Mitochondrial enhancement therapy using stem cells enriched with functional mitochondria

Abstract

The present invention provides stem cells enriched with healthy, functional mitochondria and therapeutic methods utilizing such cells to alleviate the debilitating effects of debilitating conditions, including aging and aging-related diseases, as well as anti-cancer therapies, in subjects in need thereof.

Description

[Technical field]

[0001] The present invention relates to stem cells enriched with functional mitochondria and therapeutic methods utilizing such cells to reduce the debilitating effects of treatment of debilitating conditions including aging and aging-related diseases, as well as anti-cancer therapies. [Background technology]

[0002] Mitochondria are membrane-bound organelles ranging from 0.5 to 1.0 μm in diameter found in most eukaryotic cells. Mitochondria are found in nearly all eukaryotic organisms and vary in number and location depending on the cell type. Mitochondria contain their own DNA (mtDNA) and their own machinery for synthesizing RNA and proteins. Because mtDNA contains only 37 genes, most gene products in the mammalian body are encoded by nuclear DNA.

[0003] Mitochondria carry out many important tasks within eukaryotic cells, such as pyruvate oxidation, the Krebs cycle, and metabolism of amino acids, fatty acids, and steroids. However, the primary function of mitochondria is the generation of energy as adenosine triphosphate (ATP) through the electron transport chain and the oxidative phosphorylation system (the "respiratory chain"). Additional processes involving mitochondria include heat production, calcium ion storage, calcium signaling, programmed cell death (apoptosis), and cell proliferation.

[0004] ATP concentrations in cells are typically 1-10 mM. ATP can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as the energy source. To synthesize complex fuels into ATP, they must first be broken down into smaller, simpler molecules. Complex carbohydrates are hydrolyzed into simple sugars such as glucose and fructose. Fats (triglycerides) are metabolized to yield fatty acids and glycerol.

[0005] The overall process of oxidizing glucose to carbon dioxide is known as cellular respiration, and can produce approximately 30 molecules of ATP from a single molecule of glucose. ATP is produced by several different cellular processes. The three main pathways used to generate energy in eukaryotes are glycolysis and the citric acid cycle / oxidative phosphorylation (both components of cellular respiration) and beta-oxidation. The majority of this ATP production by non-photosynthetic eukaryotes occurs in mitochondria, which can occupy nearly 25% of the total volume of a typical cell. A variety of mitochondrial disorders are known to result from defective genes in mitochondrial DNA.

[0006] Our WO2016 / 135723 discloses mammalian bone marrow cells enriched with mitochondria for the treatment of mitochondrial diseases.

[0007] US2012 / 0058091 discloses a diagnostic and therapeutic treatment for mitochondrial disorders, which involves microinjecting heterologous mitochondria into oocytes or embryonic cells, where the heterologous mitochondria are capable of achieving at least a normal level of mitochondrial membrane potential in the oocyte or embryonic cell.

[0008] WO2001 / 046401 discloses embryonic or stem-like cells produced by interspecies nuclear transfer. The efficiency of nuclear transfer is enhanced by the introduction of compatible cytoplasmic or mitochondrial DNA (same species or similar to the donor cell or nucleus).

[0009] WO2013 / 002880 describes compositions and methods comprising bioenergetic agents for restoring the quality of aged oocytes, enhancing oocyte stem cells or improving derivatives thereof (e.g., cytoplasm or isolated mitochondria) for use in capacitation procedures.

[0010] US2013 / 0022666 provides compositions comprising a lipid carrier and mitochondria, as well as methods for delivering exogenous mitochondria to cells and for treating or reversing the progression of disorders associated with mitochondrial dysfunction in a mammalian subject in need thereof.

[0011] WO2017 / 124037 relates to compositions comprising isolated mitochondria or combined mitochondrial agents and methods of using such compositions to treat disorders.

[0012] US2008 / 0275005 relates to antioxidant compounds targeted to mitochondria.The compounds of the invention contain a lipophilic cation covalently attached to an antioxidant moiety.

[0013] US9855296 discloses a method for enhancing cardiac or cardiovascular function in a human subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a sufficient amount of isolated, substantially pure mitochondria to enhance the cardiac or cardiovascular function, the mitochondria being syngeneic or allogeneic.

[0014] US9603872 provides methods, kits, and compositions for mitochondrial replacement in the treatment of disorders resulting from mitochondrial dysfunction.The present invention also features methods for diagnosing neuropsychiatric disorders (e.g., bipolar disorder) and neurodegenerative disorders based on mitochondrial structural abnormalities.

[0015] US2018 / 0071337 discloses a therapeutic composition comprising human mitochondria isolated from cells and a pharma- ceutically acceptable excipient, where the mitochondria may be in a carrier comprising a lipid bilayer, a vesicle, or a liposome, with or without at least one polypeptide or glycoprotein.

[0016] US2001 / 0021526 provides cellular and animal models of diseases associated with mitochondrial defects. Cybrid cell lines are described that have utility as model systems for the study of disorders associated with mitochondrial defects.

[0017] WO2013 / 035101 to the present inventors relates to mitochondrial compositions and therapeutic methods using same, disclosing compositions of partially purified functional mitochondria and methods of using the compositions to treat conditions that benefit from increased mitochondrial function by administering the compositions to a subject in need thereof.

[0018] Attempts to induce the transfer of mitochondria into host cells or tissues have been reported. Most methods require active transfer of mitochondria by injection (e.g., McCully et al. Am J Physiol Heart Circ Physiol. 2009, 296(1):H94-H105). Transfer of mitochondria phagocytosed into vehicles such as liposomes is also known (e.g., Shi et al. Ethnicity and Disease, 2008;18(S1):43).

[0019] It has been shown that mitochondrial transfer can occur spontaneously between cells in vitro, but it has only been established that mtDNA is transferred, and not intact, fully functional mitochondria (e.g., Plotnikov et al. Exp Cell Res. 2010,316(15):2447-55; Spees et al. Proc Natl Acad Sci,2006;103(5):1283-8). Mitochondrial transfer in vitro by endocytosis or internalization has also been demonstrated (Clark et al., Nature,1982:295:605-607; Katrangi et al., Rejuvenation Research,2007;10(4):561-570).

[0020] US2011 / 0105359 provides a cryopreserved composition of cells in the form of self-sustaining bodies, as well as cellular and subcellular fractions.On the other hand, attempts to inject isolated mitochondria during early reperfusion for cardioprotection showed that freshly isolated mitochondria are required for cardioprotection, as frozen mitochondria failed to provide cardioprotection and showed significantly reduced oxygen consumption compared to freshly isolated mitochondria (McCully et al., supra).

[0021] WO2016 / 008937 relates to a method for the intercellular transfer of mitochondria isolated from a population of donor cells to a population of recipient cells, the method showing improved efficiency of the transfer of a quantity of mitochondria.

[0022] US2012 / 0107285 is directed to mitochondrial enhancement of cells. Certain embodiments include, but are not limited to, a method of modifying stem cells or administering modified stem cells to at least one living tissue.

[0023] Old age is one of the greatest known risk factors for many human diseases. Old age-related diseases are the most frequent diseases that increase in frequency as aging progresses. Essentially, old age-related diseases are complications that arise from aging. Old age-related diseases are distinct from the aging process itself, since all adult animals age, but not all adult animals experience old age-related diseases.

[0024] The decline in mitochondrial quality and activity is associated with normal aging and correlates with the development of a wide range of age-related diseases. Mitochondria contribute to certain aspects of the aging process, including cellular senescence, chronic inflammation, and age-dependent decline in stem cell activity. Abundant supporting evidence indicates that mitochondrial dysfunction occurs with age due to the accumulation of mitochondrial DNA mutations. Various mitochondrial DNA point mutations have been shown to increase significantly with age in human brain, heart, skeletal muscle, and liver tissues. An increase in the frequency of mitochondrial DNA deletions / insertions has been reported with aging in both animal models and humans. It has been hypothesized that the replication cycle and the accumulation of mitochondrial DNA mutations may be a conserved mechanism underlying stem cell aging, such that mitochondria affect or regulate some key aspects of aging (Sun et al., Cell, 2016, 61:654-66; Srivastava, Genes, 2017, 8:398; Ren et al., Genes, 2017, 8:397).

[0025] Cancer is caused by the uncontrolled proliferation of abnormal cells in the organs or tissues of the body. Various types of cancer treatments are available, including surgery, chemotherapy, radiation therapy, immunotherapy, targeted therapy, hormone therapy, and stem cell transplantation. Cancer treatments often cause severe adverse effects, including fatigue, nausea and vomiting, anemia, diarrhea, anorexia, thrombocytopenia, delirium, hair loss, fertility problems, peripheral neuropathy, pain, and lymphedema. These debilitating effects significantly reduce the quality of life of cancer patients. It is well known to use bone marrow cells to replenish the bone marrow of cancer patients with hematopoietic malignancies who have undergone bone marrow ablation. Bone marrow transplants most often use matched healthy donors. However, in cases such as multiple myeloma, autologous bone marrow can be performed. The use of bone marrow cells to treat non-hematopoietic cancers is not customary in the treatment of those patients.

[0026] There is an unmet need to improve the quality of life of subjects suffering from various conditions such as old age and old age-related diseases, as well as cancer patients undergoing chemotherapy or radiotherapy.Reversing the decline in mitochondrial function may slow down the effects of old age, and reduce the debilitating effects of old age-related diseases and anti-cancer treatments. Summary of the Invention

[0027] The present invention provides mammalian stem cells enriched with healthy functional mitochondria and methods for reducing the debilitating effects of many conditions, including aging and aging-related diseases and adverse events of anti-cancer treatment. Surprisingly, it has now been shown for the first time that transplanting active cells enriched with healthy mitochondria can significantly slow the progression of aging symptoms and aging-related diseases. Furthermore, mitochondrial enhancement therapy using stem cells enriched with healthy mitochondria can alleviate the debilitating effects of chemotherapy, radiotherapy, and / or immunotherapy with monoclonal antibodies in cancer patients undergoing anti-cancer treatment. In particular, the present invention provides compositions comprising stem cells, including autologous or donor stem cells enriched with functional mitochondria. These cells are useful for alleviating or reducing the effects of debilitating conditions when introduced into a subject to be treated.

[0028] In certain embodiments, subjects are treated with stem cells enriched with functional mitochondria obtained from healthy donors.Convenient sources of healthy donor mitochondria include, but are not limited to, placental mitochondria or mitochondria from blood cells.Therefore, the present invention provides a method of using allogeneic, autologous, or syngeneic "mitochondrial enriched" stem cells to treat or reduce the debilitating effects of aging and aging-related diseases and anti-cancer treatment in cancer patients.

[0029] The present invention is based, in part, on the discovery that aged C57BL mice that receive healthy mitochondrial-enriched bone marrow cells from full-term mouse placentas exhibit improvements in functional, cognitive, and physiological blood tests compared to age-matched mice that receive bone marrow that is not enriched with mitochondria.

[0030] According to various embodiments, the source of stem cells can be autologous, syngeneic, or from a donor. The provision of stem cells enriched ex vivo with healthy mitochondria and returned to the same subject to a subject with a debilitating condition provides benefits over other methods involving allogeneic cell therapy. For example, the provided method eliminates the need to screen populations and find a donor that is human leukocyte antigen (HLA) compatible with the subject, which is a time-consuming and costly process that is not always successful. The method further advantageously eliminates the need for lifelong immunosuppressive therapy for the subject, such that the subject's body does not reject the allogeneic cell population. Thus, the present invention advantageously provides a unique methodology of ex vivo therapy in which human stem cells are removed from the subject's body, enriched ex vivo with healthy functional mitochondria, and returned to the same patient. Furthermore, the present invention relates to the administration of stem cells that circulate throughout the body in different tissues and increase the subject's energy levels, thereby increasing the quality of life of a subject with a debilitating condition, without being bound by any theory or mechanism.

[0031] The present invention is based, in part, on the surprising discovery that functional mitochondria can enter intact fibroblasts, hematopoietic stem cells, and bone marrow cells, and that treatment of fibroblasts, hematopoietic stem cells, and bone marrow cells with functional mitochondria increases mitochondrial content, cell survival, and ATP production.

[0032] The present invention provides, for the first time, stem cells with enhanced or strengthened mitochondrial activity to aged subjects or cancer patients. These stem cells are enriched with healthy functional mitochondria from a suitable source. Typically, mitochondria can be obtained from blood cells, placental cells, placental cell cultures, or other suitable cell lines. Each possibility is a separate embodiment of the present invention.

[0033] The present invention, in one aspect, provides methods for treating or reducing the debilitating effects of various conditions by introducing isolated or partially purified freeze-thawed functional human mitochondria into stem cells obtained or derived from a subject suffering from a debilitating condition or from a donor, and transplanting into a subject suffering from a debilitating condition at least 105 to 2 x 107 "mitochondria-enriched" human stem cells per kilogram of patient body weight in a pharma- ceutically acceptable liquid medium capable of supporting cell viability.

[0034] According to another aspect, the present invention provides a method for treating or lessening a debilitating condition in a subject, comprising parenterally administering to the subject a pharmaceutical composition comprising at least 5*105 to 5*109 human stem cells enriched with frozen-thawed healthy functional exogenous mitochondria, wherein the debilitating condition is selected from the group consisting of old age, old age-related diseases, and sequelae of anti-cancer treatment.

[0035] According to yet another aspect, the present invention provides a pharmaceutical composition for use in treating or reducing a debilitating condition in a subject, the pharmaceutical composition comprising at least 105-2x107 human stem cells per kilogram of body weight of the subject, the human stem cells suspended in a pharma- ceutically acceptable liquid medium capable of supporting cell viability, the human stem cells enriched with frozen-thawed healthy functional exogenous mitochondria, and the debilitating condition selected from the group consisting of aging, aging-associated diseases, and sequelae of anti-cancer treatment. According to some embodiments, the mitochondrial enrichment of the stem cells comprises introducing into the stem cells a mitochondrial dose of at least 0.088 and up to 176 milliunits of CS activity per million cells. According to further embodiments, the mitochondrial enrichment of the stem cells comprises introducing into the stem cells a mitochondrial dose of at least 0.088 and up to 17.6 milliunits of CS activity per million cells.

[0036] In some embodiments, the volume of isolated mitochondria is added to the recipient cells at a desired concentration. The ratio of the number of mitochondria donor cells to the number of mitochondria recipient cells is greater than 2:1 (donor cells to recipient cells). In typical embodiments, the ratio is at least 5, or at least 10 or more. In certain embodiments, the ratio of donor cells to recipient cells from which mitochondria are harvested is at least 20, 50, 100 or more. Each possibility is a separate embodiment.

[0037] In some embodiments, the subject with the debilitating condition is an elderly subject. In certain embodiments, the subject with the debilitating condition suffers from an old age-related disease(s). In other embodiments, the subject with the debilitating condition is a cancer patient undergoing chemotherapy, radiation therapy, immunotherapy with monoclonal antibodies, or a combination thereof. Each possibility represents a separate embodiment of the present invention.

[0038] In certain embodiments, the healthy, functional, human exogenous mitochondria are allogeneic mitochondria, hi other embodiments, the healthy, functional, human exogenous mitochondria are autologous or syngeneic, i.e., of the same maternal lineage.

[0039] In another aspect, the present invention provides an ex vivo method for enriching human stem cells with healthy mitochondria, the method comprising: (i) providing a first composition comprising a plurality of human stem cells obtained or derived from an individual suffering from a debilitating condition or from a healthy donor not suffering from the debilitating condition; (ii) providing a second composition comprising a plurality of isolated or partially purified freeze-thawed human functional, healthy exogenous mitochondria obtained from a healthy donor not suffering from the debilitating condition; (iii) contacting the human stem cells of the first composition with the freeze-thawed human functional mitochondria of the second composition at a ratio of 0.088-176 mU of CS activity per 10 stem cells; and (iv) incubating the composition of (iii) under conditions that allow the freeze-thawed human functional mitochondria to enter the human stem cells, thereby enriching the freeze-thawed human stem cells with the human functional mitochondria, wherein the functional mitochondrial content of the enriched human stem cells is detectably higher than the healthy functional mitochondrial content of the human stem cells in the first composition.

[0040] In certain embodiments, the subject suffering from a debilitating condition is a cancer patient after treatment with a debilitating anti-cancer therapy.Accordingly, the present invention provides an ex vivo method for enriching human stem cells with healthy functional exogenous mitochondria, the method comprising the steps of: (i) providing a first composition comprising a plurality of human stem cells from an individual suffering from a malignant disease or from a healthy subject not suffering from a malignant disease; (ii) providing a second composition comprising a plurality of isolated or partially purified freeze-thawed functional human mitochondria obtained from the same individual suffering from a malignant disease or from a healthy subject not suffering from a malignant disease prior to anti-cancer treatment; and (iii) enriching the first composition with functional human mitochondria. and (iv) incubating the composition of (iii) under conditions that permit human functional mitochondria to enter the frozen-thawed human stem cells, thereby enriching the human stem cells with the human functional mitochondria, wherein the functional mitochondrial content of the enriched human stem cells is detectably higher than the healthy functional mitochondrial content of the human stem cells in the first composition.

[0041] In some embodiments, the conditions that allow healthy functional human exogenous mitochondria to enter human stem cells include incubating human stem cells with the healthy functional exogenous mitochondria at a temperature ranging from 16-37° C. for a time ranging from 0.5-30 hours. In some embodiments, the conditions that allow healthy functional human exogenous mitochondria to enter human stem cells include incubating human stem cells with the healthy functional exogenous mitochondria in a culture medium in an environment that supports cell survival at a temperature ranging from 16-37° C. for a time ranging from 0.5-30 hours. According to some embodiments, the culture medium is saline containing human serum albumin. In some embodiments, the incubation conditions include an atmosphere containing 5% CO2. In some embodiments, the incubation conditions do not include added CO2 above the level found in air. Each possibility represents a separate embodiment of the present invention.

[0042] In some embodiments, the method further comprises centrifugation of the human stem cells and healthy, functional, exogenous mitochondria before, during, or after incubation. In some embodiments, prior to incubation, the method further comprises a single centrifugation of the human stem cells and healthy, functional, exogenous mitochondria at a centrifugal force of greater than 2500×g. Each possibility represents a separate embodiment of the present invention.

[0043] In some embodiments, mitochondria that have been subjected to freeze-thaw cycles exhibit a comparable rate of oxygen consumption after thawing compared to control mitochondria that have not been subjected to freeze-thaw cycles.

[0044] In certain embodiments, the above method further comprises freezing the mitochondrially enriched human stem cells, and optionally further comprises thawing.

[0045] In additional embodiments, the human stem cells are expanded before or after mitochondrial enhancement.

[0046] Detectable enrichment of stem cells with functional mitochondria can be determined by functional and / or enzymatic assays, including but not limited to oxygen (O2) consumption rate, activity level of citrate synthase, adenosine triphosphate (ATP) production rate, mitochondrial protein content (such as succinate dehydrogenase complex subunit A-SDHA and cytochrome C oxidase-COX1), and mitochondrial DNA content. In another method, enrichment of stem cells with healthy donor mitochondria can be confirmed by detection of donor mitochondrial DNA (mtDNA). According to some embodiments, the degree of enrichment of stem cells with functional mitochondria can be determined by the level of heteroplasmy changes and / or by the copy number of mtDNA per cell. According to certain exemplary embodiments, enrichment of stem cells with healthy functional mitochondria can be determined by conventional assays recognized in the art. For example, the presence of donor mitochondria can be determined by a method selected from (i) activity level of citrate synthase, or (ii) mtDNA sequencing, which indicates two or more sources of mtDNA. Each possibility represents a separate embodiment of the present invention.

[0047] According to some embodiments, mitochondria can be matched between the donor and the treated subject according to mtDNA haplogroups. According to other embodiments, mitochondria are selected according to specific distinct mtDNA haplogroups prior to stem cell enrichment.

[0048] In certain embodiments, the mitochondrial content of the stem cells in the first composition or the fourth composition is determined by determining the activity level of citrate synthase. Each possibility represents a separate embodiment of the present invention.

[0049] In certain embodiments, the process of enriching human stem cells with mitochondria is carried out prior to freezing the cells, while in other embodiments, the process of enriching human stem cells with mitochondria is carried out after freezing and thawing the cells.

[0050] In certain embodiments, the autologous human stem cells are frozen and stored prior to contracting the debilitating condition, hi other embodiments, the process of enriching the human stem cells with mitochondria is carried out after freezing and thawing the cells.

[0051] In certain embodiments, the stem cells are pluripotent stem cells (PSCs). In other embodiments, the PSCs are non-embryonic stem cells. In some embodiments, the stem cells are induced PSCs (iPSCs). In certain embodiments, the stem cells are derived from bone marrow cells. In certain embodiments, the stem cells express the myeloid hematopoietic progenitor cell antigen CD34 (CD34+). In certain embodiments, the stem cells are mesenchymal stem cells. In other embodiments, the stem cells are derived from adipose tissue. In still other embodiments, the stem cells are derived from blood. In further embodiments, the stem cells are derived from umbilical cord blood. In further embodiments, the stem cells are derived from oral mucosa. In further embodiments, the stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0052] In certain embodiments, the stem cells are bone marrow cells.

[0053] In certain embodiments, the stem cells are bone marrow derived stem cells, including bone marrow hematopoietic cells. In certain embodiments, the bone marrow derived stem cells include erythropoietic cells. In certain embodiments, the bone marrow derived stem cells include pluripotent hematopoietic stem cells (HSCs). In certain embodiments, the bone marrow derived stem cells include common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow derived stem cells include megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. In certain embodiments, the bone marrow derived stem cells include mesenchymal stem cells. Each possibility represents a separate embodiment of the present invention.

[0054] In certain embodiments, the stem cells are CD34+ cells. In certain embodiments, the CD34+ expressing cells are obtained from umbilical cord blood (i.e., non-bone marrow hematopoietic stem cells). In some embodiments, the cells used are autologous stem cells, which can be frozen and stored before old age or debilitating conditions related to cancer therapy. In some embodiments, the process of enriching cells with mitochondria is carried out before freezing. In alternative embodiments, the process of enriching cells with mitochondria is carried out after freezing and thawing the stem cells.

[0055] In certain embodiments, the stem cells in the first composition are obtained from an old subject or from a donor. In certain embodiments, the stem cells in the first composition are bone marrow cells obtained from the bone marrow of an old subject or from a donor. In certain embodiments, the stem cells in the first composition are obtained directly or indirectly from the bone marrow of an old subject or from the bone marrow of a donor. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of an old subject or from the bone marrow of a donor. In certain embodiments, the stem cells in the first composition are obtained from the peripheral blood of an old subject or from the peripheral blood of a donor. Each possibility represents a separate embodiment of the present invention.

[0056] In certain embodiments, the stem cells in the first composition are obtained from a subject suffering from a malignant disease. In certain embodiments, the stem cells in the first composition are obtained from a subject suffering from a non-hematopoietic malignant disease or from a healthy subject not suffering from a malignant disease. In certain embodiments, the stem cells in the first composition are obtained from the bone marrow of a subject suffering from a non-hematopoietic malignant disease or from a healthy subject not suffering from a malignant disease. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of a subject suffering from a non-hematopoietic malignant disease or from the bone marrow of a healthy subject not suffering from a malignant disease. In certain embodiments, the stem cells in the first composition are obtained directly from the bone marrow of a subject suffering from a non-hematopoietic malignant disease or from the bone marrow of a healthy subject not suffering from a malignant disease. In certain embodiments, the stem cells in the first composition are obtained indirectly from the bone marrow of a subject suffering from a non-hematopoietic malignant disease or from the bone marrow of a healthy subject not suffering from a malignant disease. In certain embodiments, the bone marrow cells of the first composition are obtained from the peripheral blood of a subject suffering from a non-hematopoietic malignancy, or from the peripheral blood of a healthy subject not suffering from a malignancy, each possibility representing a separate embodiment of the present invention.

[0057] In certain embodiments, the stem cells are at least partially purified.

[0058] In certain embodiments, the healthy functional mitochondria are derived from cells or tissues selected from the group consisting of placenta, placental cells grown in culture, and blood cells.

[0059] In certain embodiments, the pharmaceutical composition is administered to a subject suffering from a debilitating condition selected from the group consisting of old age, old age-related diseases, and sequelae of anti-cancer treatment. In further embodiments, the pharmaceutical composition is administered to a specific tissue or organ. In still further embodiments, the pharmaceutical composition is administered by systemic parenteral administration. In other embodiments, the pharmaceutical composition comprises at least about 106 mitochondrially enriched human stem cells per kilogram of patient body weight. In additional embodiments, the pharmaceutical composition comprises a total of about 5x105 to 5x109 human stem cells enriched with human mitochondria. In certain embodiments, administration of the pharmaceutical composition to the subject is by a parenteral route selected from the group consisting of intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and direct injection into a tissue or organ. Each possibility represents a separate embodiment of the invention.

[0060] In certain embodiments, the above method further comprises a previous step, which comprises administering to a subject suffering from a debilitating condition, either aging or a non-hematopoietic malignant disease, or to a healthy donor, an agent that induces stem cell mobilization from bone marrow to peripheral blood. In certain embodiments, the agent is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor), salts thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the above method further comprises isolating stem cells from the peripheral blood of a subject suffering from a debilitating condition, either aging or a non-hematopoietic malignant disease, or from the peripheral blood of a healthy subject. In certain embodiments, the isolation is performed by apheresis.

[0061] In certain embodiments, the method further comprises administering to a subject suffering from a debilitating condition selected from the group consisting of old age, old age-related diseases, and sequelae of anti-cancer treatment, an agent that prevents, delays, minimizes, or neutralizes adverse immunogenic reactions between the subject and stem cells of an allogeneic donor. In additional embodiments, the functional mitochondria in the second composition are obtained from a subject suffering from a malignant disease prior to anti-cancer treatment.

[0062] In certain embodiments, the method further comprises enriching the stem cells and functional mitochondria in the third composition before or during incubation.In certain embodiments, the method further comprises centrifuging the third composition before, during, or after incubation.Each possibility represents a separate embodiment of the present invention.

[0063] In alternative embodiments, mitochondria-enriched stem cells are transplanted into an aged subject or a subject suffering from aging-related disease(s). In certain embodiments, the stem cells are from a donor who is not afflicted with aging-related disease(s). In certain embodiments, the stem cells are autologous bone marrow stem cells. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of an aged subject or a subject suffering from aging-related disease(s), or from the bone marrow of a healthy donor who is not afflicted with aging-related disease(s). In certain embodiments, the stem cells in the first composition are obtained from the peripheral blood of an aged subject or a subject suffering from aging-related disease(s), or from the peripheral blood of a healthy donor who is not afflicted with aging-related disease(s). Each possibility represents a separate embodiment of the present invention.

[0064] In alternative embodiments, the subject suffers from a hematopoietic malignancy, and the stem cells transplanted into the subject are enriched with mitochondria. In certain embodiments, the stem cells are from a healthy donor not suffering from a malignancy. In certain embodiments, the stem cells are autologous bone marrow stem cells as used in various hematopoietic malignancies, including multiple myeloma and certain types of lymphoma. According to these embodiments, the stem cells in the first composition are obtained from the bone marrow of a subject suffering from a non-hematopoietic malignancy, or from the bone marrow of a healthy subject not suffering from a malignancy. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of a subject suffering from a hematopoietic malignancy, or from the bone marrow of a healthy subject not suffering from a malignancy. In certain embodiments, the stem cells of the first composition are obtained from the peripheral blood of a subject suffering from a hematopoietic malignancy, or from the peripheral blood of a healthy subject not suffering from a malignancy. Each possibility represents a separate embodiment of the present invention.

[0065] In certain embodiments, the above method further comprises a previous step, which comprises administering to the subject an agent that induces mobilization of bone marrow stem cells from bone marrow to peripheral blood. In certain embodiments, the agent is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor), salts thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the above method further comprises isolating stem cells from the peripheral blood of a subject suffering from a hematopoietic malignancy or from the peripheral blood of a healthy subject not suffering from a malignancy. In certain embodiments, the isolation is performed by apheresis.

[0066] In certain embodiments, the above method further comprises enriching the stem cells and functional mitochondria in composition (iii) before or during the incubation. In certain embodiments, the above method further comprises centrifuging composition (iii) before, during, or after the incubation. Each possibility represents a separate embodiment of the present invention.

[0067] In certain embodiments, the stem cells in the first composition are obtained from a subject with a debilitating condition selected from aging, aging-related disease, and malignant disease undergoing debilitating therapy, and have (i) a decreased oxygen (O2) consumption rate, (ii) a decreased citrate synthase activity level, (iii) a decreased adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii), compared to a subject not suffering from the debilitating condition. Each possibility represents a separate embodiment of the present invention.

[0068] In certain embodiments, the stem cells in the first composition are obtained from a healthy donor not suffering from a debilitating condition and have (i) a normal oxygen (O2) consumption rate, (ii) a normal citrate synthase activity level, (iii) a normal adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention. In certain embodiments, the isolated or partially purified human functional mitochondria in the second composition are obtained from a donor not suffering from a debilitating condition, having normal mitochondrial DNA. As used herein, the term "normal mitochondrial DNA" refers to mitochondrial DNA that does not have any deletions or mutations known to be associated with primary mitochondrial disease.

[0069] In certain embodiments, stem cells enriched with healthy functional mitochondria have (i) an increased rate of oxygen (O2) consumption, (ii) an increased level of citrate synthase activity, (iii) an increased rate of adenosine triphosphate (ATP) production, (iv) an increased normal mitochondrial DNA content, or (v) any combination of (i), (ii), (iii), and (iv) compared to stem cells prior to mitochondrial enrichment. Each possibility represents a separate embodiment of the present invention.

[0070] According to certain exemplary embodiments, stem cells enriched with healthy functional mitochondria have (i) increased levels of citrate synthase activity, and (ii) increased normal mitochondrial DNA content, compared to stem cells prior to mitochondrial enrichment.

[0071] In certain embodiments, the total amount of mitochondrial protein in the partially purified mitochondria is 20%-80% of the total amount of cellular protein in the sample. Exemplary methods for obtaining such compositions of isolated or partially purified mitochondria are disclosed in WO2013 / 035101.

[0072] The present invention further provides, in another aspect, a plurality of human stem cells enriched with healthy mitochondria, obtained by any one of the above-mentioned method embodiments.It should be clearly understood that the human stem cells enriched with functional mitochondria according to the present invention are not derived from subjects suffering from primary mitochondrial disease.According to some particular embodiments, the stem cells enriched with healthy mitochondria are other than bone marrow stem cells.

[0073] The invention further provides, in another aspect, a plurality of human stem cells enriched ex vivo with mitochondria, the stem cells having at least one characteristic selected from the group consisting of: (a) increased mitochondrial DNA content, (b) increased activity level of citrate synthase, (c) increased content of at least one mitochondrial protein selected from SDHA and COX1, (d) increased rate of oxygen (O2) consumption, (e) increased rate of ATP production, or (f) any combination thereof, compared to corresponding levels in the stem cells prior to mitochondria enrichment. Each possibility represents a separate embodiment of the present invention.

[0074] According to some embodiments, the stem cells are CD34+ stem cells. Human stem cells enriched ex vivo with functional mitochondria according to the present invention are not derived from a subject suffering from a primary mitochondrial disease.

[0075] In certain embodiments, the total amount of mitochondrial protein in partially purified mitochondria is between 20% and 80% of the total amount of cellular protein in the sample.

[0076] In certain embodiments, the plurality of human stem cells are CD34+ and have increased mitochondrial content, increased mitochondrial DNA content, increased oxygen (O2) consumption rate, and increased citrate synthase activity level compared to the stem cells before mitochondria enrichment.In some embodiments, the increased content or activity is higher than the content or activity in the cells at the time of isolation.

[0077] The present invention further provides, in another aspect, a pharmaceutical composition comprising a plurality of human bone marrow stem cells enriched ex vivo with healthy functional mitochondria as described above.

[0078] The present invention further provides, in another aspect, the pharmaceutical composition described above for use in treating a human subject suffering from a debilitating condition. According to certain embodiments, the subject suffering from a debilitating condition is an elderly subject. In certain embodiments, the subject suffering from a debilitating condition suffers from an old age-related disease(s). In some embodiments, the subject suffering from a debilitating condition suffers from a malignant disease undergoing a debilitating therapy. In further embodiments, the pharmaceutical composition described above is used to treat a human subject during remission or after recovery from a malignant disease.

[0079] The present invention further provides, in another aspect, a method for treating a human subject suffering from a debilitating condition, comprising administering the pharmaceutical composition to the patient. According to certain embodiments, the subject suffering from the debilitating condition is an elderly subject. In certain embodiments, the subject suffering from the debilitating condition suffers from an old age-related disease(s). In some embodiments, the subject suffering from the debilitating condition suffers from a malignant disease undergoing a debilitating therapy. In further embodiments, the pharmaceutical composition is used to treat a human subject during remission or after recovery from a malignant disease. In certain embodiments, the stem cells comprising the pharmaceutical composition are autologous or syngeneic to the subject suffering from the debilitating condition. In certain embodiments, the stem cells comprising the pharmaceutical composition are allogeneic to the subject suffering from the debilitating condition. Each possibility represents a separate embodiment of the present invention.

[0080] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0081] [Figure 1]Three micrographs obtained with a fluorescent confocal microscope showing mouse fibroblasts expressing mitochondrial GFP (left panel), incubation with isolated RFP-labeled mitochondria (middle panel), and an overlay (right panel). [Diagram 2] 1 is a bar graph showing a comparison of ATP levels in mouse fibroblasts that were either untreated (control), treated with a mitochondrial complex I irreversible inhibitor (rotenone), or treated with rotenone and mouse placental mitochondria (rotenone+mitochondria). Data are presented as mean ± SEM, (*) p-value < 0.05. RLU-Relative Luminescence Units. [Diagram 3] FIG. 1 shows four photomicrographs obtained with a fluorescent confocal microscope showing mouse bone marrow cells incubated with GFP-labeled mitochondria isolated from mouse melanoma cells. [Figure 4] 1 is a bar graph showing levels of C57BL mtDNA in the bone marrow of FVB / N mice at various time points following IV injection of bone marrow cells enriched with exogenous mitochondria from C57BL mice. [Diagram 5] FIG. 1 is a bar graph showing a comparison of citrate synthase (CS) activity in mouse bone marrow (BM) cells incubated with various amounts of GFP-labeled mitochondria isolated from mouse melanoma cells, with or without centrifugation. [Figure 6A] 1 is a bar graph showing a comparison of CS activity in mouse BM cells after enrichment with increasing amounts of GFP-labeled mitochondria. [Figure 6B] FIG. 1 is a bar graph showing a comparison of cytochrome c reductase activity (black bars) in these cells compared to the activity of GFP-labeled mitochondria (grey bars). [Figure 7A] 1 is a bar graph showing the copy number of C57BL mtDNA in FVB / N bone marrow cells after incubating the cells with various concentrations (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 mUnits of CS activity) of exogenous mitochondria from C57BL mice compared to untreated cells (NT). [Figure 7B] 1 is a bar graph showing the content of mtDNA-encoded (COX1) protein in FVB / N bone marrow cells after incubating the cells with various concentrations (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 mUnits of CS activity) of exogenous mitochondria from C57BL mice compared to untreated cells (NT), normalized to Janus levels. [Figure 7C] 1 is a bar graph showing the content of nuclear-encoded (SDHA) protein in FVB / N bone marrow cells after incubating the cells with various concentrations (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 mUnits of CS activity) of exogenous mitochondria from C57BL mice compared to untreated cells (NT), normalized to Janus levels. [Figure 8A] 1 is a bar graph showing a comparison of CS activity in control, untreated human BM cells and human BM cells incubated with GFP-labeled mitochondria isolated from human placental cells with or without centrifugation. [Figure 8B] 1 is a bar graph showing a comparison of ATP levels in control, untreated human BM cells, and human BM cells incubated with centrifuged, GFP-labeled mitochondria isolated from human placental cells. [Figure 9A] Shown are the results of FACS analysis in human BM cells that were not incubated with GFP-labeled mitochondria. [Figure 9B] FIG. 1 shows the results of FACS analysis on human BM cells incubated with GFP-labeled mitochondria after centrifugation. [Figure 10A] FIG. 1 is a bar graph showing ATP content of human CD34+ cells from healthy donors either untreated (NT) or treated with blood-derived mitochondria (MNV-BLD). [Figure 10B] FIG. 1 is a bar graph showing CS activity of human CD34+ cells from healthy donors treated or not with blood-derived mitochondria. [Figure 11]Three micrographs obtained by fluorescence confocal microscopy of CD34+ cells incubated with GFP-labeled mitochondria isolated from HeLa-TurboGFP-mitochondria cells. [Figure 12A] 1 is a diagram of mtDNA deletion in umbilical cord blood cells of a Pearson patient, as well as Southern blot analysis showing the deletion. [Figure 12B] FIG. 1 is a bar graph showing human mtDNA copy number in bone marrow of NSGS mice two months after mitochondrial enhancement therapy using Pearson cord blood cells enriched with human mitochondria (UCB+Mito) compared to mice injected with non-enhanced umbilical cord blood cells (UCB). [Figure 13A] 1 is a bar graph showing FVB / N ATP8 mutant mtDNA levels in bone marrow of FVB / N mice one month after administration of stem cells enriched with healthy functional mitochondria derived from C57BL placenta. [Figure 13B] 1 is a bar graph showing FVB / N ATP8 mutant mtDNA levels in the liver of FVB / N mice 3 months after administration of stem cells enriched with healthy functional mitochondria derived from C57BL placenta. [Figures 14A-14C] 14A-C are graphs showing biodistribution of mitochondrially enriched bone marrow cells by amount of C57BL mtDNA in the bone marrow (FIG. 14A), brain (FIG. 14B), and heart (FIG. 14C) of mice up to 3 months after MAT. White bars and associated dots indicate enhancement of bone marrow samples, gray bars are controls. [Figure 15]FIG. 11 is a bar graph showing a comparison of FVB / N ATP8 mutant mtDNA levels in the brains of FVB / N mice one month after administration of stem cells enriched with healthy functional wild type mitochondria (isolated from the liver of C57BL mice) from untreated FVB / N mice (Naive), FVB / N mice administered stem cells enriched with C57BL healthy liver mitochondria (C57BL Mito), FVB / N mice administered stem cells enriched with C57BL healthy mitochondria and received total body irradiation (TBI) prior to stem cell administration (TBI C57BL Mito), and FVB / N mice administered stem cells enriched with C57BL healthy mitochondria and received busulfan chemotherapy prior to stem cell administration (Busulfan C57BL Mito). [Figures 16A-16C] Figure 16 shows line graphs depicting the performance of 12-month-old C57BL / 6J mice treated with mitochondrial-enriched BM cells (MNV-BM-PLC, 1x106 cells), bone marrow cells (BM control, 1x106 cells), or control vehicle solution (control, 4.5% albumin in 0.9% w / v NaCl) in the open field behavioral test before and after 9 months of treatment. Figure 16A shows quantification of distance traveled during the open field test. Figure 16B shows median duration (time (sec) or % change from baseline). Figure 16C shows wall duration (time (sec) or % change from baseline). [Figure 16D] FIG. 1 is a line graph showing blood urea nitrogen (BUN) levels in 12-month-old C57BL / 6J mice treated with mitochondrial-enriched BM cells (MNV-BM-PLC, 1x106 cells), bone marrow cells (BM Control, 1x106 cells), or control vehicle solution (Control, 4.5% albumin in 0.9% w / v NaCl) before and 9 months after treatment. [Fig. 16E-16F]FIG. 16B shows a bar graph depicting the rotarod test of 12-month-old C57BL / 6J mice administered and treated with either mitochondrial-enhanced bone marrow (BM) cells (MNV-BM-PLC, 1×106 cells), bone marrow cells (BM, 1×106 cells), or control vehicle solution (VEHICLE, 4.5% albumin in 0.9% w / v NaCl). Results shown are pre-treatment and 1 and 3 months after treatment. FIG. 16E shows the rotarod scores (in seconds (s)) of the various treated test groups at the indicated time points. FIG. 16F shows the rotarod scores (expressed as a percentage from baseline) of the various treated test groups at the indicated time points. [Fig. 16G-16J] Figure 16 shows bar graphs depicting force testing of 12 month old C57BL / 6J mice administered and treated with either mitochondrial enhanced bone marrow (BM) cells (MNV-BM-PLC, 1x106 cells), bone marrow cells (BM, 1x106 cells), or control vehicle solution (VEHICLE, 4.5% albumin in 0.9% w / v NaCl). Results shown are pre-treatment and 1 and 3 months after treatment. Figures 16G-16H - Grip strength (force) (g or % change from baseline); Figures 16I-16J - Grip strength time (time (sec) or % change from baseline). [Figure 17A] FIG. 1 is a scheme showing the course of treatment and evaluation in a clinical trial performed on patient 1 (a patient with juvenile Pearson syndrome (PS) and PS-associated Fanconi syndrome (FS)), who has a deletion mutation in mtDNA, including ATP8. [Figure 17B] 1 is a bar graph showing aerobic metabolic equivalent (MET) scores prior to administration of stem cells enriched with functional mitochondria, 2.5 months and 8 months after administration of enriched stem cells. [Figure 17C] 1 is a bar graph showing blood lactate levels in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17D] 1 is a line graph showing the standard deviation scores of weight and height of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17E]1 is a line graph showing alkaline phosphatase (ALP) levels in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17F] 1 is a line graph showing the longitudinal increase in blood red blood cell (RBC) levels in PS patients before and after therapy provided by the present invention. [Figure 17G] 1 is a line graph showing the longitudinal increase in blood hemoglobin (HGB) levels in PS patients before and after therapy provided by the present invention. [Figure 17H] 1 is a line graph showing the longitudinal increase in blood hematocrit (HCT) levels in PS patients before and after therapy provided by the present invention. [Figure 17I] 1 is a line graph showing creatinine levels of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17J] 1 is a line graph showing bicarbonate levels in PS patients treated by the methods provided herein as a function of time before and after treatment. [Figure 17K] 1 is a line graph showing base excess levels of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17L] 1 is a bar graph showing blood magnesium levels as a function of time in PS patients treated by the methods provided herein, before and after therapy, and before and after magnesium supplementation. [Figure 17M] 1 is a bar graph showing urinary glucose to creatinine ratios of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17N] 1 is a bar graph showing urinary potassium to creatinine ratios in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17O] 1 is a bar graph showing urinary chloride to creatinine ratios of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 17P]1 is a bar graph showing urinary sodium to creatinine ratios in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 18A] FIG. 1 is a line graph showing normal mtDNA content of three PS patients (Pt. 1, Pt. 2, and Pt. 3) treated by the methods provided herein as a function of time before and after therapy, measured by digital PCR of the deleted regions (for each patient) compared to 18S genomic DNA, which represents the number of normal mtDNA per cell, and normalized to baseline. [Figure 18B] FIG. 1 is a line graph showing the heteroplasmy levels (deleted mtDNA compared to total mtDNA) of three PS patients (Pt.1, Pt.2, and Pt.3) at baseline after MAT. The dotted lines represent the baseline for each patient. [Figure 19A] Further provided by the present invention is another scheme of different treatment stages for Pearson Syndrome (PS) patients. [Figure 19B] 1 is a bar graph showing blood lactate levels in PS patients treated by the methods provided herein as a function of time before (B) and after (C) therapy. [Figure 19C] 1 is a bar graph showing sit-to-stand scores of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19D] 1 is a bar graph showing 6-minute walk test scores of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19E] 1 is a bar graph showing dynamometer scores for three consecutive repetitions (R1, R2, R3) of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19F] 1 is a bar graph showing urinary magnesium to creatinine ratio in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19G]1 is a bar graph showing urinary potassium to creatinine ratios in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19H] 1 is a bar graph showing urinary calcium to creatinine ratios in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19I] 1 is a bar graph showing urinary ATP8 to 18S copy number ratios of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 19J] 1 is a bar graph showing ATP levels in lymphocytes of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 20A] Further provided by the present invention is yet another scheme of different treatment stages for patients with Pearson syndrome (PS) and Kearns-Sayre syndrome (KSS). [Figure 20B] 1 is a bar graph showing blood lactate levels in PS patients treated by the methods provided herein as a function of time before (B) and after (C) therapy. [Figure 20C] 1 is a bar graph showing AST and ALT levels in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 20D] 1 is a bar graph showing triglyceride, total cholesterol, and VLDL cholesterol levels in PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 20E] 1 is a bar graph showing hemoglobin A1C (HbA1C) scores of PS patients treated by the methods provided herein as a function of time before and after therapy. [Figure 20F] 1 is a line graph showing the rise score of a PS patient (Pt. 3) treated by the methods provided herein as a function of time before and after therapy. [Figure 20G]1 is a line graph showing the 6-minute walk test scores of a PS patient (Pt. 3) treated by the methods provided herein as a function of time before and after therapy. [Figure 21] 1 is a bar graph showing ATP content in peripheral blood of KSS patients treated by the methods provided herein before and after therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] The present invention provides a cellular platform, more specifically stem cell-derived cellular platform, for targeted systemic delivery of therapeutically significant amounts of fully functional and healthy mitochondria, and methods for their utilization in subjects with debilitating conditions, including aged subjects and subjects suffering from aging-related disease(s), as well as cancer patients suffering from the sequelae of anti-cancer treatment, including chemotherapy, radiotherapy, or immunotherapy with monoclonal antibodies.The present invention is based on several surprising discoveries, including the clinical results illustrated herein, which show that intravenous injection of bone marrow-derived hematopoietic stem cells enriched with normal functional and healthy mitochondria can beneficially affect various tissues of subjects.In other words, improved function can be achieved in various organs and tissues after administration of stem cells enriched with healthy mitochondria.

[0083] The present invention is based in part on the discovery that bone marrow cells are receptive to enrichment with intact and functional mitochondria, and that human bone marrow cells are particularly receptive to enrichment with mitochondria, as disclosed, for example, in WO2016 / 135723.Without being bound by any theory or mechanism, it is hypothesized that co-incubation of stem cells with healthy mitochondria promotes the transfer of intact and functional mitochondria to stem cells.

[0084] It has also been found that the degree of enrichment of stem cells, including but not limited to bone marrow derived hematopoietic stem cells, with mitochondria and improvement of cellular mitochondrial function is dependent on the concentration of isolated or partially purified mitochondria, as well as the conditions used for mitochondrial enrichment, including but not limited to incubation conditions, and therefore can be manipulated to obtain the desired enrichment.

[0085] The present invention provides, in one aspect, methods for treating and / or reducing the debilitating effects of various conditions by introducing partially purified healthy human mitochondria ex vivo into stem cells obtained or derived from a subject suffering from a debilitating condition or from a healthy donor, and transplanting the "mitochondria-enriched" stem cells into a subject suffering from a debilitating condition.

[0086] In certain embodiments, the subject suffering from the debilitating condition suffers from aging or an aging-related disease(s). In other embodiments, the subject suffering from the debilitating effect is a cancer patient undergoing chemotherapy, radiation therapy, or immunotherapy with monoclonal antibodies. In some embodiments, the cancer patient is a subject suffering from a non-hematopoietic malignancy. In other embodiments, the cancer patient is a subject suffering from a non-hematopoietic malignancy.

[0087] In further embodiments, the human stem cells administered to the subject are autologous to the subject, hi other embodiments, the human stem cells administered to the subject are from a donor, i.e., are allogeneic to the subject.

[0088] In certain embodiments, the autologous or allogeneic human stem cell is a pluripotent stem cell (PSC) or an induced pluripotent stem cell (iPSC). In further embodiments, the autologous or allogeneic human stem cell is a mesenchymal stem cell.

[0089] According to some embodiments, the human stem cells are derived from adipose tissue, oral mucosa, blood, umbilical cord blood, or bone marrow. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the human stem cells are derived from bone marrow.

[0090] In another aspect, the present invention provides a pharmaceutical composition for use in treating or reducing a debilitating condition in a subject, the pharmaceutical composition comprising at least 105-2 x 107 human stem cells per kilogram body weight of the subject, the human stem cells suspended in a pharma- ceutical acceptable liquid medium capable of supporting cell viability, the human stem cells enriched with freeze-thawed healthy functional exogenous mitochondria, and the debilitating condition is selected from the group consisting of aging, aging-associated diseases, and sequelae of anti-cancer treatment.

[0091] In some embodiments, the pharmaceutical composition comprises at least 105 to 2x107 mitochondrial enriched human stem cells per kilogram of patient body weight. In some embodiments, the pharmaceutical composition comprises at least 5x105 to 1.5x107 mitochondrial enriched human stem cells per kilogram of patient body weight. In some embodiments, the pharmaceutical composition comprises at least 5x105 to 4x107 mitochondrial enriched human stem cells per kilogram of patient body weight. In some embodiments, the pharmaceutical composition comprises at least 106 to 107 mitochondrial enriched human stem cells per kilogram of patient body weight. In other embodiments, the pharmaceutical composition comprises at least 105 or at least 106 mitochondrial enriched human stem cells per kilogram of patient body weight. Each possibility represents a separate embodiment of the present invention. In some embodiments, the pharmaceutical composition comprises a total of at least 5x105 to up to 5x109 mitochondrial enriched human stem cells. In some embodiments, the pharmaceutical composition comprises a total of at least 106 to up to 109 mitochondrial enriched human stem cells. In other embodiments, the pharmaceutical composition comprises a total of at least 2x106 to up to 5x108 mitochondrial enriched human stem cells.

[0092] In another aspect, the present invention provides an ex vivo method for enriching human stem cells with functional mitochondria, the method comprising the steps of: (i) providing a first composition comprising a plurality of human stem cells obtained or derived from a subject suffering from a debilitating condition or from a healthy donor not suffering from the debilitating condition; (ii) providing a second composition comprising a plurality of isolated or partially purified human functional mitochondria obtained from a healthy donor not suffering from the debilitating condition; (iii) contacting the human stem cells of the first composition with the human functional mitochondria of the second composition, thus forming a third composition; and (iv) incubating the third composition under conditions that allow human functional mitochondria to enter the human stem cells, thereby enriching the human stem cells with the human functional mitochondria, thus forming a fourth composition, wherein the mitochondrial content of the enriched human stem cells in the fourth composition is detectably higher than the mitochondrial content of the human stem cells in the first composition.

[0093] The present invention in one aspect provides an ex vivo method for enriching human bone marrow cells with functional mitochondria, the method comprising the steps of: (i) providing a first composition comprising a plurality of human bone marrow cells obtained or derived from a patient suffering from a malignant disease or from a healthy subject not suffering from a malignant disease; (ii) providing a second composition comprising a plurality of isolated human functional mitochondria obtained from the same patient suffering from a malignant disease or from a healthy subject not suffering from a malignant disease prior to anti-cancer treatment; (iii) mixing the human bone marrow cells of the first composition with the human functional mitochondria of the second composition, thus forming a third composition; and (iv) incubating the third composition under conditions that allow human functional mitochondria to enter the human bone marrow cells, thereby enriching the human bone marrow cells with the human functional mitochondria, thus forming a fourth composition, wherein the mitochondrial content of the human bone marrow cells in the fourth composition is detectably higher than the mitochondrial content of the human bone marrow cells in the first composition.

[0094] As used herein, the term "ex vivo methods" refers to methods that include steps that are performed entirely outside the human body. In particular, ex vivo methods involve the manipulation of cells outside the body that are then reintroduced or transplanted into the subject to be treated.

[0095] As used herein, the term "enrichment" refers to any action designed to increase the mitochondrial content, e.g., the number of intact mitochondria, or the functionality of mitochondria in mammalian cells. In particular, stem cells enriched with functional mitochondria exhibit enhanced function compared to the same stem cells before enrichment.

[0096] As used herein, the term "stem cell" generally refers to any mammalian stem cell. Stem cells are undifferentiated cells that can differentiate into other types of cells and can divide to produce stem cells of approximately the same type. Stem cells can be either totipotent or pluripotent.

[0097] As used herein, the term "human stem cell" generally refers to all stem cells naturally found in humans, and all stem cells produced or induced ex vivo and compatible with humans. "Progenitor cells", like stem cells, tend to differentiate into a specific type of cell, but are more specific than stem cells already, and are made to differentiate into their "target" cells. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times. As used herein, the term "human stem cell" further includes "progenitor cells" and "not fully differentiated stem cells".

[0098] In some embodiments, enriching stem cells with healthy functional human exogenous mitochondria comprises incubating human stem cells with the healthy functional human exogenous mitochondria, followed by washing the mitochondria-enriched stem cells. This step provides a composition of mitochondria-enriched stem cells that is substantially devoid of cell debris or mitochondrial membrane remnants and mitochondria that did not enter the stem cells. In some embodiments, washing comprises centrifuging the mitochondria-enriched stem cells after incubating human stem cells with the healthy functional human exogenous mitochondria. According to some embodiments, the pharmaceutical composition comprising mitochondria-enriched human stem cells is separated from free mitochondria, i.e., mitochondria that did not enter the stem cells, or other cell debris. According to some embodiments, the pharmaceutical composition comprising mitochondria-enriched human stem cells does not contain detectable amounts of free mitochondria.

[0099] As used herein, the term "pluripotent stem cell (PSC)" refers to a cell that can grow indefinitely and give rise to multiple cell types in the body. A totipotent stem cell is a cell that can give rise to all other cell types in the body. Embryonic stem cells (ESCs) are totipotent stem cells, and induced pluripotent stem cells (iPSCs) are multipotent stem cells.

[0100] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to a type of pluripotent stem cell that can be generated from human adult somatic cells.

[0101] As used herein, the term "embryonic stem cell (ESC)" refers to a type of totipotent stem cell derived from the inner cell mass of a blastocyst.

[0102] As used herein, the term "bone marrow cells" generally refers to all human cells naturally found in human bone marrow and all cell populations naturally found in human bone marrow. The terms "bone marrow stem cells" and "bone marrow-derived stem cells" refer to stem cell populations derived from bone marrow.

[0103] The terms "functional mitochondria" and "healthy mitochondria" are used interchangeably herein and refer to mitochondria that exhibit normal mtDNA and parameters that indicate normal, non-pathological activity levels. Mitochondrial activity can be measured by a variety of methods well known in the art, such as membrane potential, O2 consumption, ATP production, and citrate synthase (CS) activity levels.

[0104] As used herein, the phrase "stem cells obtained from a subject suffering from a debilitating condition or from a donor not suffering from a debilitating condition" refers to cells that were the subject's / donor's stem cells at the time they were isolated from the subject.

[0105] As used herein, the phrase "stem cells derived from a subject suffering from a debilitating condition or a donor not suffering from a debilitating condition" refers to cells that have been engineered to become stem cells, not stem cells of the subject / donor. As used herein, the term "engineered" refers to using any one of the methods known in the art (Yu J. et al., Science, 2007, Vol. 318 (5858), pages 1917-1920) to reprogram somatic cells to an undifferentiated state, become induced pluripotent stem cells (iPSCs), and optionally further reprogram the iPSCs to become cells of a desired lineage or population, such as bone marrow cells (Xu Y. et al., PLoS ONE, 2012, Vol. 7 (4), page e34321) (Chen M. et al., IOVS, 2010, Vol. 51 (11), pages 5970-5978).

[0106] As used herein, the term "CD34+ cells" refers to hematopoietic stem cells characterized as being CD34 positive, whether obtained from stem cells or mobilized from bone marrow or obtained from umbilical cord blood.

[0107] As used herein, the term "subject suffering from a debilitating condition" refers to a human subject experiencing the debilitating effects caused by a particular condition. The debilitating condition may refer to old age, old age-related diseases, or cancer patients undergoing anti-cancer treatment, as well as other debilitating conditions.

[0108] The term "old age" refers to the inevitable and progressive decline in physiological function that accompanies aging and is demographically characterized by an age-dependent increase in mortality and a decline in various physical and mental capacities.

[0109] As used herein, the term "age-related disease" refers to "diseases of the elderly" and is a disease that is increasingly frequent with increasing aging. Age-related diseases include, but are not limited to, atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and dementia such as Alzheimer's disease. The incidence of all these diseases increases cumulatively with age.

[0110] As used herein, the term "subject suffering from malignant disease" refers to a human subject who has been diagnosed with a malignant disease, is suspected of having a malignant disease, or is at risk of developing a malignant disease. Because certain types of malignant tumors are hereditary, the offspring of a subject diagnosed with a malignant disease are considered at risk of developing a malignant disease.

[0111] As used herein, the term "subject / donor not afflicted with a malignant disease" refers to a human subject who has not been diagnosed with and / or is not suspected of having a malignant disease.

[0112] As used herein, the term "subject suffering from a non-hematopoietic malignancy" refers to a human subject who has been diagnosed with and / or is suspected of having a non-hematopoietic malignancy.

[0113] As used herein, the term "subject suffering from a hematopoietic malignancy" refers to a human subject who has been diagnosed with and / or is suspected of having a hematopoietic malignancy.

[0114] The terms "healthy donor" and "healthy subject" are used interchangeably and refer to a subject not suffering from the disease or condition being treated.

[0115] The term "contacting" refers to bringing a composition of mitochondria and cells into sufficient proximity to facilitate entry of the mitochondria into the cell. The term introducing mitochondria into a target cell is used interchangeably with the term contacting.

[0116] As used herein, the term "isolated or partially purified functional human mitochondria" refers to intact mitochondria isolated from cells obtained from healthy subjects not suffering from mitochondrial disease. The total amount of mitochondrial protein in partially purified mitochondria is 20%-80% of the total amount of cellular protein in the sample.

[0117] In the context of mitochondria, the term "isolated" as used herein and in the claims includes mitochondria that are at least partially purified from other components found in the source. In certain embodiments, the total amount of mitochondrial protein in the second composition comprising a plurality of isolated healthy functional exogenous mitochondria is 20%-80%, 20-70%, 40-70%, 20-40%, or 20-30% of the total amount of cellular protein in the sample. Each possibility represents a separate embodiment of the invention. In certain embodiments, the total amount of mitochondrial protein in the second composition comprising a plurality of isolated healthy functional exogenous mitochondria is 20%-80% of the total amount of cellular protein in the sample. In certain embodiments, the total amount of mitochondrial protein in the second composition comprising a plurality of isolated healthy functional exogenous mitochondria is 20%-80% of the combined weight of mitochondria and other subcellular fractions. In other embodiments, the total amount of mitochondrial protein in the second composition comprising a plurality of isolated healthy functional exogenous mitochondria is more than 80% of the combined weight of mitochondria and other subcellular fractions.

[0118] According to some embodiments, the method for enriching human stem cells with healthy, functional exogenous mitochondria does not include measuring the membrane potential of the cells.

[0119] In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.044 and up to 176 milliunits of CS activity per million cells. In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.088 and up to 176 milliunits of CS activity per million cells. In other embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.2 and up to 150 milliunits of CS activity per million cells. In other embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.4 and up to 100 milliunits of CS activity per million cells. In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.6 and up to 80 milliunits of CS activity per million cells. In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.7 and up to 50 milliunits of CS activity per million cells. In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.8 and up to 20 milliunits of CS activity per million cells. In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.88 and up to 17.6 milliunits of CS activity per million cells. In some embodiments, enriching stem cells with healthy functional exogenous mitochondria comprises introducing into the stem cells a mitochondrial dose of at least 0.44 and up to 17.6 milliunits of CS activity per million cells.

[0120] Mitochondrial dose can be expressed in CS activity units or mtDNA copy number, other quantifiable measures of the amount of healthy functional mitochondria, as described herein. A "unit of CS activity" is defined as the amount that allows the conversion of 1 micromole of substrate per minute in a 1 mL reaction volume.

[0121] In some embodiments, identification / distinguishing of endogenous mitochondria from exogenous mitochondria after the latter are introduced into a target cell can be performed by various means, including, for example and without limitation, identifying differences in mtDNA sequences, e.g., different haplotypes between endogenous and exogenous mitochondria, identifying specific mitochondrial proteins derived from the source tissue of the exogenous mitochondria, e.g., cytochrome p450 cholesterol side chain cleavage (P450SCC) from placenta, UCP1 from brown adipose tissue, etc., or any combination thereof.

[0122] The term "exogenous" with respect to mitochondria refers to mitochondria that are introduced into target cells (e.g., stem cells) from a source outside the cell.For example, in some embodiments, exogenous mitochondria can generally be derived from or isolated from donor cells that are different from the target cell.For example, exogenous mitochondria can be produced / made in donor cells, purified / isolated, obtained from donor cells, and then introduced into target cells.

[0123] The term "endogenous" with respect to mitochondria refers to mitochondria that are made / expressed / produced by a cell and are not introduced into the cell from an external source. In some embodiments, endogenous mitochondria contain proteins and / or other molecules that are encoded by the genome of the cell. In some embodiments, the term "endogenous mitochondria" is equivalent to the term "host mitochondria."

[0124] As used herein, the term "autologous cells" or "cells that are autologous" refers to cells that are the patient's own. The term "autologous mitochondria" refers to mitochondria obtained from the patient's own cells or maternally related cells. The term "allogeneic cells" or "allogeneic mitochondria" refers to cells that are from a different donor individual.

[0125] As used herein and in the claims, the term "syngeneic" refers to sufficient genetic identity or near genetic identity to permit transplantation between individuals without rejection. The term syngeneic in the context of mitochondria is used interchangeably herein with the term autologous mitochondria, which refers to the same maternal lineage.

[0126] The term "exogenous mitochondria" refers to mitochondria or mitochondrial DNA that is introduced into target cells (i.e., stem cells) from a source outside the cell. For example, in some embodiments, exogenous mitochondria can be derived from or isolated from cells different from target cells. For example, exogenous mitochondria can be produced / made in donor cells, purified / isolated, obtained from donor cells, and then introduced into target cells.

[0127] As used herein, the phrase "conditions that allow human functional mitochondria to enter human stem cells" generally refers to parameters such as time, temperature, culture medium, and proximity between mitochondria and stem cells. For example, human cells and human cell lines are routinely incubated in liquid medium and maintained in a sterile environment, such as in tissue culture incubators, at 37°C and 5% CO2 atmosphere. According to alternative embodiments disclosed and exemplified herein, cells can be incubated at room temperature in saline supplemented with human serum albumin. According to some embodiments, centrifugation is performed prior to incubation of human functional mitochondria with human stem cells. According to other embodiments, incubation occurs prior to centrifugation. In still further embodiments, centrifugation occurs during said incubation. In certain embodiments, the centrifugation speed is 8,000 g. In certain embodiments, the centrifugation speed is 7,000 g. According to further embodiments, the centrifugation is at a speed of 5,000 to 10,000 g. According to a further embodiment, the centrifugation is at a speed of 7,000 to 8,000 g.

[0128] In certain embodiments, human stem cells are incubated with healthy, functional, exogenous mitochondria at a temperature ranging from about 16° C. to about 37° C. for a time ranging from 0.5 to 30 hours. In certain embodiments, human stem cells are incubated with healthy, functional, exogenous mitochondria for a time ranging from 1 to 30 or 5 to 25 hours. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the incubation is for 20 to 30 hours. In some embodiments, the incubation is for at least 1, 5, 10, 15, or 20 hours. Each possibility represents a separate embodiment of the present invention. In other embodiments, the incubation is for up to 5, 10, 15, 20, or 30 hours. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the incubation is for 24 hours. In some embodiments, the incubation is at room temperature (16° C. to 30° C.). In other embodiments, the incubation is at 37° C. In some embodiments, the incubation is in a 5% CO2 atmosphere. In other embodiments, incubation does not include added CO2 above levels found in air. In certain embodiments, incubation is until the mitochondrial content in the stem cells is increased by an average of 1%-45% compared to their initial mitochondrial content.

[0129] In yet a further embodiment, incubation is carried out in culture medium supplemented with human serum albumin (HSA).In an additional embodiment, incubation is carried out in saline supplemented with HSA.According to a particular exemplary embodiment, the condition that allows functional mitochondria to enter human stem cells, thereby enriching said human stem cells with said human functional mitochondria, comprises incubation at room temperature in saline supplemented with 4.5% human serum albumin.

[0130] The characteristics of the product can be manipulated by manipulating the incubation conditions. In certain embodiments, the incubation is performed at 37° C. In certain embodiments, the incubation is performed for at least 6 hours. In certain embodiments, the incubation is performed for at least 12 hours. In certain embodiments, the incubation is performed for 12-24 hours. In certain embodiments, the incubation is performed at a ratio of 1*105 to 1*107 naive stem cells per amount of exogenous mitochondria having or exhibiting 4.4 milliunits of CS. In certain embodiments, the incubation is performed at a ratio of 1*106 naive stem cells per amount of exogenous mitochondria having or exhibiting 4.4 milliunits of CS. In certain embodiments, the conditions are sufficient to increase the mitochondrial content of naive stem cells by at least about 3%, 5%, or 10%, as determined by CS activity. Each possibility represents a separate embodiment of the present invention.

[0131] As used herein, the term "mitochondrial content" refers to the amount of functional mitochondria in a cell, or the average amount of functional mitochondria in multiple cells.

[0132] As used herein and in the claims, the terms "mitochondrial disease" and "primary mitochondrial disease" may be used interchangeably. As used herein, the term "primary mitochondrial disease" refers to a mitochondrial disease diagnosed by a known or obvious pathogenic mutation in mitochondrial DNA or by a mutation in a gene in nuclear DNA whose product is imported into mitochondria. According to some embodiments, the primary mitochondrial disease is a congenital disease. According to some embodiments, the primary mitochondrial disease is not a secondary mitochondrial dysfunction. The terms "secondary mitochondrial dysfunction" and "acquired mitochondrial dysfunction" are used interchangeably throughout this application.

[0133] In certain embodiments, the above method in its various embodiments further comprises centrifugation before, during, or after incubation of the stem cells with the exogenous mitochondria. Each possibility represents a separate embodiment of the present invention. In some embodiments, the above method in its various embodiments comprises a single centrifugation step before, during, or after incubation of the stem cells with the exogenous mitochondria. In some embodiments, the centrifugal force is in the range of 1000g to 8500g. In some embodiments, the centrifugal force is in the range of 2000g to 4000g. In some embodiments, the centrifugal force is greater than 2500g. In some embodiments, the centrifugal force is in the range of 2500g to 8500g. In some embodiments, the centrifugal force is in the range of 2500g to 8000g. In some embodiments, the centrifugal force is in the range of 3000g to 8000g. In other embodiments, the centrifugal force is in the range of 4000g to 8000g. In certain embodiments, the centrifugal force is 7000g. In other embodiments, the centrifugal force is 8000 g. In some embodiments, the centrifugation is carried out for a time ranging from 2 minutes to 30 minutes. In some embodiments, the centrifugation is carried out for a time ranging from 3 minutes to 25 minutes. In some embodiments, the centrifugation is carried out for a time ranging from 5 minutes to 20 minutes. In some embodiments, the centrifugation is carried out for a time ranging from 8 minutes to 15 minutes.

[0134] In some embodiments, the centrifugation is performed at a temperature ranging from 4 to 37°C. In certain embodiments, the centrifugation is performed at a temperature ranging from 4 to 10°C or 16 to 30°C. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the centrifugation is performed at 2 to 6°C. In certain embodiments, the centrifugation is performed at 4°C. In some embodiments, the above method in its various embodiments comprises a single centrifugation before, during, or after incubation of the stem cells with the exogenous mitochondria, followed by resting the cells at a temperature below 30°C. In some embodiments, the conditions that allow human functional mitochondria to enter the human stem cells comprise a single centrifugation before, during, or after incubation of the stem cells with the exogenous mitochondria, followed by resting the cells at a temperature ranging from 16 to 28°C.

[0135] In certain embodiments, the first composition is fresh. In certain embodiments, the first composition is frozen and then thawed before incubation. In certain embodiments, the second composition is fresh. In certain embodiments, the second composition is frozen and then thawed before incubation. In certain embodiments, the fourth composition is fresh. In certain embodiments, the fourth composition is frozen and then thawed before administration.

[0136] In certain embodiments, the stem cells obtained from a patient suffering from a malignant disease or from a healthy subject are bone marrow cells or bone marrow-derived stem cells.

[0137] The term "mammalian stem cells enriched with functional mitochondria" refers to human and non-human mammals.

[0138] According to the principles of the present invention, healthy functional human exogenous mitochondria are introduced into human stem cells, thus enriching these cells with healthy functional human mitochondria. It should be understood that such enrichment alters the mitochondrial content of human stem cells. Naive human stem cells have essentially one population of host / autologous mitochondria, whereas human stem cells enriched with exogenous mitochondria have essentially two populations of mitochondria, a first population of host / autologous / endogenous mitochondria and another population of introduced mitochondria (i.e., exogenous mitochondria). Thus, the term "enriched" refers to the state of the cells after receiving / uptake of exogenous mitochondria. It is straightforward to determine the number and / or ratio between the two populations of mitochondria, since the two populations may differ in some aspects, e.g., mitochondrial DNA. Thus, the phrase "human stem cells enriched with healthy functional human mitochondria" is equivalent to the phrase "human stem cells comprising endogenous mitochondria and healthy functional exogenous mitochondria". For example, human stem cells containing at least 1% healthy functional exogenous mitochondria of the total mitochondria are considered to contain a 99:1 ratio of host / autologous / endogenous mitochondria and healthy functional exogenous mitochondria. For example, "3% of total mitochondria" means that after enrichment, the original (endogenous) mitochondrial content is 97% of the total mitochondria and the introduced (exogenous) mitochondria is 3% of the total mitochondria, which is equivalent to an enrichment of (3 / 97=) 3.1%. As another example, "33% of total mitochondria" means that after enrichment, the original (endogenous) mitochondrial content is 67% of the total mitochondria and the introduced (exogenous) mitochondria is 33% of the total mitochondria, which is equivalent to an enrichment of (33 / 67=) 49.2%.

[0139] Heteroplasmy is the presence of two or more types of mitochondrial DNA in a cell or individual. Heteroplasmy level is the ratio of mutant mtDNA molecules to wild-type / functional mtDNA molecules and is an important factor in considering the severity of mitochondrial disease. Low levels of heteroplasmy (sufficient amount of mitochondria functioning) are associated with a healthy phenotype, while high levels of heteroplasmy (sufficient amount of mitochondria not functioning) are associated with disease states. In certain embodiments, the heteroplasmy level of the stem cells in the fourth composition is at least 1% lower than the heteroplasmy level of the stem cells in the first composition. In certain embodiments, the heteroplasmy level of the stem cells in the fourth composition is at least 3% lower than the heteroplasmy level of the stem cells in the first composition. In certain embodiments, the heteroplasmy level of the stem cells in the fourth composition is at least 5% lower than the heteroplasmy level of the stem cells in the first composition. In certain embodiments, the heteroplasmy level of the stem cells in the fourth composition is at least 10% lower than the heteroplasmy level of the stem cells in the first composition. In certain embodiments, the heteroplasmy level of stem cells in the fourth composition is at least 15% lower than the heteroplasmy level of stem cells in the first composition.In certain embodiments, the heteroplasmy level of stem cells in the fourth composition is at least 20% lower than the heteroplasmy level of stem cells in the first composition.In certain embodiments, the heteroplasmy level of stem cells in the fourth composition is at least 25% lower than the heteroplasmy level of stem cells in the first composition.In certain embodiments, the heteroplasmy level of stem cells in the fourth composition is at least 30% lower than the heteroplasmy level of stem cells in the first composition.

[0140] In certain embodiments, the mitochondrial content of human stem cells enriched with healthy mitochondria (also referred to herein as the cells of the fourth composition) is detectably higher than the mitochondrial content of human stem cells in the first composition.According to various embodiments, the mitochondrial content of the fourth composition is at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200% or more higher than the mitochondrial content of the first composition.In certain embodiments, the first composition is used fresh.

[0141] In certain embodiments, the first composition is frozen, then stored, and used after thawing. In other embodiments, the second composition comprising a plurality of functional human mitochondria is used fresh. In further embodiments, the second composition is frozen and thawed before use. In further embodiments, the fourth composition is used without freezing and storage. In yet further embodiments, the fourth composition is used after freezing, storage, and thawing. Suitable methods for freezing and thawing cell preparations to maintain viability are well known in the art. Suitable methods for freezing and thawing mitochondria to maintain structure and function are disclosed in our WO2013 / 035101 and WO2016 / 135723 and the references cited therein.

[0142] Citrate synthase (CS) is localized in the mitochondrial matrix but is encoded by nuclear DNA. Citrate synthase is involved in the first step of the Krebs cycle and is commonly used as a quantitative enzymatic marker for the presence of intact mitochondria (Larsen S. et al., J. Physiol., 2012, Vol. 590(14), pages 3349-3360; Cook GA et al., Biochim. Biophys. Acta., 1983, Vol. 763(4), pages 356-367).

[0143] In certain embodiments, the mitochondrial content of stem cells in the first composition, the second composition, or the fourth composition is determined by determining the content of citrate synthase.In certain embodiments, the mitochondrial content of stem cells in the first composition, the second composition, or the fourth composition is determined by determining the activity level of citrate synthase.In certain embodiments, the mitochondrial content of stem cells in the first composition, the second composition, or the fourth composition is correlated with the content of citrate synthase.In certain embodiments, the mitochondrial content of stem cells in the first composition, the second composition, or the fourth composition is correlated with the activity level of citrate synthase.CS activity can be measured by using a commercially available kit, for example, CS activity kit CS0720 (Sigma).

[0144] Eukaryotic NADPH-cytochrome C reductase (cytochrome C reductase) is a flavoprotein localized in the endoplasmic reticulum. It transfers electrons from NADPH to several oxygenases, the most important of which are the cytochrome P450 family of enzymes involved in the detoxification of xenobiotics. Cytochrome C reductase is widely used as an endoplasmic reticulum marker. In certain embodiments, the second composition is substantially free of cytochrome C reductase or cytochrome C reductase activity. In certain embodiments, the fourth composition is not enriched in cytochrome C reductase or cytochrome C reductase activity compared to the first composition.

[0145] In certain embodiments, the stem cells are pluripotent stem cells (PSCs). In other embodiments, the PSCs are non-embryonic stem cells. According to some embodiments, embryonic stem cells are expressly excluded from the scope of the present invention. In some embodiments, the stem cells are induced PSCs (iPSCs). In certain embodiments, the stem cells are embryonic stem cells. In certain embodiments, the stem cells are derived from bone marrow cells. In certain embodiments, the stem cells are CD34+ cells. In certain embodiments, the stem cells are mesenchymal stem cells. In other embodiments, the stem cells are derived from adipose tissue. In yet other embodiments, the stem cells are derived from blood. In further embodiments, the stem cells are derived from umbilical cord blood. In further embodiments, the stem cells are derived from oral mucosa.

[0146] In certain embodiments, bone marrow-derived stem cells include bone marrow hematopoietic cells. As used herein, the term "bone marrow hematopoietic cells" refers to cells involved in myelopoiesis, e.g., the bone marrow and all cells arising therefrom, i.e., the production of all blood cells.

[0147] In certain embodiments, bone marrow-derived stem cells include erythropoietic cells. As used herein, the term "erythropoietic cells" refers to cells involved in erythropoiesis, e.g., the production of red blood cells (erythrocytes).

[0148] In certain embodiments, bone marrow-derived stem cells include multipotent hematopoietic stem cells (HSCs). As used herein, the term "multipotent hematopoietic stem cells" or "hematopoietic blast cells" refers to stem cells that give rise to all other blood cells through the process of hematopoiesis.

[0149] In certain embodiments, the bone marrow derived stem cells include common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow derived stem cells include mesenchymal stem cells. As used herein, the term "common myeloid progenitor cells" refers to cells that give rise to myeloid cells. As used herein, the term "common lymphoid progenitor cells" refers to cells that give rise to lymphocytes.

[0150] In certain embodiments, the bone marrow-derived stem cells of the first composition further comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0151] In certain embodiments, bone marrow-derived stem cells include mesenchymal stem cells. As used herein, the term "mesenchymal stem cells" refers to multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells).

[0152] In certain embodiments, the bone marrow derived stem cells consist of bone marrow hematopoietic cells. In certain embodiments, the bone marrow derived stem cells consist of erythropoietic cells. In certain embodiments, the bone marrow derived stem cells consist of pluripotent hematopoietic stem cells (HSCs). In certain embodiments, the bone marrow derived stem cells consist of common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow derived stem cells consist of megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. In certain embodiments, the bone marrow derived stem cells consist of mesenchymal stem cells. Each possibility represents a separate embodiment of the present invention.

[0153] Hematopoietic progenitor antigen CD34, also known as CD34 antigen, is a protein that is encoded by CD34 gene in humans. CD34 is a differentiation cluster of cell surface glycoprotein and functions as a cell-cell adhesion factor. In certain embodiments, bone marrow stem cells express bone marrow progenitor antigen CD34 (are CD34+). In certain embodiments, bone marrow stem cells present bone marrow progenitor antigen CD34 on their outer membrane. In certain embodiments, CD34+ cells are from umbilical cord blood.

[0154] In certain embodiments, the stem cells in the first composition are directly derived from a subject suffering from a debilitating condition. In certain embodiments, the stem cells in the first composition are directly derived from a donor who is not suffering from a debilitating condition. As used herein, the term "directly derived" refers to stem cells that are directly derived from other cells. In certain embodiments, the hematopoietic stem cells (HSCs) are derived from bone marrow cells. In certain embodiments, the hematopoietic stem cells (HSCs) are derived from peripheral blood.

[0155] In certain embodiments, the stem cells in the first composition are indirectly derived from a subject suffering from a debilitating condition.In certain embodiments, the stem cells in the first composition are indirectly derived from a donor who is not suffering from a debilitating condition.As used herein, the term "indirectly derived" refers to stem cells derived from non-stem cells.In certain embodiments, the stem cells are derived from somatic cells that are manipulated to become induced pluripotent stem cells (iPSCs).

[0156] In certain embodiments, the stem cells in the first composition are directly obtained from the bone marrow of the subject suffering from debilitating condition.In certain embodiments, the stem cells in the first composition are directly obtained from the bone marrow of a donor who does not suffer from debilitating condition.As used herein, the term "directly obtained" refers to the stem cells obtained from the bone marrow itself, for example, by surgical operation or needle aspiration by syringe.

[0157] In certain embodiments, the stem cells in the first composition are indirectly obtained from the bone marrow of a patient suffering from a debilitating condition.In certain embodiments, the stem cells in the first composition are indirectly obtained from the bone marrow of a donor who does not suffer from a debilitating condition.As used herein, the term "indirectly obtained" refers to bone marrow cells obtained from a location other than bone marrow itself.

[0158] In certain embodiments, the stem cells in the first composition are obtained from the peripheral blood of a subject suffering from a debilitating condition.In certain embodiments, the stem cells in the first composition are obtained from the peripheral blood of a subject not suffering from a debilitating condition or from the peripheral blood of a subject not suffering from a debilitating condition.As used herein, the term "peripheral blood" refers to the blood that circulates in the blood system.

[0159] In certain embodiments, a first composition comprises a plurality of human bone marrow stem cells obtained from peripheral blood, the first composition further comprising megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0160] In certain embodiments, the method further comprises a previous step of administering to a subject suffering from a debilitating condition an agent that induces mobilization of bone marrow cells into the peripheral blood. In certain embodiments, the method further comprises a previous step of administering to a donor not suffering from a debilitating condition an agent that induces mobilization of bone marrow cells into the peripheral blood.

[0161] In certain embodiments, the agent that induces mobilization of bone marrow cells / stem cells produced in bone marrow to peripheral blood is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor, CAS number 155148-31-5), salts thereof, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0162] In certain embodiments, the method further comprises isolating stem cells from the peripheral blood of a subject suffering from a debilitating condition. In certain embodiments, the method further comprises isolating stem cells from the peripheral blood of a donor not suffering from a debilitating condition. As used herein, the term "isolate from peripheral blood" refers to the isolation of stem cells from other components of blood.

[0163] During apheresis, the subject's or donor's blood is passed through a device that separates one specific component and returns the rest to the circulation. It is therefore a medical procedure performed outside the body. In certain embodiments, the isolation is performed by apheresis.

[0164] In certain embodiments, the above method further comprises enriching the stem cells and functional mitochondria in the third composition before incubation.In certain embodiments, the above method further comprises enriching the stem cells and functional mitochondria in the third composition during incubation.

[0165] In certain embodiments, the method further comprises centrifuging the third composition before incubation. In other embodiments, the method further comprises centrifuging the third composition during incubation. In certain embodiments, the method further comprises centrifuging the third composition after incubation.

[0166] In certain embodiments, the stem cells in the first composition are obtained from a subject suffering from a debilitating condition, and the stem cells have (i) a normal oxygen (O2) consumption rate, (ii) a normal citrate synthase content or activity level, (iii) a normal adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the present invention.

[0167] In certain embodiments, the stem cells in the first composition are obtained from a subject suffering from a debilitating condition, and the stem cells have (i) a decreased oxygen (O2) consumption rate, (ii) a decreased citrate synthase content or activity level, (iii) a decreased adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii), compared to a subject not suffering from the debilitating condition. Each possibility represents a separate embodiment of the present invention.

[0168] It should be emphasized that any reference to any measurable characteristic or property or aspect of a plurality of cells or mitochondria refers to the average measurable characteristic or property or aspect of the plurality of cells or mitochondria.

[0169] In certain embodiments, the stem cells in the first composition are obtained from a donor not suffering from a debilitating condition and have (i) a normal oxygen (O2) consumption rate, (ii) a normal citrate synthase content or activity level, (iii) a normal adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the present invention.

[0170] In certain embodiments, the isolated human functional mitochondria in the second composition are obtained from a healthy subject with normal mitochondrial DNA and have (i) a normal oxygen (O2) consumption rate, (ii) a normal citrate synthase content or activity level, (iii) a normal adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the present invention.

[0171] In certain embodiments, the stem cells in the fourth composition have, compared to the stem cells in the first composition, (i) an increased oxygen (O2) consumption rate, (ii) an increased citrate synthase content or activity level, (iii) an increased adenosine triphosphate (ATP) production rate, (iv) an increased mitochondrial DNA content, or (v) any combination of (i), (ii), (iii), and (iv). Each possibility represents a separate embodiment of the present invention.

[0172] As used herein, the term "increased oxygen (O2) consumption rate" refers to an oxygen (O2) consumption rate that is detectably higher than the oxygen (O2) consumption rate in the first composition prior to mitochondrial enrichment.

[0173] As used herein, the term "increased citrate synthase content or activity level" refers to a citrate synthase content or activity level that is detectably higher than the citrate synthase content or activity level in the first composition prior to mitochondrial enrichment.

[0174] As used herein, the term "increased adenosine triphosphate (ATP) production rate" refers to an adenosine triphosphate (ATP) production rate that is detectably higher than the adenosine triphosphate (ATP) production rate in the first composition prior to mitochondrial enrichment.

[0175] As used herein, the term "increased mitochondrial DNA content" refers to a content of mitochondrial DNA that is detectably higher than the mitochondrial DNA content in the first composition prior to mitochondrial enrichment. The mitochondrial content may be determined by measuring the content of SDHA or COX1. In the context of this specification and claims, "normal mitochondrial DNA" refers to mitochondrial DNA that does not have / does not have mutations or deletions that are known to be associated with mitochondrial disease. As used herein, the term "normal oxygen (O2) consumption rate" refers to the average O2 consumption of cells from a healthy individual. As used herein, the term "normal activity level of citrate synthase" refers to the average activity level of citrate synthase in cells from a healthy individual. As used herein, the term "normal adenosine triphosphate (ATP) production rate" refers to the average ATP production rate in cells from a healthy individual.

[0176] According to some aspects, the present invention provides a method of treating a debilitating condition or a symptom thereof in a subject in need thereof, the method comprising administering to the patient a pharmaceutical composition comprising a plurality of human stem cells, the human stem cells being enriched with frozen-thawed healthy, functional, exogenous mitochondria free of pathogenic mutations in their mitochondrial DNA.

[0177] In certain embodiments, the symptoms are selected from the group consisting of impaired walking ability, impaired motor skills, impaired language skills, impaired memory, weight loss, cachexia, low blood alkaline phosphatase levels, low blood magnesium levels, high blood creatinine levels, low blood bicarbonate levels, low blood base excess levels, high urine glucose / creatinine ratio, high urine chloride / creatinine ratio, high urine sodium / creatinine ratio, high blood lactate levels, high urine magnesium / creatinine ratio, high urine potassium / creatinine ratio, high urine calcium / creatinine ratio, glycosuria, magnesiumuria, high blood urea levels, low C-peptide levels, high HbA1C levels, hypoparathyroidism, ptosis, hearing loss, cardiac conduction disorders, low ATP content, and oxygen consumption in lymphocytes, bipolar disorder, obsessive-compulsive disorder, depressive disorder, and mood disorders including personality disorders. Each possibility represents a separate embodiment of the present invention. It will be understood that defining symptoms as "high" and "low" corresponds to "detectably higher than normal" and "detectably lower than normal," respectively, with normal levels being the corresponding levels in subjects not afflicted with mitochondrial disease.

[0178] In certain embodiments, the pharmaceutical composition is administered to a specific tissue or organ. In certain embodiments, the pharmaceutical composition comprises at least 10 4 mitochondrial-enriched human stem cells. In certain embodiments, the pharmaceutical composition comprises about 10 4 to about 10 8 mitochondrial-enriched human stem cells.

[0179] In certain embodiments, the pharmaceutical composition is administered by parenteral administration. In certain embodiments, the pharmaceutical composition is administered by systemic administration. In certain embodiments, the pharmaceutical composition is administered by intravenous injection. In certain embodiments, the pharmaceutical composition is administered by intravenous infusion. In certain embodiments, the pharmaceutical composition comprises at least 105 mitochondrial-enriched human stem cells. In certain embodiments, the pharmaceutical composition comprises about 106 to about 108 mitochondrial-enriched human stem cells. In certain embodiments, the pharmaceutical composition comprises at least about 105 to 2*107 mitochondrial-enriched human stem cells per kilogram of patient body weight. In certain embodiments, the pharmaceutical composition comprises at least about 105 mitochondrial-enriched human stem cells per kilogram of patient body weight. In certain embodiments, the pharmaceutical composition comprises about 105 to about 2*107 mitochondrial-enriched human stem cells per kilogram of patient body weight. In certain embodiments, the pharmaceutical composition comprises about 106 to about 5*106 mitochondrial-enriched human stem cells per kilogram of patient body weight.

[0180] Mitochondrial DNA content can be measured by performing quantitative PCR of mitochondrial genes before and after mitochondrial enrichment, normalized to nuclear genes.

[0181] In certain circumstances, the same cells prior to mitochondrial enrichment serve as a control for measuring CS and ATP activity and determining the enrichment level.

[0182] In certain embodiments, the term "detectably high" as used herein refers to a statistically significant increase between normal and increased values. In certain embodiments, the term "detectably high" as used herein refers to a non-pathological increase, i.e., a level that does not reveal pathological symptoms associated with substantially high values. In certain embodiments, the term "increased" as used herein refers to a value that is 1.05-fold, 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold or more higher than the corresponding value found in the corresponding cells or corresponding mitochondria of a healthy subject(s), or in the stem cells of the first composition before mitochondrial enrichment. Each possibility represents a separate embodiment of the present invention.

[0183] In certain embodiments, the stem cells in the fourth composition have at least one of the following: (i) increased normal mitochondrial DNA content compared to the mitochondrial DNA content in stem cells before mitochondrial enrichment; (ii) increased oxygen (O2) consumption rate compared to the oxygen (O2) consumption rate in stem cells before mitochondrial enrichment; (iii) increased citrate synthase content or activity level compared to the citrate synthase content or activity level in stem cells before mitochondrial enrichment; (iv) increased adenosine triphosphate (ATP) production rate compared to the adenosine triphosphate (ATP) production rate in stem cells before mitochondrial enrichment; or (v) any combination of (i), (ii), (iii), and (iv).Each possibility represents a separate embodiment of the present invention.

[0184] In certain embodiments, the total amount of mitochondrial protein in the second composition is between 20% and 80% of the total amount of cellular protein in the sample.

[0185] As used herein, the term "about" refers to ±10% of the indicated numerical value. Typically, as used herein, numerical values ​​refer to ±10% of the indicated numerical value.

[0186] In certain embodiments, the method further comprises freezing the fourth composition. In certain embodiments, the method further comprises freezing and then thawing the fourth composition.

[0187] The present invention further provides, in another aspect, a plurality of human stem cells enriched with functional mitochondria, obtainable by the above method.

[0188] In certain embodiments, the plurality of stem cells is frozen before being enriched with functional mitochondria. In further embodiments, the plurality of stem cells is frozen and then thawed before being enriched with functional mitochondria. In other embodiments, the plurality of stem cells enriched with functional mitochondria is frozen. In other embodiments, the plurality of stem cells enriched with functional mitochondria is frozen and then thawed before use.

[0189] The invention further provides, in another aspect, a plurality of human stem cells, the stem cells having at least one characteristic selected from the group consisting of: (a) increased mitochondrial content, (b) increased oxygen (O2) consumption rate, (c) increased citrate synthase content or activity level, (d) increased mitochondrial DNA content, or (e) any combination of (a), (b), (c), and (d) compared to human stem cells from the same source prior to enrichment with healthy mitochondria in accordance with the principles of the invention. Each possibility represents a separate embodiment of the invention. According to some embodiments, the stem cells are CD34+ stem cells.

[0190] As used herein, the term "increased mitochondrial content" refers to a mitochondrial content that is detectably higher than the mitochondrial content of the first composition prior to mitochondrial enrichment.

[0191] In certain embodiments, the plurality of cells is frozen. In certain embodiments, the plurality of cells is frozen and then thawed prior to use.

[0192] In certain embodiments, the plurality of human stem cells are CD34+ and have increased mitochondrial content, increased levels of normal mitochondrial DNA, increased oxygen (O2) consumption rates, and increased levels of citrate synthase activity. Each possibility represents a separate embodiment of the present invention.

[0193] In certain embodiments, the plurality of human stem cells have increased mitochondrial content, increased levels of normal mitochondrial DNA, increased oxygen (O2) consumption rates, and increased levels of citrate synthase activity.

[0194] The present invention further provides, in another aspect, a pharmaceutical composition comprising a plurality of human bone marrow stem cells enriched with functional mitochondria as described above.

[0195] As used herein, the term "pharmaceutical composition" refers to any composition comprising cells that further comprises a medium or carrier in which the cells are maintained in a viable state.

[0196] In certain embodiments, the pharmaceutical composition is frozen. In certain embodiments, the pharmaceutical composition is frozen and then thawed prior to use.

[0197] In certain embodiments, the pharmaceutical composition is for use in a method for treating a certain symptom of a human subject having a debilitating condition. As used herein, the term "treat" includes the reduction, alleviation, or amelioration of at least one symptom associated with or induced by the debilitating effect of the condition afflicting the subject.

[0198] The present invention further provides, in another aspect, a method of alleviating or reducing the debilitating effects of a condition, including but not limited to anti-cancer therapy, in a human subject suffering from aging, an aging-related disease, or a malignant disease, comprising administering to the subject a pharmaceutical composition as described above.

[0199] As used herein, the term "method" generally refers to methods, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those methods, means, techniques, and procedures that are either known to practitioners of the chemical, pharmacological, biological, biochemical, and medical arts or readily developed from known methods, means, techniques, and procedures by such practitioners.

[0200] In certain embodiments, the pharmaceutical composition is frozen and the above method further comprises thawing the frozen pharmaceutical composition prior to use.

[0201] In certain embodiments, the stem cells are autologous to the subject suffering from the debilitating condition.

[0202] Contacting functional mitochondria with autologous stem cells to a subject suffering from a debilitating condition results in rejuvenation / regeneration of the stem cells.

[0203] In some embodiments, the above method in various embodiments further comprises expanding the stem cells of the first composition by culturing the stem cells in a growth medium capable of expanding the stem cells. In other embodiments, the method further comprises expanding the mitochondria-enriched stem cells of the fourth composition by culturing the cells in a culture or growth medium capable of expanding the stem cells. As used throughout this application, the term "culture or growth medium" refers to a fluid medium, such as a cell culture medium, a cell growth medium, a buffer that provides nutrients to the cells, and the like. As used throughout this application and in the claims, the term "pharmaceutical composition" refers to a fluid carrier, such as a cell culture medium, a cell growth medium, a buffer that provides nutrients to the cells, and the like.

[0204] In certain embodiments, administration of stem cells rejuvenated with functional mitochondria in a subject suffering from debilitating effects can reduce these effects. In some embodiments, administration of rejuvenated stem cells can restore the organization and distribution of epithelial cells in the intestinal villi of a subject suffering from a debilitating condition. In other embodiments, administration of rejuvenated stem cells can restore the activity of epithelial stem cells in the intestinal crypts of a subject. In further embodiments, administration of rejuvenated stem cells can restore the thickness of the dermis of a subject. In yet further embodiments, administration of rejuvenated stem cells can restore hair follicle activity of a subject. In additional embodiments, administration of rejuvenated stem cells can restore wound healing activity in dermal tissue of a subject. According to some embodiments, stem cells enriched with functional mitochondria can rejuvenate blood progenitor cells in an autologous hematopoietic stem cell graft. According to other embodiments, stem cells enriched with functional mitochondria can rejuvenate blood progenitor cells in an allogeneic hematopoietic stem cell graft. According to still other embodiments, stem cells enriched with functional mitochondria can rejuvenate progenitor cells of the dermis or intestinal epithelium. In additional embodiments, administration of rejuvenated stem cells can restore pancreatic function of beta cells in a subject. According to some embodiments, stem cells enriched with functional mitochondria can rejuvenate hepatocytes in the liver. According to other embodiments, stem cells enriched with functional mitochondria can slow kidney function decline. According to yet other embodiments, stem cells enriched with functional mitochondria can reduce macular degeneration.

[0205] In certain embodiments, the stem cells are allogeneic to the subject suffering from the debilitating condition. The terms "allogeneic to the subject", "from a donor" and "from a healthy donor" are used interchangeably herein and refer to stem cells or mitochondria that are from different donor individuals. If possible, the donor stem cells are preferably HLA-matched or at least partially HLA-matched to the patient's cells. According to certain embodiments, the donor is matched to the patient by identifying a specific mitochondrial DNA haplogroup.

[0206] As used herein, the term "HLA-matched" refers to the desire that the patient and the stem cell donor are as closely HLA-matched as possible, at least to the extent that the patient does not develop an acute immune response against the donor's stem cells. Such immune response prevention and / or therapy can be achieved with or without the acute or chronic use of immunosuppressants. In certain embodiments, the stem cells from the donor are HLA-matched to the patient to the extent that the patient does not reject the stem cells.

[0207] In certain embodiments, the patient is further treated with immunosuppressive therapy to prevent immune rejection of the stem cell graft.

[0208] In certain embodiments, the mitochondria are from the same haplogroup.

[0209] In other embodiments, the mitochondria are from different haplogroups.

[0210] In certain embodiments, the above method further comprises the step of administering a pre-transplant conditioning agent to the subject prior to administration of the pharmaceutical composition. As used herein, the term "pre-transplant conditioning agent" refers to any agent capable of killing bone marrow cells in the bone marrow of a human subject. In certain embodiments, the pre-transplant conditioning agent is busulfan.

[0211] In certain embodiments, the pharmaceutical composition is administered systemically. In certain embodiments, the administration of the pharmaceutical composition to the subject is by a route selected from the group consisting of intravenous, intraarterial, intramuscular, subcutaneous, intravitreal, and direct injection into tissue or organ. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the pharmaceutical composition is directly injected into the tissue and organ affected by the debilitating condition of the present invention. Specific tissues or organs that are known to show dysfunction associated with reduced mitochondrial quality and activity include, but are not limited to, the eye, kidney, liver, pancreas, brain, and heart.

[0212] In certain embodiments, functional mitochondria are obtained from human cells or tissue selected from the group consisting of placenta, placental cells grown in culture, and blood cells, with each possibility representing a separate embodiment of the present invention.

[0213] According to certain embodiments, the functional mitochondria have been subjected to freeze-thaw cycles. Without wishing to be bound by any theory or mechanism, mitochondria subjected to freeze-thaw cycles exhibit a comparable oxygen consumption rate after thawing compared to control mitochondria that have not been subjected to freeze-thaw cycles.

[0214] According to some embodiments, the freeze-thaw cycle comprises freezing the functional mitochondria for at least 24 hours before thawing. According to another embodiment, the freeze-thaw cycle comprises freezing the functional mitochondria for at least one month before thawing, or for several months or more before thawing. Each possibility represents a separate embodiment of the invention. According to another embodiment, the oxygen consumption of the functional mitochondria after the freeze-thaw cycle is equal to or higher than the oxygen consumption of the functional mitochondria before the freeze-thaw cycle.

[0215] As used herein, the term "freeze-thaw cycle" refers to freezing functional mitochondria to a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a predetermined period of time, and thawing the mitochondria to room temperature or body temperature or any temperature above 0°C that allows the mitochondria to process stem cells. Each possibility represents a separate embodiment of the invention. As used herein, the term "room temperature" typically refers to a temperature between 18°C ​​and 25°C. As used herein, the term "body temperature" refers to a temperature between 35.5°C and 37.5°C, preferably 37°C. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are functional mitochondria.

[0216] In another embodiment, the mitochondria subjected to freeze-thaw cycles are frozen at a temperature of at least -70°C or lower. In another embodiment, the mitochondria subjected to freeze-thaw cycles are frozen at a temperature of at least -20°C or lower. In another embodiment, the mitochondria subjected to freeze-thaw cycles are frozen at a temperature of at least -4°C or lower. According to another embodiment, the freezing of mitochondria is gradual. According to some embodiments, the freezing of mitochondria is by flash freezing. As used herein, the term "flash freezing" refers to the rapid freezing of mitochondria by subjecting them to cryogenic storage temperatures.

[0217] In another embodiment, the mitochondria subjected to the freeze-thaw cycle are frozen for at least 30 minutes before thawing. According to another embodiment, the freeze-thaw cycle includes freezing the functional mitochondria for at least 30, 60, 90, 120, 180, 210 minutes before thawing. Each possibility represents a separate embodiment of the present invention. In another embodiment, the mitochondria subjected to the freeze-thaw cycle are frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48, 72, 96, or 120 hours before thawing. Each freezing time represents a separate embodiment of the present invention. In another embodiment, the mitochondria subjected to the freeze-thaw cycle are frozen for at least 4, 5, 6, 7, 30, 60, 120, 365 days before thawing. Each freezing time represents a separate embodiment of the present invention. According to another embodiment, the freeze-thaw cycle includes freezing the functional mitochondria for at least 1, 2, 3 weeks before thawing. Each possibility represents a separate embodiment of the present invention. According to alternative embodiments, the freeze-thaw cycle comprises freezing the functional mitochondria for at least 1, 2, 3, 4, 5, 6 months prior to thawing, with each possibility representing a separate embodiment of the present invention.

[0218] In another embodiment, mitochondria subjected to freeze-thaw cycles were frozen at -70°C for at least 30 minutes before thawing. Without wishing to be bound by any theory or mechanism, the possibility to freeze mitochondria and thaw them after an extended period of time allows for easy storage and use of mitochondria with reproducible results even after long periods of storage.

[0219] According to certain embodiments, the thawing is at room temperature. In another embodiment, the thawing is at body temperature. According to another embodiment, the thawing is at a temperature that allows for administration of mitochondria according to the methods of the present invention. According to another embodiment, the thawing is performed gradually.

[0220] According to another embodiment, the mitochondria that have undergone freeze-thaw cycles are frozen in a freezing buffer. According to another embodiment, the mitochondria that have undergone freeze-thaw cycles are frozen in an isolation buffer. As used herein, the term "isolation buffer" refers to the buffer in which the mitochondria of the present invention are isolated. In a non-limiting example, the isolation buffer is a sucrose buffer. Without wishing to be bound by any mechanism or theory, freezing mitochondria in an isolation buffer saves time and isolation steps, since there is no need to exchange isolation buffer for freezing buffer before freezing or exchange freezing buffer upon thawing.

[0221] According to another embodiment, the freezing buffer comprises a cryoprotectant. According to some embodiments, the cryoprotectant is a sugar, an oligosaccharide, or a polysaccharide. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the sugar concentration in the freezing buffer is a sufficient sugar concentration that acts to preserve mitochondrial function. According to another embodiment, the isolation buffer comprises a sugar. According to another embodiment, the sugar concentration in the isolation buffer is a sufficient sugar concentration that acts to preserve mitochondrial function. According to another embodiment, the sugar is sucrose.

[0222] In certain embodiments, the method further comprises the prior steps of (a) freezing the human stem cells enriched with healthy functional human exogenous mitochondria, (b) thawing the human stem cells enriched with healthy functional human exogenous mitochondria, and (c) administering the human stem cells enriched with healthy functional human exogenous mitochondria to the patient.

[0223] In certain embodiments, healthy functional exogenous mitochondria constitute at least 3% of the total mitochondria in the mitochondria-enriched cells. In certain embodiments, healthy functional exogenous mitochondria constitute at least 10% of the total mitochondria in the mitochondria-enriched cells. In some embodiments, healthy functional exogenous mitochondria constitute at least about 3%, 5%, 10%, 15%, 20%, 25%, or 30% of the total mitochondria in the mitochondria-enriched cells. Each possibility represents a separate embodiment of the present invention.

[0224] The degree of enrichment of stem cells with functional mitochondria can be determined by functional and / or enzymatic assays, including but not limited to oxygen (O2) consumption rate, citrate synthase content or activity level, adenosine triphosphate (ATP) production rate. In another method, enrichment of stem cells with healthy donor mitochondria can be confirmed by detection of donor mitochondrial DNA. According to some embodiments, the degree of enrichment of stem cells with functional mitochondria can be determined by the level of heteroplasmy change and / or by the copy number of mtDNA per cell. Each possibility represents a separate embodiment of the present invention.

[0225] TMRM (tetramethylrhodamine methyl ester) or the related TMRE (tetramethylrhodamine ethyl ester) are cell-permeable fluorogenic dyes commonly used to assess mitochondrial function in live cells by identifying changes in mitochondrial membrane potential. According to some embodiments, the level of enrichment can be determined by staining with TMRE or TMRM.

[0226] According to some embodiments, mitochondrial membrane integrity can be determined by any method known in the art. In a non-limiting example, mitochondrial membrane integrity is measured using tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE) fluorescent probes. Each possibility represents a separate embodiment of the present invention. Mitochondria observed under a microscope and showing TMRM or TMRE staining have an intact mitochondrial outer membrane. As used herein, the term "mitochondrial membrane" refers to a mitochondrial membrane selected from the group consisting of mitochondrial inner membrane, mitochondrial outer membrane, and both.

[0227] In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched human stem cells is determined by sequencing at least a statistically representative portion of the total mitochondrial DNA in cells and determining the relative levels of host / endogenous mitochondrial DNA and exogenous mitochondrial DNA.In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched human stem cells is determined by single nucleotide polymorphism (SNP) analysis.In certain embodiments, the largest mitochondrial population and / or the largest mitochondrial DNA population is a host / endogenous mitochondrial population and / or a host / endogenous mitochondrial DNA population, and / or the second largest mitochondrial population and / or the second largest mitochondrial DNA population is an exogenous mitochondrial population and / or an exogenous mitochondrial DNA population.Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the enrichment of stem cells with healthy functional mitochondria can be determined by conventional assays recognized in the art.In certain embodiments, the level of enrichment of mitochondria in mitochondria-enriched human stem cells is determined by (i) the level of host / endogenous mitochondrial DNA and exogenous mitochondrial DNA, (ii) the level of mitochondrial protein selected from the group consisting of citrate synthase (CS), cytochrome C oxidase (COX1), succinate dehydrogenase complex flavoprotein subunit A (SDHA), and any combination thereof, (iii) the level of CS activity, or (iv) any combination of (i), (ii), and (iii).Each possibility represents a separate embodiment of the present invention.

[0228] In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched human stem cells is determined by at least one of the following: (i) the level of host mitochondrial DNA and exogenous mitochondrial DNA in the case of allogeneic mitochondria; (ii) the level of citrate synthase activity; (iii) the level of succinate dehydrogenase complex flavoprotein subunit A (SDHA) or cytochrome C oxidase (COX1); (iv) oxygen (O2) consumption rate; (v) adenosine triphosphate (ATP) production rate; or (vi) any combination thereof. Each possibility represents a separate embodiment of the present invention. Methods for measuring these various parameters are well known in the art.

[0229] In some aspects, the present invention provides a pharmaceutical composition comprising human stem cells enriched with healthy functional mitochondria for use in treating or reducing the debilitating effects of a condition in a subject, the debilitating effects of the condition being selected from the group consisting of, but not limited to, aging, aging-related diseases, and sequelae of anti-cancer treatment.

[0230] In some embodiments, the present invention provides a method for treating or reducing the debilitating effects of a condition in a subject, comprising administering to the subject a pharmaceutical composition comprising human stem cells enriched with healthy functional mitochondria, wherein the debilitating effects of the condition are selected from the group consisting of, but not limited to, old age, old age-related diseases, and sequelae of anti-cancer treatment. In certain embodiments, the anti-cancer treatment is selected from the group consisting of radiation therapy, chemotherapy, immunotherapy with monoclonal antibodies, or any combination thereof.

[0231] According to certain embodiments, healthy functional mitochondria are isolated from donors selected from specific mitochondrial haplogroups according to the subject's debilitating condition.For example, for aged subjects, administration of stem cells enriched with functional mitochondria from J mitochondrial haplogroup is preferred because it is associated with longevity and low blood pressure (De Benedictis et al., FASEB J.1999;13(12):1532-6; Rea et al., AGE 2013;34(4):1445-56). H and N haplogroups are associated with better muscle functionality and strength (Larsen et al., Biochim Biophys Acta.2014;1837(2):226-31; Fuku et al., Int J Sports Med.2012;33(5):410-4). The D4b haplogroup may protect against stroke (Yang et al., Mol Genet Genomics. 2014; 289(6): 1241-6), the K, U, H, and V haplogroups may confer protection against cognitive impairment (Colicino et al., Environ Health. 2014; 13(1): 42), and the R haplogroup has been shown to confer a better prognosis for recovery from septic encephalopathy (Yang et al., Intensive Care Med. 2011; 37(10): 1613-9). Haplogroup N9a confers resistance to diabetes (Fuku et al., Am J Hum Genet. 2007; 80(3): 407-15) and metabolic syndrome (Tanaka et al., Diabetes 2007; 56(2): 518-21). The H haplogroup protects against the development of eye diseases, including age-related macular degeneration (AMD) (Mueller et al., PloS one 2012;7(2):e30874).

[0232] According to certain embodiments, the stem cells of the first composition are from donors that are selected from certain mitochondrial haplogroups according to the debilitating condition of the subject.For example, for subjects suffering from the debilitating effects of anti-cancer treatment, J, K2 and U haplogroups can be considered, because they have been shown to be better donors for allogeneic hematopoietic stem cell transplantation, with less GVHD and / or relapse (Ross et al.Biol Blood Marrow Transplant 2015;21:81-88).

[0233] As used herein, the term "haplogroup" refers to a genetic population of people who share a common ancestor along the maternal line. Mitochondrial haplogroups are determined by sequencing.

[0234] In certain cases, one may wish to match haplotypes between the donor and acceptor.

[0235] As used herein, the term "about" refers to a range of less than 10% to more than 10% of a given integer, number, or amount. For example, "about 1*105" means "1.1*105 to 9*104."

[0236] Although the invention has been described with reference to certain specific embodiments, those skilled in the art will recognize that various modifications can be made and equivalents can be substituted without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is not intended that the invention be limited to the specific embodiments disclosed, but the invention is intended to include all embodiments falling within the scope of the appended claims.

[0237] The following examples are presented to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions, and reported data described to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention. EXAMPLES

[0238] Example 1. Isolated human mitochondria: preparation and cryopreservation. Mitochondria can be isolated and preserved as previously disclosed in WO2013 / 035101 and WO2016 / 135723. Below are exemplary protocols used for the isolation of mitochondria from peripheral blood cells (MNV-BLD) and enrichment of CD34+ cells (MNV-BM-BLD). Stage 1 - Production of MNV-BLD: Buffy coat is isolated from peripheral blood (500 mL) obtained from the patient or donated by a donor. The buffy coat is then layered on top of Lymphoprep™ and centrifuged. White blood cells (buffy coat on top of Lymphoprep™) are collected and centrifuged. The cell pellet (lymphocytes) is washed, the cell pellet is frozen and suspended in ice-cold 250 mM sucrose buffer (250 mM sucrose, 10 mM Tris, 1 mM EDTA) pH=7.4. The cell suspension is collected and homogenized after passing three times through a 30G needle. The homogenate is centrifuged. The supernatant is collected and placed on ice, the pellet is washed with sucrose solution, homogenized and centrifuged. The second supernatant from the washed pellet is collected and combined with the previous one. The combined supernatant is filtered through a 5 μm filter and centrifuged at 8000 g. The pellet is washed with sucrose solution and resuspended in 1 ml of cold 250 mM sucrose buffer pH = 7.4. The resulting mitochondrial solution (herein referred to as MNV-BLD) is stored frozen in a vapor nitrogen tank until use. Phase 2 - Generation of MNV-BM-BLD: After bone marrow cells are mobilized to peripheral blood, the CliniMACS™ system is used to isolate patient or donor CD34+ cells from blood collected by leukapheresis. The pellet of CD34+ cells is suspended in 4.5% HSA in 0.9% NaCl solution to a final concentration of 1×106 cells / ml. MNV-BLD (mitochondrial suspension) is thawed at room temperature and added to the CD34+ cells at 4.4 citrate synthase (CS) activity milliunits (1×106 cells) per ml of cell suspension. MNV-BLD and CD34+ cells are mixed in a 2 mL tube and centrifuged at 7000 g for 5 minutes at 4° C. After centrifugation, the cells are suspended in the same 4.5% HSA in 0.9% NaCl solution, combined, seeded into flasks and incubated at room temperature for 24 hours. After incubation, the enriched CD34+ cells are washed twice with 4.5% HSA solution and centrifuged at 300 g for 10 minutes. The cell pellet is resuspended in 100 ml of 4.5% HSA in 0.9% NaCl and loaded into an infusion bag.

[0239] Example 2. Isolated mitochondria can enter fibroblast cells. Mouse fibroblasts (3T3) expressing green fluorescent protein (GFP) in mitochondria (left panel) were incubated for 24 h with red fluorescent protein (RFP)-labeled mitochondria isolated from mouse fibroblasts (3T3) expressing RFP in mitochondria (middle panel). Fluorescence confocal microscopy was used to identify fibroblasts labeled with both GFP and RFP, which appear yellow (right panel), as previously described in WO2016 / 135723 (Figure 1).

[0240] The results shown in FIG. 1 demonstrate that mitochondria can enter fibroblasts.

[0241] Example 3. Mitochondria increase ATP production in cells with inhibited mitochondrial activity. Mouse fibroblast cells (104, 3T3) were either not treated (control) or treated with 0.5 μM rotenone (rotenone, mitochondrial complex I irreversible inhibitor, CAS number 83-79-4) for 4 h, washed, and further treated with 0.02 mg / ml mouse placental mitochondria (rotenone + mitochondria) for 3 h. Cells were washed and ATP levels were determined using a Perkin Elmer ATPlite kit (Figure 2), as previously shown in WO2016 / 135723. As can be seen in Figure 2, ATP production was fully rescued in cells incubated with mitochondria compared to the control.

[0242] The results shown in Figure 2 clearly show that while rotenone alone reduced ATP levels by approximately 50%, the addition of mitochondria is able to substantially negate the inhibitory effect of rotenone and reach ATP levels of control cells. This experiment provides evidence of the ability of mitochondria to increase mitochondrial ATP production in cells with impaired or impaired mitochondrial activity.

[0243] Example 4. Mitochondria can enter mouse bone marrow cells. Mouse bone marrow cells (10) were incubated for 24 h with GFP-labeled mitochondria isolated from mouse melanoma cells. Fluorescence confocal microscopy was used to identify GFP-labeled mitochondria in bone marrow cells as previously described in WO2016 / 135723 (Figure 3). The results shown in FIG. 3 indicate that mitochondria can enter bone marrow cells.

[0244] Bone marrow cells from wild-type (ICR) and mutant mitochondrial (FVB / N, carrying a mutation in ATP8) mice were incubated with isolated mitochondria of different origins in DMEM for 24 h at 37 °C and 5% CO2 atmosphere to increase mitochondrial content and activity. Table 1 shows representative results of the mitochondrial enhancement process, as determined by the relative increase in CS activity of cells after the process compared to CS activity of cells before the process. [Table 1]

[0245] To examine the effects of mitochondrial enhancement therapy in vivo, FVB / N bone marrow cells (1x106) enriched with 4.4 mUnitsCS activity of C57 / BL placental mitochondria were IV injected into FVB / N mice. Bone marrow was collected from mice 1 day, 1 week, 1 month, and 3 months after treatment, and levels of WT mtDNA were detected using dPCR. As seen in Figure 4, significant amounts of WT mtDNA were detected in bone marrow 1 day after treatment.

[0246] Example 5. Mitochondria enter bone marrow cells in a concentration-dependent manner. Mouse bone marrow cells (106) were either untreated or incubated for 15 h with different amounts of GFP-labeled mitochondria isolated from mouse melanoma cells. Before plating the cells, mitochondria were either mixed with the cells and left at room temperature for 5 min ((-)Cent) or centrifuged at 40 C and 8,000 g for 5 min ((+)Cent). The cells were then plated in 24 wells (106 cells / well). After 15 h of incubation, the cells were washed twice to remove any mitochondria that did not enter the cells. Citrate synthase activity was determined using the CS0720 Sigma kit as previously described in WO2016 / 135723 (Figure 5). CS activity levels measured under the conditions specified above are summarized in Table 2. [Table 2]

[0247] The results shown in Figure 5 indicate that the addition of mitochondria increases cellular CS activity in a dose-dependent manner, and that increasing the concentration, and thus presumably increasing the contact between mitochondria and cells, for example by centrifugation, further increases CS activity.

[0248] Mouse bone marrow cells (106) were either untreated or incubated for 24 h with GFP-labeled mitochondria (17 milliunits or 34 milliunits, indicating the level of citrate synthase activity as a marker of mitochondrial content) isolated from mouse melanoma cells. Cells were mixed with mitochondria, centrifuged at 8000 g, and resuspended. After 24 h of incubation, cells were washed twice with PBS and the levels of citrate synthase (CS) activity (Figure 6A) and cytochrome c reductase activity (Figure 6B) were measured using CS0720 and CYOIOO kits (Sigma), respectively, as previously described in WO2016 / 135723.

[0249] FVB / N bone marrow cells (carrying a mutation in mtDNA ATP8) were incubated with C57 / BL wild-type (WT) mitochondria isolated from placenta at various doses (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 mUnits CS activity per M cells in 1 mL). As seen in Figure 7A, dPCR using WT-specific sequences showed a dose-dependent increase in WT mtDNA at most dosages. Enriched cells also showed a dose-dependent increase in the content of mtDNA-encoded (COX1) (Figure 7B) and nuclear-encoded (SDHA) (Figure 7C).

[0250] Example 6. Mitochondria can enter human bone marrow cells. Human CD34+ cells (1.4*105, ATCC PCS-800-012) were either untreated or incubated for 20 h with GFP-labeled mitochondria isolated from human placental cells. Before plating the cells, mitochondria were mixed with the cells, centrifuged at 8000g, and resuspended. After incubation, cells were washed twice with PBS and CS activity was measured using the CS0720 Sigma kit (Figure 8A). ATP content was measured using ATPlite (Perkin Elmer) (Figure 8B). CS activity levels measured under the conditions specified above (Figure 8A) are summarized in Table 3. [Table 3]

[0251] The results shown in FIG. 8 (see Table 3) clearly demonstrate that the mitochondrial content of human bone marrow cells can be increased many-fold by interaction and co-incubation with isolated human mitochondria, exceeding the capacity of either human or mouse fibroblasts or mouse bone marrow cells.

[0252] The cell populations shown in Figure 8B were further evaluated by FACS analysis. As previously shown in WO2016 / 135723, in CD34+ cells not incubated with GFP-labeled mitochondria, only a small fraction of cells (0.9%) were fluorescent (Figure 9A), whereas CD34+ cells incubated with GFP-labeled mitochondria after centrifugation were substantially fluorescent (28.4%) (Figure 9B).

[0253] Example 7. Mitochondria can enter human CD34+ bone marrow cells. Human CD34+ cells from healthy donors treated with GCS-F were obtained by apheresis, purified using the CliniMACS system, and frozen. Cells were thawed and treated with blood-derived mitochondria (MNV-BLD) (4.4 milliunits of mitochondrial CS activity per 1x106 cells) or left untreated (NT), centrifuged at 8000g, and incubated for 24 hours. Cells were then washed with PBS and CS activity (Figure 10B) and ATP content (Figure 10A) were measured (using the CS0720 Sigma kit and ATPlite Perkin Elmer, respectively).

[0254] CD34+ cells treated with blood-derived mitochondria showed a significant increase in mitochondrial activity, as measured by CS activity (FIG. 10B) and ATP content (FIG. 10A).

[0255] Using mitochondria isolated from HeLa-TurboGFP-Mitochondria cells (CellTrend GmbH), CD34+ cells from healthy donors were treated with MitotrackerOrange (MTO) and washed prior to MAT. Cells were fixed with 2% PFA for 10 min and fixed with DAPI. Cells were scanned using a confocal microscope equipped with a 60X / 1.42 oil immersion objective.

[0256] As seen in FIG. 11, exogenous mitochondria entered CD34+ cells 0.5 h after MAT (bright, nearly white, intracellular specks) and continued over the 8 and 24 h periods examined.

[0257] Example 8. Culturing CD34+ cells with saline at room temperature improves viability. CD34+ cells were either untreated (NT) or incubated with blood-derived mitochondria (MNV-BLD). Cells were cultured at room temperature (RT) or 37°C in culture medium (CellGro™) or saline (Zenalb™) containing 4.5% human serum albumin (HSA).

[0258] Cell viability under different culture conditions is summarized in Table 4. [Table 4]

[0259] The results, shown in Table 4, indicate that CD34+ cell viability is improved when cultured at RT with human serum albumin in saline rather than culture medium.

[0260] Example 9. Bone marrow from NSGS mice transplanted with human umbilical cord blood contains more human mtDNA 2 months after MAT After incubating Pearson patient cord blood cells with 0.88mU of human mitochondria for 24 hours, the medium was removed, the cells were washed and resuspended in 4.5% HSA, and the enriched cells were injected IV into NSGS mice (100,000 CD34+ cells per mouse).

[0261] FIG. 12A is a diagram of mtDNA deletion in cord blood cells of Pearson patients, showing the 4978 kb deleted UCB mtDNA region (left) as well as Southern blot analysis demonstrating the deletion (right).

[0262] Bone marrow was collected from mice 2 months after MAT, and the copy number of non-deleted WT mtDNA was analyzed by dPCR using primers and probes that identify UCB non-deleted WT mtDNA sequences.

[0263] As seen in Figure 12B, two months after mitochondrial enhancement therapy, the bone marrow of the mice contained approximately 100% more human mtDNA compared to the bone marrow of mice injected with non-enhanced cord blood cells.

[0264] Example 10. In vivo safety and biodistribution animal studies Mitochondria are introduced into bone marrow cells of control healthy mice from two different backgrounds: the source of mitochondria is from mice with different mtDNA sequences (Jenuth JP et al., Nature Genetics, 1996, Vol. 14, pages 146-151).

[0265] Mitochondria were isolated from wild-type mouse (C57BL) placentas. Bone marrow cells were isolated from FVB / N mice. Mutant FVB / N bone marrow cells (106) were loaded with healthy functional C57BL mitochondria (4.4 mU) and administered IV to FVB / N mice.

[0266] The steps of the method are: (1) isolate mitochondria from placenta of C57BL mice, freeze at -80℃, thaw or use fresh, (2) obtain bone marrow cells from mtDNA mutant FVB / N mice, (3) contact mitochondria and bone marrow cells, centrifuge at 8000g for 5 min, resuspend and incubate for 24 h, (4) wash bone marrow cells twice with PBS and inject into tail vein of FVB / N mice. At various time points after transplantation, e.g., 24 h, 1 week, 1 month, 3 months, tissues (blood, bone marrow, lymphocytes, brain, heart, kidney, liver, lung, spleen, skeletal muscle, eye, ovary / testis) were collected and DNA was extracted for further sequence analysis.

[0267] The decreased levels of FVB / N in bone marrow one month after transplantation is shown in Figure 13 A. As seen in Figure 13 B, mtDNA levels were also decreased in liver of FVB / N mice three months after transplantation.

[0268] Bone marrow harvested from FVB / N females was enriched with C57BL / 6 placental mitochondria (4.4mU CS activity per 1X10^6 cells). Recipient mice received IV administration of 1 million augmented cells per animal. Digital PCR was used to detect C57BL / 6-specific SNPs. Figure 14A shows the presence of C57BL / 6 mtDNA in the bone marrow of FVBN mice 1 day after MAT, with some mice showing persistence up to 3 months after treatment. Figures 14B and 14C show the presence of C57BL / 6-derived mtDNA in the heart and brain of mice 3 months after MAT.

[0269] Example 11. In vivo preclinical animal studies: Effect of preconditioning on transplantation of foreign mitochondria Mitochondria were isolated from wild-type mouse (C57BL) liver. Bone marrow cells were isolated from mice with mutated mitochondria (FVB / N mice). Mutated FVB / N bone marrow cells were loaded with healthy functional C57BL mitochondria. Untreated FVB / N mice (controls), FVB / N mice administered enriched mitochondria, FVB / N mice treated with a chemotherapeutic agent (busulfan) prior to administration of enriched mitochondria, and FVB / N mice that underwent total body irradiation (TBI) prior to administration of enriched mitochondria were compared.

[0270] The steps of the method are: (1) isolate mitochondria from the liver of C57BL mice, freeze at -80°C, thaw or use fresh; (2) obtain bone marrow cells from mtDNA mutant FVB / N mice; (3) contact mitochondria and bone marrow cells, centrifuge at 8000g for 5 min, resuspend and incubate for 24 h; (4) wash bone marrow cells twice with PBS; (5) administer busulfan or total body irradiation (TBI) to the desired group; (6) inject bone marrow cells of FVB / N mice enriched with healthy mitochondria of C57BL mice into the tail vein of FVB / N mice. One month after transplantation, tissues (blood, bone marrow, lymphocytes, brain, heart, kidney, liver, lung, spleen, pancreas, skeletal muscle, eye, ovary / testis) were collected and DNA was extracted for further sequence analysis.

[0271] One month after transplantation, the decreased levels of FVB / N in the brains of mice treated with mitochondria, TBI, and busulfan are shown in FIG.

[0272] Example 12. Effects of mitochondrial enrichment on aged mice. Mitochondria were isolated from the placenta of full-term C57BL mice. Bone marrow cells were obtained from 12-month-old C57BL mice. Mitochondria-enriched bone marrow cells (MNV-BM-PLC, 1x106 cells), bone marrow cells alone (BM, 1x106 cells), or control vehicle solution (VEHICLE, 4.5% albumin in 0.9% w / v NaCl) were injected IV into the tail vein of 12-month-old C57BL mice at the start of the experiment and again at approximately 15, 18, and 21 months of age. BUN blood tests were performed 1, 3, 4, and 6 months after the first IV injection. Open field tests were performed 9 months after the first IV injection. BUN blood tests were performed 2, 4, and 6 months after IV injection.

[0273] As seen in Figures 16A-16D, aged mice (12 months) transplanted with bone marrow cells enriched with healthy mitochondria (MNV-BM-PLC) showed improved physical activity and exploratory behavior compared to age-matched mice transplanted with bone marrow not enriched with mitochondria (BM control) and mice not transplanted at all (control). MNV-BM-PLC-treated mice showed a typical behavioral pattern of young mice, traveling longer distances (Figure 16A), spending more time in the center of the cage (Figure 16B), and spending less time next to the wall (Figure 16C) compared to controls. Also, as shown in Figure 16D, administration of bone marrow enriched with functional mitochondria to aged mice halted kidney deterioration. Increased time spent in the central zone of the arena indicates extensive exploratory behavior in mice receiving mitochondrial augmentation treatment, which, together with reduced thigmotaxis associated with anxiety-like behavior, demonstrates the anxiolytic effect of mitochondrial augmentation. Gross motor skills and coordination were also assessed using a rotarod device in these mice. As shown in Figures 16E-16F, after 1 month of treatment, the VEHICLE and BM control groups showed a decrease in latency to fall off the rotating rod (-2.82% and -2.18% from baseline, ns), which further decreased by 14.15% and 21.79% compared to baseline after 3 months of treatment (***p=0.0008). MNV-BM-PLC mice showed a 16.17% decrease in latency to fall off the rod after 1 month of mitochondrial enrichment therapy (*p=0.0464), which ceased after 3 months of enrichment (-8.72% from baseline, ns). The results showed that mitochondrially enriched middle-aged mice had more mitochondrial function than age-matched controls. Reduced These results indicate impaired motor function, suggesting that mitochondrial enrichment therapy may alleviate age-related decline in motor function. Skeletal muscle function was also assessed by the forelimb grip strength test in these mice. As shown in Figures 16G-16H, MNV-BM-PLC mice maintained stable grip strength scores after 1 and 3 months of mitochondrial enrichment therapy (-1.29% and -1.40% of baseline, respectively) and showed a slower deterioration in grip strength time (latency to release grasp) after 3 months of treatment (+6.07% and -0.69% of baseline after 1 and 3 months of treatment). As shown in Figures 16I-16J, compared to the VEHICLE and BM control groups, the decline from baseline of -4.80% and -0.9% observed after 1 month of treatment was further increased after 2 months (-15.3% and -6.35% of baseline, respectively, ns). The baseline grip strength of the VEHICLE and BM control mice increased after 1 month of treatment (+6.01% and +4.06% from baseline, ns) and declined by -6.03% (**p=0.0084) and -17.77% (*p=0.0404) of baseline, respectively, after 2 months. These results indicate slower / reduced deterioration of grip strength and holding time in mitochondrial-enriched treated mice, suggesting that mitochondrial-enrichment therapy may ameliorate old-age-associated impairments in muscle function.

[0274] Example 13. Reducing the debilitating effects of aging and aging-related diseases in human subjects The steps of the method for reducing the debilitating effects of aging human subjects or subjects suffering from aging-related disease(s) include: (1) administering G-CSF to the aged subject or donor five times at a dosage of 10-16 μg / kg; (2) on day 5, considering administering Mozovir to the subject for 1-2 days; (3) on day 6, performing apheresis on the subject's blood to obtain bone marrow cells. If the amount of stem cells is insufficient, apheresis can be performed again on day 7; (4) in parallel, isolating functional mitochondria from a blood sample or placenta of a healthy donor. Isolation of functional mitochondria can also be performed prior to this process, storing mitochondria frozen at -80°C (at least) and thawing before use; (5) incubating bone marrow cells with functional mitochondria for 24 hours; (6) washing the bone marrow cells; and (7) injecting the bone marrow cells enriched with mitochondria into the aged subject. Throughout the period, the patients are assessed for changes in food consumption, body weight, lactic acidosis, blood counts, and biochemical blood markers.

[0275] Another method for reducing the debilitating effects of aged human subjects or subjects suffering from aging-related disease(s) involves (1) obtaining adipose tissue from aged subjects using a surgical procedure such as liposuction, (2) isolating mesenchymal stem cells (MSCs), expanding the cells in culture, and optionally cryopreserving the cells, and (3) in parallel isolating functional mitochondria from blood samples or placenta of healthy donors. Isolation of functional mitochondria can also be performed prior to this process, storing frozen mitochondria at -80°C (at least) and thawing before use, (5) incubating MSCs with functional mitochondria for 24 hours, (6) washing the MSCs, and (7) injecting the mitochondria-enriched MSCs into the subject. Throughout the entire period, changes in the patient's food consumption, body weight, lactic acidosis, blood counts, and biochemical blood markers are evaluated.

[0276] Example 14. Therapy of human patients suffering from non-hematopoietic neoplastic diseases. The steps of the method for therapy of human patients suffering from non-hematopoietic neoplastic diseases include: (1) administering G-CSF to the patient suffering from neoplastic diseases at a dosage of 10-16 μg / kg for 5 days; (2) on the 6th day, performing apheresis on the patient's blood to obtain bone marrow cells; (3) in parallel, isolating functional mitochondria from a blood sample of a healthy donor; (4) incubating the bone marrow cells with functional mitochondria for 24 hours; (5) washing the bone marrow cells; (6) injecting the mitochondria-loaded bone marrow cells into the patient. Throughout the entire period, the changes in the patient's food consumption, body weight, lactic acidosis, blood counts, and biochemical blood markers are evaluated.

[0277] Example 15. Compassionate treatment of a young patient with Pearson Syndrome (PS) using autologous CD34+ cells enriched with MNV-BLD (blood-derived mitochondria). A 6.5-year-old male patient (Patient 1) was diagnosed with Pearson syndrome and had a deletion of nucleotides 5835-9753 in mtDNA. Prior to mitochondrial augmentation therapy (MAT), he weighed 14.5 KG and was unable to walk more than 100 meters or climb stairs. His growth was significantly delayed for 3 years prior to treatment, with a baseline weight of -4.1 standard deviation score (SDS) and height of -3.2 SDS (compared to the population) and did not improve despite being fed by a gastrostomy tube (G-tube) for over 1 year. He had renal failure (GFR 22 ml / min) and proximal tubulopathy requiring electrolyte supplementation. He had hypoparathyroidism requiring calcium supplementation, and incomplete right bundle branch block (ICRBB) on electrocardiogram.

[0278] Mobilization of hematopoietic stem and progenitor cells (HSPCs) was performed by subcutaneous administration of GCSF (10 μg / kg) alone for 5 days. Leukapheresis (n=2) was performed via peripheral venous access according to institutional guidelines using the Spectra Optia system (TerumoBCT). CD34 positive selection was performed on mobilized peripheral blood-derived cells by using CliniMACS CD34 reagent according to the manufacturer's instructions. Mitochondria were isolated by differential centrifugation from maternal peripheral blood mononuclear cells (PBMCs) using 250 mM sucrose buffer pH 7.4. For mitochondrial enhancement therapy (MAT), autologous CD34+ cells were incubated with healthy mitochondria (1*106 cells per mitochondrial amount with 4.4 milliunits of citrate synthase (CS)) from the patient's mother, resulting in a 1.56-fold increase in cellular mitochondrial content (56% increase in mitochondrial content as indicated by CS activity). Incubation with mitochondria was performed in saline containing 4.5% HSA for 24 hours at RT. Enriched cells were suspended in 4.5% human serum albumin in saline. Following the timeline shown in Figure 17A, patients received a single treatment of 1.1*106 autologous CD34+ cells enriched with healthy mitochondria per kilogram of body weight by IV infusion.

[0279] As seen in FIG. 17B, the patient's aerobic metabolic equivalent (MET) score increased 4 months after transplantation of mitochondrial-enriched cells, and the effect was unchanged 8 months after transplantation. The data show that the patient's aerobic MET score increased significantly over time after therapy from 5 (moderate intensity activities such as walking and cycling) to 8 (vigorous intensity activities such as running, jogging, and jumping rope). MET is a physiological measure that represents the energy cost of physical activity. Because aerobic MET scores decline with age, the ability of enriched cell transplantation to improve this parameter is promising for aging subjects.

[0280] FIG. 17C shows the level of lactate found in the patient's blood as a function of time after IV injection. Blood lactate is lactate that appears in the blood as a result of anaerobic metabolism when mitochondria are damaged or when oxygen transport to tissues is insufficient to support normal metabolic demands, one of the hallmarks of mitochondrial dysfunction. As seen in FIG. 4C, after MAT, patient 1's blood lactate level was reduced to normal values. Lactate is oxidized in mitochondria, which are partially responsible for lactate turnover in the human body. As mitochondrial quality and activity decline with age, lactate levels rise. Thus, the ability of enriched bone marrow stem cells to reduce lactate levels suggests potential action on aging subjects.

[0281] Table 5 shows the results of the Pediatric Mitochondrial Disease Scale (IPMDS)-Quality of Life (QoL) questionnaire for patients as a function of time after cell therapy. In both categories of "Complaints and symptoms" and "Physical examination", 0 represents "normal" for the relevant attribute, while a worsened condition is scored from 1 to 5 depending on the severity. [Table 5]

[0282] It should be noted that the patient had not gained weight in the three years prior to treatment, i.e., had not gained weight since age 3.5. The data shown in Figure 17D shows the growth as measured by the patient's weight and height standard deviation scores, beginning four years prior to MAT and during the follow-up period. The data shows that approximately 15 months after one treatment, this patient gained height and weight.

[0283] Another evidence of the patient's growth comes from alkaline phosphatase levels. The alkaline phosphatase level test (ALP test) measures the amount of alkaline phosphatase enzyme in the bloodstream. Lower than normal ALP levels in the blood may indicate malnutrition, which may be due to deficiencies of certain vitamins and minerals. The data shown in Figure 17E show that a single treatment was sufficient to raise the patient's alkaline phosphatase levels from 159 to 486 IU / L in just 12 months. The reversal of the trend of weight loss as well as the rise in ALP is associated with both old age and anti-cancer treatment, which may lead to weight loss and malnutrition.

[0284] As seen in Figures 17F-H, treatment resulted in significant improvements in red blood cell levels (Figure 17F), hemoglobin levels (Figure 17G), and hematocrit levels (Figure 17H). These results indicate that a single treatment is sufficient to ameliorate the symptoms of anemia.

[0285] FIG. 17I shows the cessation of renal deterioration as indicated by urinary creatinine levels after cell transplantation. As further seen in FIG. 17J and 17K, cell therapy also resulted in a notable improvement in bicarbonate (FIG. 17J) and base excess (FIG. 17K) levels without bicarbonate supplementation. FIG. 17L shows the magnesium supplementation and blood magnesium levels of the patient as a function of time after cell therapy. The data shows that magnesium supplementation is no longer necessary as the patient's blood levels of magnesium increased significantly over time. Achieving high levels of magnesium without magnesium supplementation is evidence of improved magnesium absorption as well as reabsorption in the kidney proximal tubules. As seen in FIG. 17M-17P, one treatment resulted in a notable decrease in the levels of several renal tubulopathy indicators such as urinary glucose levels (FIG. 17M) and certain salt levels (FIG. 17N-potassium; FIG. 17O-chloride; FIG. 17P-sodium). FIG. 17I-17P are all relevant to aged subjects as renal function deteriorates with age.

[0286] The genetic indicator of the success of the therapy used is the prevalence of normal mtDNA compared to the total mtDNA per cell. As shown in Figure 18A (Pt.1), the prevalence of normal total mtDNA in the patient's peripheral blood increased from about 1 at baseline to 1.6 (+60%) in just 4 months, to 1.9 (+90%) after 20 months of treatment, and above baseline levels at the majority of time points. Notably, normal mtDNA levels were above baseline levels at most time points.

[0287] Another indication of the efficacy of transplanting cells enriched with healthy functional mitochondria is shown in Figure 18B. Patient 1, who had relatively high levels of heteroplasmy at baseline, experienced a small decrease in heteroplasmy (less deleted mtDNA) after MAT, which continued throughout the follow-up period.

[0288] Neurologists at the hospital reported neurological improvement following transplantation of autologous cells with healthy mitochondria that did not carry the deletion mutation. The patient improved his ability to walk, climb stairs, use scissors, and draw. There was also substantial improvement in execution and response time to commands, as well as motor and language skills. The mother also reported improved memory. These findings are particularly relevant and important for geriatric subjects, as neurological deterioration in motor skills and memory often occurs in old age.

[0289] As the data presented above demonstrate, a one-time treatment regimen of bone marrow stem cells enriched with functional mitochondria successfully treated many debilitating conditions that affect aging.

[0290] Example 16. Compassionate treatment of a young person with Pearson Syndrome (PS) using autologous CD34+ cells enriched with MNV-BLD (blood-derived mitochondria). A 7-year-old female patient (Patient 2) was diagnosed with Pearson syndrome and has a deletion of 4977 nucleotides in mtDNA. The patient also suffers from anemia, endocrine pancreatic insufficiency, and is diabetic (HbA1C 7.1%). Patient 2 has high lactate levels (>25 mg / dL), is underweight, and has problems eating and gaining weight. The patient also suffers from hypermagnesuria (high levels of magnesium in urine, low levels of magnesium in blood). The patient has memory and learning problems, astigmatism, and low mitochondrial activity in peripheral lymphocytes as determined by TMRE, ATP content, and O2 consumption rate (compared to the healthy mother). Bone marrow mobilization was performed using G-CSF (10 μg / kg) and one dose of Plerixafor Mozobil™ (0.24 mg / ml). The patient began treatment with 1.8*106 cells / kg of autologous CD34+ cells enriched with healthy mitochondria isolated from her mother according to the timeline shown in HSPC mobilization, and performed leukapheresis and CD34 positive selection similar to Patient 1 (Example 18) with the addition of plerixafor (n=2) administration 1 day prior to leukapheresis. Mitochondria were isolated by differential centrifugation from maternal peripheral blood mononuclear cells (PBMCs) using 250 mM sucrose buffer pH 7.4. For MAT, autologous CD34+ cells were incubated with healthy mitochondria (106 cells per amount of mitochondria with 4.4 milliunits of citrate synthase (CS)) from the patient's mother, resulting in a 1.62-fold increase in the mitochondrial content of the cells (62% increase in mitochondrial content as indicated by CS activity). Incubation with mitochondria was performed for 24 h at RT in saline containing 4.5% HSA. It should be noted that after mitochondrial enrichment, CD34+ cells from the patient increased their colony-forming efficiency by 26%.

[0291] Patient 2 (15KG on treatment day) was treated with 1.8*106 autologous CD34+ cells enriched with healthy mitochondria per kilogram of body weight via IV infusion according to the timeline shown in Figure 19A.

[0292] FIG. 19B shows the beneficial effect of transplantation of mitochondrial-enriched cells on blood lactate levels, which are reduced after 5 months of treatment.

[0293] Muscle strength and muscle mass are known to deteriorate with old age. Figures 19C-19E show the striking effect of transplantation of enriched cells on these parameters in a series of functional tests. Figure 19C shows the results of a sit-to-stand test. Elderly people who are unable to rise from a chair without support are at risk of becoming even less active and therefore at risk for further motor impairment. The tested subjects are asked to perform as many sit-to-stand cycles as they can within a 30-second time frame. Patient 2 was able to perform more sit-to-stand cycles 5 months after transplantation. Figure 19D shows the 6-minute walk test (6MWT), which measures the distance in meters traversed by the subject within the allotted 6 minutes. Patient 2 exceeded the normal distance 5 months after transplantation. Figure 19E shows the improvement in muscle strength 5 months after cell transplantation, as evidenced by the rise in dynamometer units, even after three consecutive repetitions against the resistance of the dynamometer.

[0294] Figures 19F, 19G, and 19H show the improvement in kidney function as indicated by the ratios of magnesium, potassium, and calcium relative to creatinine found in the patient's urine as a function of time after IV injection, respectively. Figure 19I shows the ratio between ATP8 and 18S in the patient's urine as a function of time after IV injection. The immune system deteriorates with age. Among the components of the immune system most affected by old age are T lymphocytes. Young naive T cells are able to metabolize glucose, amino acids, and lipids to catabolically promote ATP production in mitochondria. It is also known that mitochondrial function is impaired by old age, so a possible relationship between T cells and mitochondrial decline has been suggested and studied. Figure 19J shows an increase in ATP content in the patient's lymphocytes. Figure 18A (Pt. 2) shows the prevalence of normal mtDNA as a function of time after IV injection. As can be seen in Figure 18A (Pt. 2), the prevalence of normal mtDNA increased from approximately 1 at baseline to 2 (+100%) in just 1 month and remained relatively high up to 10 months after treatment. Notably, normal mtDNA levels were above baseline levels at all time points. Figure 18B (Pt. 2) shows the change in heteroplasmy levels as a function of time after MAT. It can be seen that in patient 2, heteroplasmy (less deleted mtDNA) decreased after MAT. This continued throughout the follow-up period.

[0295] Example 17. Compassionate treatment of young patients with Pearson Syndrome (PS) and PS-associated Fanconi Syndrome (FS) using autologous CD34+ cells enriched with MNV-BLD (blood-derived mitochondria). A 10.5-year-old female patient (Patient 3) was diagnosed with Pearson syndrome, with a deletion of nucleotides 12113-14421 in mtDNA. The patient also suffers from anemia and Fanconi syndrome that has progressed to stage 4 renal failure. The patient is treated with dialysis three times a week. Recently, the patient has also suffered from severe visual impairment, narrowing of the visual field, and loss of near vision. The patient is unable to perform any physical activity (does not walk, sits in a stroller).

[0296] The patient had high lactate levels (>50 mg / dL) and pancreatic dysfunction that was treated with insulin. Brain MRI showed many lesions and areas of atrophy. The patient was fed exclusively by gastrostomy. The patient had memory and learning problems. The patient had low mitochondrial activity in peripheral lymphocytes as determined by tetramethylrhodamine ethyl ester (TMRE), ATP content, and O2 consumption rate (compared to the healthy mother) tests. Mobilization of hematopoietic stem and progenitor cells (HSPCs), as well as leukapheresis and CD34-positive selection, were performed similarly to Patient 1 (Example 3) with the addition of plerixafor (n=1) on day -1 prior to leukapheresis. Leukapheresis was performed via a permanent dialysis catheter. Mitochondria were isolated by differential centrifugation from maternal peripheral blood mononuclear cells (PBMCs) using 250 mM sucrose buffer pH 7.4. For MAT, autologous CD34+ cells were incubated with healthy mitochondria (1*106 cells per amount of mitochondria with 4.4 milliunits of citrate synthase (CS)) from the patient's mother, resulting in a 1.14-fold increase in cellular mitochondrial content (14% increase in mitochondrial content as indicated by CS activity). Cells were incubated with mitochondria for 24 hours at RT in saline containing 4.5% HSA. It should be noted that after mitochondrial enrichment, CD34+ cells from patients had a 52% increase in plating efficiency.

[0297] Patient 3 (21KG) was treated with 2.8*106 autologous CD34+ cells enriched with healthy mitochondria from her mother per kilogram of body weight via IV infusion according to the timeline shown in Figure 20A.

[0298] Figure 202B shows the beneficial effect of transplantation of mitochondrial-enriched cells on blood lactate levels, which declined 2 and 3 months after transplantation. The line below 20 mg / dl indicates normal levels of blood lactate.

[0299] Figure 20C shows the levels of AST and ALT liver enzymes in the patient's blood as a function of time before and after cell therapy. Achieving low levels of blood liver enzymes is evidence of reduced liver damage.

[0300] Figure 20D shows the levels of triglycerides, total cholesterol, and very low density lipoprotein (VLDL) cholesterol in the patient's blood as a function of time before and after cell therapy. Achieving low levels of triglycerides, total cholesterol, and VLDL cholesterol in the blood is evidence of increased liver function and improved lipid metabolism. Glycosylated hemoglobin (sometimes called hemoglobin A1c, HbA1c, A1C, Hb1c, Hb1c, or HGBA1C) is a form of hemoglobin that is primarily measured to determine a three-month average plasma glucose concentration. The life span of a red blood cell is four months (120 days), so the test is limited to an average of three months. Figure 20E shows the results of a patient's A1C test as a function of time before and after therapy.

[0301] 20F and 20G show the results of the patient's "Stand Up" (20F) and "6 Minute Walk" (20G) tests as a function of time after IV injection, showing improvement in both parameters after 5 months of treatment. Figure 18A (Pt. 3) shows the prevalence of normal mtDNA as a function of time after IV injection. As seen in Figure 18A (Pt. 3), the prevalence of normal mtDNA increased by 50% after 7 months of treatment. Notably, normal mtDNA levels were above baseline levels at most time points. Figure 18B (Pt. 3) shows the change in heteroplasmy levels as a function of time after MAT. Patient 3, who had a relatively low level of heteroplasmy at baseline, showed a decrease in heteroplasmy (less deleted mtDNA) after MAT. This continued throughout the follow-up period.

[0302] Example 18. Compassionate treatment of a young person with Kearns-Sayre Syndrome (KSS) using autologous CD34+ cells enriched with MNV-BLD (blood-derived mitochondria). Patient 4 was a 14-year-old, 19.5 kg female patient diagnosed with Kearns-Sayre syndrome and experiencing tunnel vision, ptosis, ophthalmoplegia, and retinal atrophy. The patient had vision problems, CPEO, epileptic seizures, pathological EEG, severe myopathy with sitting or gait disturbances, and cardiac arrhythmias. The patient had a 7.4 Kb deletion in mitochondrial DNA that includes the following genes: TK, NC8, ATP8, ATP6, CO3, TG, ND3, TR, ND4L, TH, TS2, TL2, ND5, ND6, TE, NC9, and CYB.

[0303] Mobilization of hematopoietic stem and progenitor cells (HSPCs), as well as leukapheresis and CD34 positive selection, were performed as in Patient 3 (Example 5). For MAT, autologous CD34+ cells were incubated for 24 hours at RT with healthy mitochondria (1*106 cells per amount of mitochondria with 4.4 milliunits of citrate synthase (CS)) from the patient's mother in saline containing 4.5% HSA. Enrichment resulted in a 1.03-fold increase in cellular mitochondrial content (3% increase in mitochondrial content as indicated by CS activity).

[0304] Patient 4 was treated with 2.2*106 autologous CD34+ cells enriched with healthy mitochondria per kilogram of body weight according to the timeline shown in Figure 20A. Unexpectedly, after 4 months of one treatment with CD34+ enriched by only 3% with healthy mitochondria, the patient showed improvement in EEG and was seizure-free. After 5 months of treatment, the patient suffered from disease-related atrioventricular (AV) block and was fitted with a pacer. The patient recovered and continued to improve. As shown in Figure 21, the ATP content in peripheral blood was measured after 6 months of treatment, showing an approximately 100% increase in ATP content compared to before treatment. After 7 months of treatment, the patient was able to sit alone, walk with assistance, and talk, had an increased appetite, and gained 3.6KG.

[0305] The foregoing description of the specific embodiments fully reveals the general nature of the present invention, so that others can easily modify and / or adapt to various uses of such specific embodiments by applying current knowledge without undue experimentation and without departing from the general concept, and therefore such adaptations and modifications should be understood within the meaning and range of equivalents of the embodiments of the present disclosure, and are intended to be so. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. The means, materials, and steps for carrying out the various functions of the present disclosure may take various alternative forms without departing from the present invention.

Claims

1. 1. A pharmaceutical composition for use in treating or reducing a wasting condition in a subject, said pharmaceutical composition comprising at least 10 mg / kg of body weight of said subject. ５ ~2×10 ７ a stem cell suspension in a pharma- tically acceptable liquid medium capable of supporting viability of the cells; The stem cells are enriched with exogenous mitochondria and have an increased mitochondrial DNA content compared to the mitochondrial DNA content of the stem cells prior to mitochondrial enrichment; and The pharmaceutical composition, wherein the debilitating condition is selected from the group consisting of age-related moderate motor dysfunction, age-related decline in kidney function, age-related decline in muscle function, age-related decline in muscle strength, and age-related loss of coordination, and the stem cells are CD34+ cells.

2. said enriching comprising contacting said stem cells with a mitochondrial dose of at least 0.044 and up to 176 milliunits of CS activity per million cells; or 2. The pharmaceutical composition of claim 1, wherein said enrichment comprises contacting said stem cells with a mitochondrial dose of 0.044 up to 17.6 milliunits of CS activity per million cells.

3. the stem cells are autologous, syngeneic, or from a donor; the stem cells are allogeneic to the subject suffering from the debilitating condition; or 2. The pharmaceutical composition of claim 1, wherein the stem cells are allogeneic to the subject suffering from the debilitating condition, and the treatment further comprises administering to the subject suffering from the debilitating condition an agent that prevents, delays, minimizes, or reverses an adverse immunogenic reaction between the subject and the stem cells of the allogeneic donor.

4. The pharmaceutical composition is administered to a specific tissue or organ, 10. The pharmaceutical composition of claim 1, which is administered by systemic parenteral administration or by a parenteral route selected from the group consisting of intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and direct injection into a tissue or organ.

5. the mitochondrially enriched stem cells exhibit a mitochondrial enrichment level relative to the corresponding level in the stem cells prior to mitochondrial enrichment, (i) increased levels of CS activity; (ii) increased content of at least one mitochondrial protein selected from succinate dehydrogenase complex subunit A (SDHA) and cytochrome C oxidase (COX1); (iii) Increased O ２ consumption rate, (iv) an increased rate of ATP production, or (v) any combination thereof The pharmaceutical composition according to any one of claims 1 to 4, comprising:

6. 2. The pharmaceutical composition of claim 1, further comprising unenriched stem cells, megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof.

7. The pharmaceutical composition according to claim 5 or 6, wherein the increased mitochondrial DNA content is derived from endogenous and / or exogenous mitochondria.

8. 2. The pharmaceutical composition of claim 1, wherein the exogenous mitochondria constitute at least 1% of the total mitochondria in the mitochondria-enriched stem cells.

9. 2. The pharmaceutical composition of claim 1, wherein the stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof.

10. The pharmaceutical composition of claim 1 , wherein the stem cells are at least partially purified.

11. 2. The pharmaceutical composition of claim 1, wherein the mitochondria are derived from a cell or tissue selected from the group consisting of placenta, placental cells grown in culture, and blood cells.

12. The pharmaceutical composition has a dosage of at least about 10 mg / kg of the subject's body weight. ６ or the pharmaceutical composition comprises a total of 5×10 mitochondria-enriched stem cells. ５ ~5×10 ９ The pharmaceutical composition of claim 1 , comprising stem cells.

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