were transfused in the patient to maintain hemoglobin levels above 8 g / dl and platelet count above 50,000. The total blood volume of the patient was treated 2-3 times using the Cobe Spectra component collection device and leukocyte separation kit (both from Gambro BCT) to collect the components. The components were collected by injecting a catheter with one lumen into the jugular vein and jugular vein.

After collecting 150 to 200 ml of buffy coat, the cell fraction was aseptically collected for CD34 analysis. Buffy coats were then reprogrammed under hematopoietic induction culture conditions. In summary, the reprogramming procedure involves aseptically adding 1000 μg of purified CR3 / 43 (prepared specifically by DakoCytomation for TriStem Corp.) diluted in 30 ml of Iscove's modified medium in a white blood cell bag. Then, the bag was incubated in a sterile tissue culture medium is set to 37 ℃ and 5% CO ₂ for 3 hours. At the end of the reprogramming process, the transformed cells were analyzed for CD34 ⁺ cell content. The cells were then washed twice with saline using a COBE cell processor 2991. After shaking and resuscitation in saline, the cell suspension was injected into the patient through the jugular vein under gravity using an infusion set. Continuous monitoring of vital signs including CBC counts of patients before and after injection of self reprogrammed cells.

Clinical and therapeutic power of patients with aplastic anemia until autologous reprogrammed stem cell (HRSC) injection patient Clinical power up to HRSC infusion Treatment until HRSC injection
Patient A
25 year old male
Severe Aplastic Anemia ㆍ Diagnosed as SAA in 2002
ㆍ show weakness and breathing difficulties
ㆍ Frequent occurrence of rectal and gum bleeding and vomiting
ㆍ Receiving 4 units of blood and 2 units of platelets per month
Signs of jaundice with serious eye congestion
ㆍ Anabolic steroids (TabMenabol was administered for 8 months but not significantly improved.
Patient B
26 year old woman
Hypoplastic anemia ㆍ diagnosis of hypoplastic anemia in January 2004
ㆍ Emergence of frequent menstruation for 6 months
Patient with a single pre-RSC infusion in which 4 units of erythrocyte packs and 6 units of platelets were transfused

ㆍ Received 3 months Haematics dose before RSC injection, but no response at all
Patient C
19 year old male
Most severe aplastic anemia ㆍ Diagnosed with the most severe Aplastic Anemia in 2003
ㆍ Anemia, weakness, dyspnea during exercise
ㆍ Severe febrile neutropenia leads to persistent fever and chills for 10-15 days.
ㆍ Numerous infections, nausea and vomiting
Hip abscess
ㆍ Stroke twice due to cerebral hemorrhage
ㆍ Receives 5 units of blood and 2 units of platelets every 2 weeks.
ㆍ Last March, 2003, I received cyclosporine treatment for 6 months, but had no effect.

Patient D
35 year old man
Most severe aplastic anemia
Anemia, weakness, difficulty breathing
ㆍ Severe severe neutrophil leukopenia accompanied by fever, chills and numerous infections and bleeding
ㆍ Diagnosis 3 years ago
ㆍ Transfusion of 4 units of blood and 2 units of platelet per month
Cyclosporine and anti-tuberculosis therapy
There was no response to 6 months of immunosuppression

All clinical monitoring was performed by IIH / KEM staff. Transfusion requirements were determined before and after transfusion from transfusion records obtained after transfusion of any unit of blood product. The transfusion unit is used only after obtaining the consent of the immediate family member as a patient and witness. All blood products were examined at Tata Memorial Hospital prior to transfusion. In addition, the patient was questioned about happiness before and after the injection of reprogrammed cells. All patients received a record or a copy of their condition and all of the experimental and clinical follow-up. The monitoring period for these patients was initially extended to two years but extended. During the first month after transfusion, patients were admitted to the aseptic pressurization room of the hospital.

The patient, date of injection, body weight and kidney, collected mononuclear cells, and injected CD34 ⁺ cells Patient ID and date of injection weight kidney Collected mononuclear cells Injected CD34 ⁺ / kg Patient A
7/6/2004 48 152 1x10 ⁹ 11.7x10 ⁶ Patient B
7/14/2004 38 153 3.6 x ^{10 9} 20x10 ⁶ Patient C
7/27/2004 52 166 3.9x10 ⁹ 25x10 ⁶ Patient D
8/17/2004 52.5 160 4.3x10 ⁹ 23x10 ⁶

One million cells were stained according to the manufacturer's instructions using the following monoclonal antibody (all antibodies are DakoCytomation products):

Panel 1 - Isotype Negative Control Consists of IgG1-FITC, IgG1-PE-Cy5 and IgG1-RPE conjugates

Panel 2 - composed of anti-human CD45-FITC and CD34-RPE-Cy5

Panel 3 - composed of anti-human CD38-FITC and CD34-RPE-Cy5

Panel 4 - Consists of CD61-FITC and CD34-RPE-Cy5

Panel 5 - Consists of CD33 / 13 RPE and CD7-FITC

Panel 6 - composed of CD45 and glycophorin-A-RPE

Panel 7 - Consists of CD3-FITC and CD19-RPE.

Cell analysis was performed with a FACSCalibur system (BD bioscience) using BD cell Quest software.

For clone assays, patient bone marrow mononuclear cells (MNC) were inoculated before and after injection of reprogrammed cells into Methocult GFH4434 supplemented with recombinant growth factors (Stem Cell Technologies) according to the manufacturer's instructions. Differentiation into hematopoietic cell colonies was assessed according to the elapsed time using a phase contrast inverted microscope.

CBC, liver enzymes and hemoglobin variants of the patients were monitored continuously before and after the procedure. CBC, liver enzymes, hemoglobin variants and peripheral blood karyotype and G-fraction were monitored in independent laboratories for revalidation of patients after discharge. These tests were performed frequently after self-reprogrammed cell infusion.

Peripheral blood samples and bone marrow cells were analyzed before and after injection of self reprogrammed cells. This test was repeated every 6 months for the first year and every year for 2 years after initiation of self-reprogrammed stem cell therapy. In order to determine the stability of the cells, the reprogrammed cells were analyzed before injection, and further after a 3-hour transformation step as well as a maximum of one month of transformed cells. Karyotype and G-fraction were monitored in a third independent laboratory.

Bone marrow smears and perforation cuts were made before and after injection of self reprogrammed cells. This test was carried out 14 to 20 days after the injection of self-reprogrammed cells, and then every year thereafter.

All plated and perforated sections were scanned using a microscope hung on a camcorder before and after injection of the reprogrammed stem cells to evaluate and preserve engrafting records.

result

All patients underwent component harvesting and single reprogrammed stem cell injection procedures without side effects. Patients A and D did not need transfusion after a single infusion of reprogrammed hematopoietic stem cells (RHSC) (see Table 6 and Table 7). Platelet, neutrophil and red blood cell engraftment took 3 days and 6 days after injection in patients A and D, respectively. Patient A and Patient D showed fetal hemoglobin replacement (see Table 4 and Table 5), but not Patient B and Patient C (data not shown). Prior to infusion, liver enzymes were elevated in patients A and D although they were found negative for HCV (as measured by ELISA). Liver enzymes began to normalize after the injection of RHSC and reached the normal level 4 years after the injection of RHSC. Patients B and C died 2 and 6 months after injection, respectively. Patient 001 and 004 showed fetal hemoglobin replacement (see Table 6 and Table 7). These two patients showed long-term engraftment after a single injection of autologous HRSC.

Hemoglobin variants and hepatic enzymes in patients with severe Aplastic Anemia before and after injection of autologous HRSC
Blood test
Before injection 07/19/2004
After injection 05/26/2005
After injection 03/07/2006
After injection 01/05/2008
After injection WBC [10 ³ / μL] 1.3 3.4 3.1 3.7 5.4 HB [g / dL] 2.6 7.1 9 12 14 RBC [10 ⁶ / μL] 0.9 2 2.3 2.9 3.86 RETIC [%] One 4 1.7 MCV [fL] 101.5 101.5 121.74 100 101.4 MCH [Pg] 33.6 35.5 39.13 30.8 36.3 MCHC [g / dL] 33.8 35 32.14 30.8 35.8 Platelets [10 ³ / μL] 18 26 37 58 189 Neutrophils [10 ³ / μL] 0.32 1,000 1.054 1.184 1.994 ESO [10 ³ / μL] 0.62 0.74 0.65 BASO [10 ³ / μL] 0 0 0.054 LYMPH [10 ³ / μL] 1.500 1.922 2.220 2.214 MONO [10 ³ / μL] 0.500 0.062 0.222 0.324 HBA [g / dL] 5.1 6.7 9.2 12 HBA2 [g / dl] 0.13 0.14 0.24 0.39 HBF [g / dl] ND 1.2 2.4 2.6 1.82 SGOT [U / L] 150 127 74 32 30 SGPT [U / L] 145 181 49 37 44

WBC = leukocyte; HB = hemoglobin; RBC = red blood cells; RETIC = reticulocyte; MCV = mean erythrocyte volume; MCH = mean erythrocyte hemoglobin; MCHC = mean erythrocyte hemoglobin concentration; ESO = eosinophil; BASO = basophil; LYMPH = lymphocyte; MONO = monocytes; HB A = hemoglobin A; HBA2 = hemoglobin A2; HBF = fetal hemoglobin; SGOT = serum glutamic acid oxaloacetic acid transaminase; SGPT = serum glutamic acid pyruvic acid transaminase.

Hemoglobin variants and hepatic enzymes before and after the injection of autologous HRSC in patients with severe aplastic anemia D
Blood test
Before injection 09/28/2004
After injection 05/30/2005
After injection 03/07/2006
After injection 01/05/2008
After injection WBC [10 ³ / μL] 1.7 3 2.2 2.7 4 HB 3 11.1 7.7 11.5 13 RBC 3.6 2.4 3.2 3.45 RETIC [%] 2.4 3.4 1.7 MCV 90.3 108.33 113 110.3 MCH 30.9 32 35.9 37.8 MCHC 34.2 29.62 31.9 34.2 Platelets [10 ³ / μL] 5 30 20 25 68 Neutrophils [10 ³ / μL] 0.1 0.72 0.50 1.0 1.24 ESO [10 ³ / μL] 0.90 0.22 0.54 0.64 BASO [10 ³ / μL] 0 0 0 0 LYMPH [10 ³ / μL] 2.01 1.67 1.59 1.88 MONO [10 ³ / μL] 0.18 0.44 0.54 0.12 HBA 10.6 7.13 10.33 12.1 HBA2 0.33 0.19 0.28 0.36 HBF 0.11 0.2 0.38 0.9 0.42 SGOT 160 46 46 69 34 SGPT 150 57 32 60 46

WBC = leukocyte; HB = hemoglobin; RBC = red blood cells; RETIC = reticulocyte; MCV = mean erythrocyte volume; MCH = mean erythrocyte hemoglobin; MCHC = mean erythrocyte hemoglobin concentration; ESO = eosinophil; BASO = basophil; LYMPH = lymphocyte; MONO = monocytes; HB A = hemoglobin A; HBA2 = hemoglobin A2; HBF = fetal hemoglobin; SGOT = serum glutamic acid oxaloacetic acid transaminase; SGPT = serum glutamic acid pyruvic acid transaminase.

Flow cytometry analysis of collected mononuclear cells 3 hours before and after induction of hematopoietic reprogramming is shown in FIG. The number of CD34-positive cells generated after hematopoietic programming is listed in Table 4. A representative flow cytometric analysis of the harvested mononuclear cells before and after hematopoietic reprogramming showed significant increases in CD34-positive cells with and without expression of CD45, CD38 and CD7 (FIG. 1). At the time of injection, CD34 cells circulated peripheral blood for 3 to 6 days and then CD34 cells were maintained at a sustainable level as shown by a significant increase in CD33 & 13 expressing cells with and without CD7 with high anterior and lateral scattering (Fig. 2). This pattern of reprogramming was observed in all patients.

No colony was formed from the patient's bone marrow aspirate prior to injection of autologous HRSC after inoculation into methylcellulose cell culture (Figure 3). In the clone assay, only patient B suffering from hypomorphic anemia showed decreased hematopoietic function. However, 14 to 20 days after injection, all the bone marrow aspirates obtained from the patients were reduced and formed a normal range of various hematopoietic colonies with a slight increase in the number of striae-forming red blood cells. Between 14 and 20 days after injection of the autologous HRSC, bone marrow smear and perforation slice (FIG. 3) showed significant increases in bone marrow cells at various stages of differentiation with mature and immature megakaryocytes in all patients when compared to baseline . In addition, erythrocyte hypermethylation appeared at various stages of differentiation in all samples.

The karyotype and G-partition pattern of peripheral blood or bone marrow samples did not change before and after injection of autologous HRSC (in Patient A and Patient D for up to 4 years) in all patients (see FIG. 4).

After implantation of autologous HRSCs in patients with aplastic anemia, primordial (CD38 negative) and murine (CD38 positive) CD34 cells circulated peripheral blood for 3 days before reprogramming into bone marrow cells (FIG. 2). Bone marrow engraftment after injection of HRSC in patient A, patient B and patient D took 3 days. On the other hand, in patient 003, bone marrow engraftment was observed on day 20 when analyzed by flow cytometry. Single infusion of HRSC without any pretreatment regimen leads to prolonged engraftment in two of four patients with aplastic anemia. Patients A and B showed prolonged engraftment without any blood transfusion after HRSC injection. Neutrophils, red blood cells, and platelet engraftment in these patients are accompanied by a shift or increase in Hb F hemoglobin levels (Table 6 and Table 7). This did not appear in the other two patients who died. The conversion of Hb F in these two patients demonstrates the reprogramming ability and consequential engraftment and reconstitution of injected HRSCs towards the inflammatory Hb phenotype as observed in cord blood stem cell transplants (Elhasid et al., Leukemia 2000, 14 : 931-934; Locatelli et al., Bone Marrow Transplant 1996, 18: 1095-101).

Long-term engraftment with preservation of chromosome numbers and chromosome breakdown reflects the fact that it is obviously safe to inject HRSC into blood conditions where clonal evolution rarely occurs in conventional therapeutic regimens.

Importantly, as seen in winter stem cells, autologous HRSC could induce long-term engraftment and survival in patients with severe poorly regenerated anemia, even if no immunosuppressive therapy was used.

Briefly, bone marrow analysis of injected patients at 14 days after injection shows that the adipocytes and stromal cells, which are the dominant species of severe aplastic anemia, are reduced, and at various stages of differentiation, the bone marrow cell integrity, Showed an increase in the giant nuclear system. There was a significant increase in the red blood cell volume of the bone marrow with a corresponding increase in reticulocyte count as well as hemoglobin level. There was also a steady increase in fetal hemoglobin (an important component when sickle cell anemia and beta mediterranean anemia relaxed) and fetal hemoglobin-expressing red blood cells after infusion. In addition, there was a significant improvement in red cell counts as determined by red cell size, hemoglobin content, and concentration. Reprogrammed cells showed normal karyotype and gene stability after injection. Finally, engraftment and prolonged repopulation were observed in three or more patients with aplastic anemia.

Example  2

Materials and methods

Self-reprogrammed hematopoietic cells (target cells) were tested in 21 patients with beta-mediated anemia. 19 are beta - mediated anemia and 2 are beta - mediated anemia. One of the mid-stage beta thalassemia patients was Mediterranean anemia / Hb E aneurism (common in Far Eastern and Indian patients) and the other one was Mediterranean anemia / sickle cell anemia.

The whole blood of each patient was treated 2 to 3 times to collect the components. The leukocytes were reprogrammed to produce self reprogrammed cells until the target cells were able to attain the characteristics described above. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

After reprogrammed cells were injected into the patients with beta - mediterranean anemia, they were measured by bio - signal monitoring, echocardiography, bone mineral density, liver and kidney enzymes, karyotype and G - fraction, and no toxicity or side effects were observed. In the case of severe anemia of beta aneurysm, the mean reduction rate (50%) was statistically significant when compared with the reference value at 9 months after the injection of reprogrammed cells. Two midterm beta-mediastinum patients (one with Mediterranean anemia / HBE and the other with Mediterranean anemia / sickle cell anemia) are now in a state where they are not required to receive a transfusion nine months after the injection of reprogrammed cells.

The mean weight and height after injection of the reprogrammed cells were significantly increased compared to baseline and the organ size of spleen and / or liver enlarged Mediterranean anemia patients was normalized. The absolute mean concentration of fetal hemoglobin was significantly increased when compared to baseline levels of reprogrammed cells in both severe and mid-season patients (Fig. 5). In addition, the red blood cell mean index reflected by the hemoglobin content (FIG. 6) and the concentration (FIG. 7) by the red cell size was significantly improved as compared with the reference value.

Finally, the mean serum ferritin (biomarker for iron overload) was significantly reduced in patients with Mediterranean anemia following the injection of reprogrammed cells (Figure 8). Iron overload is a major cause of mortality and morbidity in patients with Mediterranean anemia, patients with sickle cell anemia, and iron overload-induced disorders after transfusion.

Example  3

Materials and methods

The whole blood of 2 diabetic patients was treated 2-3 times to collect the components. The collected leukocyte cells were reprogrammed to produce self-reprogramming mesenchymal stem cells, pluripotent stem cells and islet cells (target cells) until the target cells exhibiting the above-mentioned characteristics were generated. Self-reprogrammed cells were administered intravenously through the patient's arm or thigh jugular vein.

result

After infusion of self-reprogrammed cells, the patient synthesized normal levels of insulin as a result of fasting and 90-minute feeding stimulated c-peptide measurements. This normal level of c-peptide was maintained for up to 3 months after the injection of reprogrammed cells (Figure 9). In addition, Hb A1C levels indicating glucose control were normalized after injection of reprogrammed cells (Figure 10). For example, patients with Hb A1c> 10% prior to injection show a 5.8% Hb A1C present after injection of reprogrammed cells. In addition, blood glucose levels in these patients have reached normal levels after infusion compared to baseline. Also, insulin intake / injection in diabetic patients significantly reduced after reprogrammed cell infusion.

Example  4

Materials and methods

We injected self reprogrammed cells into four patients with amyotrophic lateral sclerosis (ALS). Diagnosis of this disease is not related to specific biomarkers. The disease is diagnosed by clinical exclusion of other similar disorders.

The whole blood of the patient was treated 2 to 3 times to collect the components. The collected leukocyte cells were reprogrammed to obtain autonomously reprogrammed pluripotent stem cells, alveolar epithelial cells and neurons (target cells) until the target cells exhibiting the above-mentioned characteristics were expressed. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

Patients treated with self-reprogrammed cells who had ALS had a significant improvement in lung function tests (PFT). This obstacle in lung function tests is one of the causes of premature death. Most of the patients showed decreased glandular sensation in the limbs and neck, and some patients reported improved language abilities. Other patients showed improvement in their ability to lift their heads as well as ability to walk.

Example  5

Materials and methods

Whole blood of four patients with Parkinson's disease was treated 2-3 times to collect the components. The collected leukocyte cells were reprogrammed to produce autonomously reprogrammed pluripotent stem cells and neurons (target cells) until the target cells exhibiting the characteristics described above were generated. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

Patients with marked tremors due to Parkinson's disease had a significant reduction in tremor and reduced the usual drug input used to manage the disease. The first patient received four doses of cinemetin (a dopaminergic agent) every day, and only one tablet daily after four months of transfusion.

Example  6

Materials and methods

The whole blood of two spinal cord injury patients was treated 2-3 times to collect the components. The collected leukocyte cells were reprogrammed until the target cells exhibiting the above-described characteristics were generated to obtain self-reprogrammed pluripotent stem cells and neurons (target cells). Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

One patient was not followed up. Another patient was a C5-C6 spinal cord injured limb paralysis patient. This patient could not sit or turn on the bed. After treatment, the patient could sit upright for a very long time and turn the body on the bed. Also, the patient could stand alone by leaning against the wall. In addition, the patient was able to move the toes and report the sensation of the bladder.

MRI analysis before and after implantation of reprogrammed cells showed a slight reduction in lesion size after injection of reprogrammed cells. Patients started to receive active physical therapy and feel much better than before.

Example  7

Materials and methods

Reprogramming the collected leukocyte cells until the expression of the target cells exhibited the above-mentioned characteristics, self-reprogrammed pluripotent stem cells, mesenchymal stem cells, and skeletal muscle cells (target cells) MD) patients were treated. The first patient had a zone MD, and the patient's disease was the most severely affected muscle, a group of MDs that generally had hips and shoulders, and the second patient had four malignant MDs.

The whole blood of the patient was treated 2 to 3 times to collect the components. Self-reprogrammed cells were generated by reprogramming in accordance with the present invention. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

The level of muscle enzyme creatine phosphokinase (CPK) can be monitored to measure MD-associated atrophy. The enzyme decreased in response to the injection of degenerated stem cells (Fig. 11). In addition, the lactate dehydrogenase system, which is an enzyme that rises upon tissue damage, is decreased (Fig. 11). In addition, liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are associated with inflammation and damage of hepatocyte and bone cell, have been reduced in patients.

In addition, patients showed improved mobility as a result of camming before and after injection of reprogrammed cells and improved lung function tests after reprogrammed cell infusion compared to baseline.

Example  8

Materials and methods

Reprogramming the collected leukocyte cells until the target cells exhibiting the above-mentioned characteristics are reproduced to obtain self-reprogrammed pluripotent stem cells, mesenchymal stem cells, and kidney cells (target cells) . The whole blood of the patient was treated 2 to 3 times to collect the components. Self-reprogrammed cells were generated by reprogramming in accordance with the present invention. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

Patients who were injected with self reprogrammed cells had improved levels of various body fluid markers (eg, urinary excretion, serum creatinine and BUN or UREA) as indicators of healthy kidney function. For example, a 75-year-old male patient with diabetes showed improved kidney function at 24 months after treatment with self-reprogrammed cells (see Table 8). This patient showed elevated levels of hemoglobin and insulin-like growth factor-1 (IGF-1), the protein molecules in the red blood cells that carry oxygen. The patient also showed a decrease in phosphorus, an organic compound, urea, creatinine, a degradation product of creatine phosphate in muscle, uric acid, a secreted organic compound in urine, and bone; The high amount of these markers is an indicator of poor kidney function. In addition, the patient was a form of hemoglobin and showed a decrease in glycated hemoglobin (HbA1C), an indicator of plasma glucose levels in people with diabetes.

Similarly, a 51-year-old diabetic male showed a similar improvement at 12 days after treatment (Table 8). This patient showed reduced levels of hemoglobin and reduced levels of creatinine, HbA1C and blood urea nitrogen (BUN) (measuring the amount of nitrogen in the blood in the form of urea).

Renal function marker levels in two diabetic male patients
75-year-old diabetic male 51-year-old diabetic male Reference value 24 months after treatment Reference value 12 months after treatment Hemoglobin [g / dl] 8 12.9 12 14.5 Element [g / dl] 190 38 n / a n / a Creatinine [mg / dl] 12 1.9 15 4.5 Uric acid [mg / dl] 8.5 4 n / a n / a Phosphorus [mg / dl] 6.1 3.8 n / a n / a HbA1C [%] 14.4 6.1 12 6 IGF-1 [ng / dl] 45 112 n / a n / a BUN [mg / dl] n / a n / a 79 19

HbA1C = glycosylated hemoglobin; IGF-1 = insulin-like growth factor 1; BUN = blood urea nitrogen

Analysis of 12 type II diabetic patients after self-reprogrammed cell therapy showed reduced levels of microalbumin (Fig. 13), which is associated with urinary albumin excretion and not only renal disease but also vascular endothelial cells Dysfunction and cardiovascular disease. In addition, 12 patients showed reduced levels of HbA1C (Figure 14).

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

A 45-year-old female patient with autoimmune glomerulonephritis Reference value 18 months after treatment Hemoglobin [g / dl] 9 11.5 Creatinine [mg / dl] 9.3 2.1 BUN [mg / dl] 89 15

BUN = blood urea nitrogen

In addition, a 59-year-old female patient with end-stage renal disease showed improved levels of creatinine, urea, hemoglobin and parathyroid hormone (PTH) following treatment with reprogrammed cells (Table 10). Kidney function marker levels in a 45-year-old female patient with autoimmunity. In addition, the patient had previously undergone hemodialysis 12 times per month but was reduced by about 8 after treatment.

Kidney function marker levels before and after treatment of a 59-year-old female patient with end-stage renal disease with reprogrammed cells. Kidney function marker
Before Treatment After treatment 03/02/2009 04/29/2009 05/23/2009 08/05/2009 08/11/2009 Hemoglobin [g / dl] 9.8 9.2 11.5 TLC / WBC (per 占 퐇) 3800 6800 5100 PLT (per 占 퐇) 361000 273000 348000 HbA1C [%] 5.6 AST [U / l] 25 16 14 ALT [U / l] 49 11 12 Element [mg / dl] 90 70 22.13 Creatinine [mg / dl] 14.5 11.2 11.86 8.86 9.58 Serum albumin [g / dl] 3 3.4 3.3 3 PT [sec] 31.2 INR 2.71 Calcium [mg / dl] 8.4 9.5 9.4 8.7 9.5 Phosphorus [mg / dl] 8 9.2 5.9 Potassium [mmol / dl] 5.9 5.2 6.9 4.1 Sodium [mmol / dl] 133 136 Uric acid [mg / dl] 7.9 6.1 7.5 4.8 PTH [pg / ml] 195.6 108.2

TLC = total leukocyte count; WBC = leukocyte; PLT = platelets; HbA1C = glycosylated hemoglobin; AST = aspartate aminotransferase; ALT = alanine aminotransferase; PT = prothrombin time; INR = International normalization ratio (blood coagulation); PTH = parathyroid hormone

In addition, a 46-year-old patient with chronic renal failure due to diabetes had improved creatinine, urea and heroglobin levels after treatment with reprogrammed cells (Table 11). The patient received hemodialysis 12 times per month, but received only about 5 times after the treatment, and received 4,000 units of epoetin alpha (EPREX ^® ) twice daily for 4,000 units.

A 46-year-old patient with chronic renal failure was treated with renal function marker levels before and after treatment with reprogrammed cells Kidney function marker
Before Treatment After treatment 09/21/2008 02/24/2009 04/20/2009 Hemoglobin [g / dl] 6.5 10.3 9.4 TLC / WBC (per 占 퐇) 5500 8200 10100 PLT (per 占 퐇) 159000 246000 300000 FBS [mg / dl] 133 120 121 2 hours PP [mg / dl] 170 172 169 AST [U / L] 9 34 32 ALT [U / L] 5 22 15 Element [mg / dl] 109 51 65 Creatinine [mg / dl] 7.5 4.9 4 Serum albumin [g / dl] 3.5 3.7 4 PT [sec] 12.7 12 11 INR One One One Calcium [mg / dl] 10 9.9 11 Phosphorus [mg / dl] 6 5.3 5 Potassium [mmol / L] 6.2 5.1 4.7 Magnesium [mg / dl] 2.3 2.4 2.8

TLC = total leukocyte count; WBC = leukocyte; PP = postprandial; PLT = platelets; FBS = fasting blood glucose; AST = aspartate aminotransferase; ALT = alanine aminotransferase; PT = prothrombin time; INR = International normalization ratio (blood coagulation); PTH = parathyroid hormone

Example  9

Materials and methods

Multiple sclerosis patients were treated with self reprogrammed pluripotent stem cells, mesenchymal stem cells and neurons (target cells). Target cells were obtained by reprogramming the collected leukocyte cells until the target cells exhibiting the above-mentioned characteristics were generated. The whole blood of the patient was treated 2 to 3 times to collect the components. Self-reprogrammed cells were generated by reprogramming in accordance with the present invention. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

Patients treated with self-reprogrammed cells showed less brain and spinal cord lesions. Reduction of lesion occurred within 3 months after treatment (Fig. 15a-b). The reduction of brain tissue damage occurred within 6 months (Fig. 16a-b).

In addition, patients treated with self-reprogrammed cells showed spinal cord lesions removed (Fig. 17a-b). In addition, these patients showed improved Kurtzke Comprehensive Disability Scale (EDSS) scores, alleviated the disease, and did not receive conventional therapy for four years after a single infusion.

Example  10

Materials and methods

HIV female patients were treated with self-reprogrammed hematopoietic stem cells. The whole blood of the patient was treated 2 to 3 times to collect the components. Target cells were obtained by reprogramming the collected leukocyte cells until the target cells exhibiting the above-mentioned characteristics were generated. Self-reprogrammed cells were injected intravenously through the patient's arm or thigh jugular vein.

result

Screening tests for HIV-1 and HIV-2 antibodies prior to treatment showed an assay value of 3.68. A test value of 1.0 or higher is found to be positive.

At 2 months after treatment of self-reprogrammed cells, the screening test for HIV-1 and HIV-2 antibodies was 0.46, indicating that the patient had no positive results for HIV-1 and HIV-2 . At 6 months after treatment, the test for HIV-1 and HIV-2 screening was 0.48, which also proves that the patient is not positive for HIV.

Example  11

Therapeutic effects of self reprogrammed cells have also been demonstrated in patients with other conditions and diseases. The target cells listed herein were reprogrammed to obtain target cells until the target cells exhibiting the above-mentioned characteristics were expressed.

Congestive heart failure

A 61 year old male patient with congestive heart failure was injected with self reprogrammed myocardial cells, pluripotent stem cells, mesenchymal stem cells and endothelial cells. This treatment is called ejection fraction (EF); Prenatal sodium natriuretic peptide precursor levels (Pro BNP), a predictor associated with coronary artery disease; Fasting blood glucose level; And HbA1C levels (see Table 12). In addition, previously dilated hearts were demonstrated in Table 12 by reduction in end-diastolic LV ID / D, end-systolic LV ID / L, and end-diastolic LV septal thickness (IVSD) As shown in Fig.

A 61-year-old patient with congestive heart failure was treated with coronary artery disease markers Coronary artery disease
Marker Before Treatment
1 month After treatment
1 month After treatment
2 months After treatment
4 months After treatment
6 months After treatment
10 months LVID / D [cm] 6.5 6.3 5.9 6.9 6.5 5.7 LVID / S [cm] 6.1 5.6 5.4 5.9 5.7 4.9 EF 15 18 22 29 33 45 IVSD [cm] 1.3 1.3 1.2 1.2 0.6 1.0 LVPWD [cm] 0.7 0.6 0.6 0.8 0.6 1.0 Left ventricular mass
Index [gm / m] 283
141.5 251
133.5 209.5
111.4 167
88.8 151.5
81 Pro BNP
[pg / ml] 2969 800 600 600 600 497 Blood glucose (fasting)
[mg / dl] 244 118 145 87 95 80 HbA1C [%] 9.2 nd nd 7.2 6.5 6

LVID / D = end-diastolic left ventricular internal diameter; LVID / S = end-diastolic LV diameter; EF = ejection fraction; IVSD = end-diastolic left ventricular septal thickness; LVPWD = posterior wall thickness of left ventricle; Pro-BNP = brain sodium natriuretic peptide precursor; HbA1C = glycosylated hemoglobin

Hepatitis C

13 reprogrammed hematopoietic cells, pluripotent stem cells, mesenchymal stem cells and hepatocytes were injected into 13 patients with hepatitis C virus (HCV) infection and with beta median anemia. The therapeutic effects not only showed reduced or eliminated HCV levels (Table 13), but also improved blood markers such as liver enzymes, bilirubin, albumin, prothrombin time and international normalization ratio (blood coagulation) (see Table 14) . In particular, patients were normalized for liver enzyme alanine aminotransferase (ALT) and aspartate aminotransferase (AST) associated with both hepatocyte inflammation and injury (Figs. 18a-b).

After 12 patients infected with hepatitis C virus were treated with self-reprogrammed stem cells,
Patient ID Reference value
Virus volume x 1000 3 months after injection
Virus volume x 1000 One 0 0 2 164 82 3 3 0 4 123 16 5 16 0 6 2 0 7 0 0 8 7.7 million 7.7 million 9 152 106 10 63 27 11 387 97 12 0 0 13 17 0

The blood markers of infected patients with hepatitis C virus and those with liver cirrhosis and the post-treatment screening of reprogrammed cells Blood marker Reference value 3 months after injection Hemoglobin [g / dl] 9.8 12.9 Platelets (per 占 퐇) 65000 120000 WBC (per 占 퐇) 4300 7200 Blood sugar (fasting) [mg / dl] 210 110 Blood sugar (2 hours after meals) [mg / dl] 190 134 ALT [U / L] 50 24 AST [U / L] 32 31 Bilirubin (whole) [mg / dl] 6.9 3.2 Bilirubin (direct) [Mg / dm] 3.1 1.8 Serum albumin [g / l] 1.9 3.1 Element [mg / dl] 60 36 Creatinine [mg / dl] 1.6 1.1 INR 1.8 1.0 PT [sec] 18 12.8

WBC = leukocyte; PP = postprandial; ALT = alanine aminotransferase; AST = aspartate aminotransferase; INR = International normalization rate (blood clotting); PT = prothrombin time

For example, a 44-year-old male patient with liver cirrhosis due to hepatitis C virus infection has been shown to improve liver enzymes alanine aminotransferase and aspartate aminotransferase, international normalization ratio (blood coagulation), bilirubin and albumin, (See Table 15). In fact, the patient was on albumin therapy before being treated with reprogrammed cells, but was not treated with albumin after treatment with reprogrammed cells.

The blood markers of a 44-year-old male patient with hepatic cirrhosis due to hepatitis C virus infection and a post-treatment test of reprogrammed cells Blood marker
Reference value After treatment 03/30/2009 04/04/2009 04/23/2009 05/06/2009 06/22/2009 hemoglobin
[g / dl] 10.9 10.4 11.6 13.4 10.2 TLC (per 占 퐇) 2500 2700 6100 4700 2800 PLT (per 占 퐇) 20000 17000 43000 50000 27000 AST [U / L] 212 170 131 141 120 ALT [U / L] 62 59 48 51 53 Alkaline phosphatase [U / L] 196 147 58 GGT [U / L] 49 70 63 35 Bilirubin T [mg / L] 12.7 10.9 11.2 8.1 8.8 Bilirubin D [mg / Dl] 6.8 5.4 4.71 4.9 4 Element [mg / dl] 13 18 20 16 25 Creatinine [mg / dl] 0.7 0.7 0.8 0.7 0.7 Serum albumin [g / L] 2.8 2.5 2.9 2.4 1.9 RBS [mg / dl] 212 154 158 188 174 INR 1.9 1.8 1.72 1.69 1.72 Potassium [mmol / L] 3.2 3.8 4.4 Sodium [mmol / L] 134 141 137 Uric acid [Mg / dl] 3.5 2.4 3 Alpha-petrotein normal Cryoglobulin voice

TLC = total leukocyte count; PLT = platelets; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma-glutamyltransferase; RBS = random blood sugar; INR = International normalization ratio (blood coagulation)

Head trauma

The head trauma patients were treated with self - reprogrammed all - embryonic stem cells. This treatment induced regeneration of damaged brain tissue (Fig. 19a-b), ejection fraction (EF), brain sodium natriuretic peptide precursor level (Pro BNP), a predictor associated with coronary artery disease; Fasting blood glucose level; And HbA1C levels (see Table 10).

Lung disease

Patients suffering from inhibitory pulmonary disease associated with motor neuron disease were treated with autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, alveolar epithelial cells and endothelial cells. After 6 months of treatment, the maximum FVC, which is the amount of air exhaled out of the lungs, increased from 50% to 71%, while the amount of air that can be released in one second The FEV ₁ increased from 64% to 68%. The percentage of FEV ₁ to FVC, which is about 75-80% in healthy adults, decreased from 100% to 82%. X-ray scan of the lungs revealed that the opacity of the lungs after treatment was reduced (Figure 20).

Menopause

51 - year - old menopausal patients were administered self - reprogrammed pluripotent stem cells, pluripotent germ cells and oocytes. After treatment, the patient was found to have increased levels of several hormones and proteins, including insulin-like growth factor (IGF-1), ester diol and low density lipoprotein (LDL) (see Table 16).

Effects of Self-Reprogrammed Stem Cells on Menopausal Patients Blood marker
Before Treatment After treatment 11/23/2006 12/09/2006 01/01/2007 02/17/2009 IGF-1 [ng / ml] 91 53 100 143 IGF-1-binding [mg / L] 5.3 4 4.5 5.9 GH [占 퐂 / L] 2.4 3.5 3.5 1.5 Progesterone [ng / ml] 1.2 1.5 17.5 2.25 Ester diol [pg / ml] 26 82 162 168.82 Cortisol [[mu] g / dl] 17.8 24 4.3 24.5 DHEA-S [Nmol / l] 2.2 3.6 0.4 5.3 Adrenocort [pmol / l] 21.1 10 10 Thioglobulin [ng / ml] 47 18 31 1.4 Calcitonin [ng / ml] 28 40 50 FSH [IU / l] 41.2 5.4 3.88 LH [IU / l] 15.1 1.8 6.76 Prolactin [/ / l] 16.2 11.1 10.5 0.6 Glass test [pmol / l] 8 6.4 7.5 Cholesterol [mg / dl] 242 258 280 268 HDL [mg / dl] 70 76 83 63.22 LDL [mg / dl] 22 162 173 179.9 Triglycer [mg / dl] 111 102 122 125 HbA1C [%] 6.5 5.3 5.5 5.4 Fbs [Mg / dl] 94 90 102 103 Inhibin [U] 40 30 Osteocalcin [ng / mL] 6.9 TSH [nu / ml] 0.84 0.7 0.5 FT3 [pg / ml] 3.1 2.7 2.7 1.54 Calcitonin [ng / l] 28 40 50 FT4 [pg / ml] 1.4 0.94 0.71 0.82

IGF-1 = insulin-like growth factor 1; IGF-1-bind = insulin-like growth factor binding; GH = growth hormone; DHEA-S = dehydroepiandrosterone sulfate ester; adrenocort = corticosteroid hormone; FSH = follicle stimulating hormone; LH = luteinizing hormone; HDL = high density lipoprotein; LDL = low density lipoprotein; triglycer = triglyceride; HBA1C = glycosylated hemoglobin; FBS = fetal blood collection; TSH = thyroid stimulating hormone; FT3 = free triiodothyronine; FT4 = glassylaoxine

depression

Patients with depression were injected with self-reprogrammed pluripotent stem cells and nerves. After treatment, the patient was admitted with increased levels of several hormones and proteins including insulin-like growth factor-1 (IGF-1), cortisol and testosterone (See Table 17).

The effect of self-reprogrammed cells in menopausal patients Blood marker
Before Treatment After treatment 06/11/2007 07/23/2007 GH [/ / l] &Lt; 0.05 0.06 IGF-1 [ng / ml] 93.4 140 IGF-bp [mg / ml] 4.4 5.6 Cortisol [[mu] g / dl] 4 20.9 Adrenocort [pg / ml] <10 17.1 Shbg [nmol / l] 37.9 39.8 FSH [IU / L] 4.94 4.98 LH [IU / L] 10.1 3.72 e2 [pg / ml] 29 <28 Testosterone [pmol / l] 28.4 57.8 Prolactin [/ / l] 15.7 1.6 DHEA-S [μmol / l] 2.3 2.9 TSH [μU / ml] 1.707 3.318 FT3 [pg / ml] 1.69 1.87 FT4 [ng / dl] 0.81 0.89

GH = growth hormone; IGF-1 = insulin-like growth factor 1; IGF-bp = insulin-like growth factor binding protein; Adrenocort = Corticosteroids; Sh-bg = sex hormone-binding globulin; FSH = follicle stimulating hormone; LH = luteinizing hormone; e2 = estradiol; DHEA-S = dehydroepiandrosterone sulfate ester; TSH = thyroid stimulating hormone; FT3 = free triiodothyronine; FT4 = glassylaoxine

Non-clogging  Amenorrhea

Allogeneic stem cells, pluripotential cells and spermatozoa were injected and self-reprogrammed into patients with non-obstructive azoospermia. After treatment, the patient showed an increase in testosterone throughout the 8 months (Figure 21).

Visual impairment

Patients who had lost sight in a previously benign benign tumor were treated with autologous reprogrammed pluripotent stem cells and neurons. After treatment, the patient showed increased retin sensitivity and improved vision (Fig. 22).

It is to be understood that the specification sets forth the preferred embodiments of the invention in detail and that such specific embodiments are not intended to limit the invention as defined herein and that many obvious modifications of the invention may be possible within the spirit and scope of the invention shall.

Claims (55)

The cells or tissues of the patient's cell line, including the reprogrammed cells obtained by reprogramming the comitted cells of the first cell lineage, are supplemented to produce aplastic anemia, beta-mediterranean anemia, Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and / or prophylaxis of atopic dermatitis (ALS), Parkinson's disease, spinal cord injury, muscular dystrophy, renal disease, multiple sclerosis, congestive heart failure, hepatitis C virus, human immunodeficiency virus, head trauma, Disorder or condition characterized by tissue damage, wherein the disorder is selected from the group consisting of:
The reprogrammed cell
(i) culturing the feed cells in a tissue culture medium,
(ii) contacting the population of cells with an inverse dividing agent that engages a receptor mediating capture, recognition, or presentation of the antigen on the surface of the population of cells to obtain a degenerated cell population of the patient; and
(iii) contacting the de-differentiating cell clone with an agent selected from the group consisting of an anticoagulant, a chelating agent, an antibiotic, a vitamin, an inorganic compound, a natural or synthetic hormone, an amino acid, a chemical compound, a nucleoside or an analogue thereof, an acid or a salt thereof, Comprising contacting the cell with a selected differentiation promoter to obtain a reprogrammed cell population of the patient, wherein the reprogramming cell population is obtained by a method of reverse differentiation and regeneration,
Wherein the differentiation promoter is independent of the reverse &lt; RTI ID = 0.0 &gt; agent. &Lt; / RTI &gt; The method according to claim 1, wherein the reprogrammed cells are selected from the group consisting of pluripotent stem cells, hematopoietic stem cells, neural stem cells, epithelial stem cells, mesenchymal stem cells, endoderm and neuroectodermal stem cells, Neuronal cells, melanocytes, osteocytes, dermal papilla cells, cartilage cells, adipocytes, endothelial cells, myocardial cells, endothelial cells, endothelial cells, endothelial cells, Cell and nutritive mother cells. The method according to claim 1 or 2, wherein said feeder cells are selected from the group consisting of T cells, B cells, eosinophils, basophils, neutrophils, guk cells, mononuclear cells, erythrocytes, granulocytes, mast cells, lymphocytes, leukocytes, platelets, &Lt; / RTI &gt; 3. The pharmaceutical composition according to claim 1 or 2, wherein the receptor is an MHC class I antigen or an MHC class II antigen. 3. The pharmaceutical composition according to claim 1 or 2, wherein said de-differentiating agent is a monoclonal antibody to a receptor. 6. The pharmaceutical composition according to claim 5, wherein said antibody is selected from the group consisting of monoclonal antibody CR3 / 43 and monoclonal antibody TAL1B5. 3. The pharmaceutical composition according to claim 1 or 2, wherein the reprogrammed cell is suitable for injection or implantation. 8. The pharmaceutical composition according to claim 7, wherein the reprogrammed cells are suitable for parenteral, intramuscular, intravenous, subcutaneous, intravaginal, oral, percutaneous or injection into spinal fluid. 3. The method according to claim 1 or 2, wherein the tissue culture medium is selected from the group consisting of Iscove transformed Dulbecco's medium (IMDM), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential (EME) Wherein the pharmaceutical composition is selected from the group consisting of a culture medium (e.g., MEM), a Roswell Park Memorial Institute (RPMI; Media Development Agency) 1640, Ham-F-12, E199, MCDB, Lobovitz L- Gt; The method according to claim 1 or 2, wherein the vitamin, the inorganic substance or a derivative thereof is selected from the group consisting of vitamin A, vitamin B ₃ , vitamin C, vitamin D ₃ , vitamin K, retinoic acid, nicotinamide, zinc or zinc compound, &Lt; / RTI &gt;
The natural or synthetic hormone is hydrocortisone or dexamethasone;
Wherein said amino acid or derivative thereof is selected from the group consisting of L-glutamine (L-glu), ergothionein (EGT), proline and non-essential amino acid (NEAA);
Wherein said chemical compound or derivative thereof is selected from the group consisting of? -Mercaptoethanol, dibutylcyclic adenosine monophosphate (dbcAMP), monothioglycerol (MTG), putrescine, dimethylsulfoxide (DMSO), hypoxanthine, adenine, Xylostamide and 3-isobutyl-1-methyl xanthine;
The nucleoside or its analog is 5-azacytidine;
Wherein said acid or its salt is selected from the group consisting of ascorbic acid, pyruvate, okadinic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), disodium EDTA, ethylene glycol tetraacetic acid (EGTA), anticoagulant citrate dextrose A (ACDA) And glycerophosphate;
Wherein said antibiotic or drug is selected from the group consisting of G418, gentamycin, pentoxifylline (1- (5-oxohexyl) -3,7-dimethylxanthine) and indomethacin;
Wherein the protein is a tissue plasminogen activator (TPA). 3. The method of claim 1 or 2, wherein the tissue culture medium is autologous plasma; Platelets; serum; Or serum of mammalian origin. 3. The pharmaceutical composition according to claim 1 or 2, wherein the cells are cultured in a blood bag, a scaffold, a tissue culture bag or a plastic tissue culture container. 13. The method of claim 12, wherein the tissue culture vessel is an adsorptive or non-adsorptive tissue culture vessel, or the tissue culture vessel is coated or uncoated, or the tissue culture vessel is selected from the group consisting of gelatin, collagen, &Lt; / RTI &gt; is coated with the agent of choice. 3. The method according to claim 1 or 2,
(a) cultured at a temperature of 18 to 40 캜; or
(b) cultured at a carbon dioxide level of 4 to 10%; or
(c) cultured at an oxygen level of 10 to 35%. comprising: (i) reprogramming a first cell line population to obtain a reprogrammed cell; and (ii) optionally, admixing the reprogrammed cell with one or more pharmaceutical excipients. CLAIMS 1. A method for obtaining a reprogrammed target cell for administration to a patient,
Said patient is suffering from a condition selected from the group consisting of myelogenous deficiency, hematologic condition, aplastic anemia, beta-mediterranean anemia, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney disease, multiple sclerosis, congestive heart failure, , Head trauma, lung disease, depression, obesity, menopause, menopause, rejuvenation, arousal ulcer, psoriasis, wrinkles, liver cirrhosis, autoimmune disease, hair loss, retinitis pigmentosa, crystalline dystrophy / blindness, diabetes and infertility Disorder or condition characterized by tissue damage selected from the group consisting of:
The reprogrammed cell may be a cell,
(i) culturing the feed cells in a tissue culture medium,
(ii) contacting the population of cells with an inverse splicing agent that binds to a receptor mediating capture, recognition, or presentation of an antigen at the surface of the population of cells to obtain a degenerated cell population of the patient, and
(iii) contacting the de-differentiating cell clone with an agent selected from the group consisting of an anticoagulant, a chelating agent, an antibiotic, a vitamin, an inorganic compound, a natural or synthetic hormone, an amino acid, a chemical compound, a nucleoside or an analogue thereof, an acid or a salt thereof, And obtaining the reprogrammed cell population of the patient by contacting the cell with a selected differentiation promoter, wherein the regenerated cell population is obtained by a method of reverse differentiation and regeneration, or conversion differentiation,
Wherein the differentiation promoter is independent of the reverse &lt; RTI ID = 0.0 &gt; reagent. &Lt; / RTI &gt; delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

Images