Compositions and methods for improving stem cell function

Abstract

This invention provides further approaches to improve stem cell function in hematopoietic stem cells in vivo and in vitro, particularly approaches to improve engraftment of progenitor cell populations, and approaches to improve the self-renewal and differentiation capabilities of hematopoietic stem cells. [Solution] A composition is provided for improving the function of stem cells in a population of hematopoietic stem cells and / or progenitor cells (HSPCs), comprising a combination of urolithin, an NAD+ precursor, and vitamin B12, wherein the NAD+ precursor is selected from the group consisting of nicotinic acid, nicotinamide, nicotinamide riboside (NR), reduced nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid riboside, and mixtures thereof.

Description

Technical Field

[0001] The present invention relates to hematopoietic stem cells and progenitor cells (HSPC). Specifically, the present invention relates to compositions and methods for improving stem cell function in hematopoietic stem cells, for example, for improving engraftment of a cell population of HSPC, and / or for improving self-renewal ability and differentiation ability.

Background Art

[0002] The hematopoietic system is a complex hierarchy of cells consisting of various mature cell lineages. Mature cells include cells of the immune system that provide protection from pathogens, cells that carry oxygen throughout the body, and cells involved in wound healing. All of these mature cells are derived from a pool of hematopoietic stem cells (HSC) that are capable of self-renewal and differentiation into any blood cell lineage.

[0003] HSCs differ from committed progeny cells in that they mainly rely on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production (Simsek, T. et al. (2010) Cell Stem Cell 7:380-90; Takubo, K. et al. (2013) Cell Stem Cell 12:49-61; Vannini, N. et al. (2016) Nat Commun 7:13125; Yu, W. M. et al. (2013) Cell Stem Cell 12:62-74). This characteristic metabolic state is thought to protect HSCs from cell damage by reactive oxygen species (ROS) in active mitochondria, thereby maintaining the long-term in vivo function of the cells (Chen, C. et al. (2008) J Exp Med 205:2397-408; Ito, K. et al. (2004) Nature 431:997-1002; Ito, K. et al. (2006) Nat Med 12:446-51; Tothova, Z. et al. (2007) Cell 128:325-39).

[0004] Mitochondrial membrane potential, indicated by the fluorescence of tetramethylrhodamine methyl ester (TMRM), has traditionally been used as an alternative indicator of the metabolic state of cells. Phenotypic hematopoietic stem cells (HSCs) have been shown to have lower mitochondrial membrane potentials compared to progenitor cells (Vannini, N. et al. (2016) Nat Commun 7:13125). In the same study, it was found that artificially lowering the mitochondrial membrane potential by chemical uncoupling of the mitochondrial electron transport chain allowed HSCs to be maintained under culture conditions that would normally induce rapid differentiation (Vannini, N. et al. (2016) Nat Commun 7:13125). Importantly, a similar mechanism has been observed in human HSCs, where artificially lowering the mitochondrial membrane potential by adding nicotinamide riboside (NAD and vitamin B3 precursor) to the culture medium resulted in significantly higher engraftment levels and enabled long-term hematopoietic production in both first and second transplanted humanized recipient mice.

[0005] However, there is still a great need for further approaches to improve stem cell function in HSCs in vivo and in vitro, particularly approaches to improve engraftment of HSPC cell populations (e.g., during hematopoietic stem cell transplantation), as well as approaches to improve the self-renewal and differentiation capabilities of HSCs. [Overview of the project]

[0006] The applicant has found that a combination of urolithin A (UroA), nicotinamide adenine dinucleotide (NAD+) precursor, preferably nicotinamide riboside, and vitamin B12 (preferably methylcobalamin) has a synergistic effect on improving hematopoietic stem cell (HSC) function through enhancements such as engraftment and self-renewal.

[0007] While we do not wish to be bound by theory, it is thought that the improvement of stem cell function is achieved through the regulation of mitochondrial membrane potential via mitophagy induction in cells exposed to this combination.

[0008] In one embodiment, the present invention provides the use of a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for improving stem cell function in a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs).

[0009] In some embodiments, use is in vitro. In some embodiments, use is ex vivo.

[0010] In some embodiments, the cell population is a population of isolated HSPC cells.

[0011] In some embodiments, the HSPC has a CD34+ phenotype.

[0012] In some embodiments, the HSPC has a CD34+CD38- phenotype.

[0013] In another aspect, the present invention provides a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in improving hematopoietic stem cell function.

[0014] In some embodiments, compositions comprising urolithin, an NAD+ precursor, and vitamin B12 are intended for use in improving hematopoietic stem cell function in a subject.

[0015] In some embodiments, stem cell function includes engraftment. In some embodiments, stem cell function includes self-renewal. In some embodiments, stem cell function includes differentiation.

[0016] In some embodiments, the stem cell function is engraftment. In some embodiments, the stem cell function is self-renewal. In some embodiments, the stem cell function is differentiation.

[0017] In another embodiment, the present invention provides a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in increasing blood cell levels in a subject.

[0018] In another aspect, the present invention provides a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in the treatment or prevention of (a) anemia, leukopenia and / or thrombocytopenia, (b) infections, and / or (c) cancer in a subject.

[0019] In another embodiment, the present invention provides a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in the treatment or prevention of anemia, leukopenia, and / or thrombocytopenia.

[0020] In another aspect, the present invention provides a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in the treatment or prevention of infectious diseases.

[0021] In another aspect, the present invention provides a composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in the treatment or prevention of cancer.

[0022] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is leukemia, lymphoma, or myeloma.

[0023] In a preferred embodiment, urolithin is urolithin A.

[0024] In preferred embodiments, the NAD+ precursor is selected from the group consisting of nicotinic acid, nicotinamide, nicotinamide riboside (NR), reduced nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid riboside, and mixtures thereof.

[0025] In a preferred embodiment, the combination comprises urolithin A, nicotinamide riboside, and vitamin B12.

[0026] In some embodiments, a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 is administered enterally or parenterally to a subject, preferably enterally. In a preferred embodiment, the combination is administered orally to the subject.

[0027] In some embodiments, a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 is in the form of a pharmaceutical composition or a nutritional composition.

[0028] In some embodiments, a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 is in the form of a food, a dietary supplement, a nutraceutical, a food for special medical purposes (FSMP), a nutritional supplement, a milk beverage, a small-dose liquid nutritional supplement, or a meal replacement beverage.

[0029] In some embodiments, the subject has or is at risk of having hematopoietic cells below normal levels, such as red blood cells, white blood cells, and / or platelets.

[0030] In some embodiments, the subject has or is at risk of having anemia, leukopenia, and / or thrombocytopenia.

[0031] In some embodiments, the subject has undergone an intervention selected from the group consisting of hematopoietic stem cell transplantation, bone marrow transplantation, myeloablative pretreatment, chemotherapy, radiotherapy, and surgery.

[0032] In some embodiments, the subject is a subject with a reduced immune system.

[0033] In some embodiments, the subject is 3 to 4 weeks after an intervention.

[0034] In some embodiments, the subject is a human or a non-human mammal, preferably a human, and optionally a human adult, child, or infant.

[0035] In some embodiments, urolithin, NAD+ precursor, and vitamin B12 are administered to the subject simultaneously, sequentially, or separately, preferably at the same time.

[0036] In some embodiments, compositions comprising urolithin, an NAD+ precursor, and vitamin B12 are present in combination formulations for simultaneous use, separate use, or sequential use with agents selected from the group consisting of G-CSF analogs, TPO receptor analogs, SCF, TPO, Flt3-L, FGF-1, IGF1, IGFBP2, IL-3, IL-6, G-CSF, M-CSF, GM-CSF, EPO, and combinations thereof.

[0037] In another embodiment, the present invention provides a method for growing a population of isolated hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs), comprising the step of contacting the population with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0038] In some embodiments, contact involves culturing the cell population in the presence of a composition comprising urolithin, an NAD+ precursor, and vitamin B12.

[0039] In some embodiments, the method is (a) A step to prepare a population of HSPC cells, (b) Optionally, a step of culturing a population of HSPC cells, preferably in HSPC growth medium or maintenance medium; (c) A step of optionally isolating a cell subpopulation of HSPCs characterized by a low mitochondrial membrane potential, (d) The method involves contacting the group of (a) or (b), or the subgroup of (c), with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0040] In some embodiments, the population prepared in step (a) is obtained from bone marrow, mobilized peripheral blood, or umbilical cord blood.

[0041] In some embodiments, the product of step (d) is enriched with cells that have long-term multiseries blood reconstitution ability.

[0042] In another aspect, the present invention provides a cell population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) that can be obtained by the method of the present invention.

[0043] In another aspect, the present invention provides a pharmaceutical composition comprising a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) according to the present invention.

[0044] In another embodiment, the present invention provides a cell culture medium comprising a composition containing urolithin, nicotinamide riboside, and vitamin B12.

[0045] In a preferred embodiment, urolithin is urolithin A.

[0046] In preferred embodiments, the NAD+ precursor is selected from the group consisting of nicotinic acid, nicotinamide, nicotinamide riboside (NR), reduced nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid riboside, and mixtures thereof.

[0047] In a preferred embodiment, the combination includes urolithin A, nicotinamide riboside, and vitamin B12.

[0048] In some embodiments, the culture medium is a culture medium for hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs).

[0049] In some embodiments, the culture medium is either a growth medium or a maintenance medium.

[0050] In another embodiment, the present invention provides a method for engrafting hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) in a target, comprising the steps of: contacting an isolated population of HSPC cells with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12; and administering the population of HSPC cells to a target requiring the population.

[0051] In another embodiment, the present invention provides a method for improving hematopoietic stem cell function, comprising the step of contacting a cell population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0052] In another embodiment, the present invention provides a method for improving hematopoietic stem cell function in a subject, comprising the steps of: contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12; and administering the HSPC population to a subject requiring the population.

[0053] In another embodiment, the present invention provides a method for improving engraftment of hematopoietic stem cells, comprising the step of contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12. In another embodiment, the present invention provides a method for improving self-renewal of hematopoietic stem cells, comprising the step of contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12. In another embodiment, the present invention provides a method for improving differentiation of hematopoietic stem cells, comprising the step of contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12. In some embodiments, engraftment, self-renewal, and / or differentiation are improved in a subject, and the method further comprises the step of administering a population of HSPC cells to a subject that requires the population.

[0054] In another aspect, the present invention provides a method for (a) increasing blood cell levels in a subject, (b) treating or preventing anemia, leukopenia and / or thrombocytopenia, (c) treating or preventing an infection, and / or (d) treating or preventing cancer, the method comprising contacting a population of hematopoietic stem cells and / or progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor and vitamin B12, and administering the population of HSPCs to a subject in need.

[0055] In another embodiment, the present invention provides a method for increasing blood cell levels, comprising the step of contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0056] In another embodiment, the present invention provides a method for treating or preventing anemia, leukopenia and / or thrombocytopenia, comprising contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0057] In another embodiment, the present invention provides a method for treating or preventing an infection, comprising contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0058] In another embodiment, the present invention provides a method for treating or preventing cancer, comprising contacting a population of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0059] In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vivo method.

[0060] In some embodiments, the cell population is an isolated population of HSPC cells. In some embodiments, the method further includes the step of administering the HSPC cell population to a subject that requires the population.

[0061] In another aspect, the present invention provides a method for improving hematopoietic stem cell function, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject in need thereof.

[0062] In another aspect, the present invention provides a method for improving the engraftment of hematopoietic stem cells, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject requiring such treatment. In another aspect, the present invention provides a method for improving the self-renewal of hematopoietic stem cells, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject requiring such treatment. In another aspect, the present invention provides a method for improving the differentiation of hematopoietic stem cells, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject requiring such treatment.

[0063] In another embodiment, the present invention provides a method for increasing blood cell levels, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject in need thereof.

[0064] In another embodiment, the present invention provides a method for treating or preventing anemia, leukopenia and / or thrombocytopenia, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor and vitamin B12 to a subject in need thereof.

[0065] In another aspect, the present invention provides a method for treating or preventing an infectious disease, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject in need thereof.

[0066] In another aspect, the present invention provides a method for treating or preventing cancer, comprising administering a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12 to a subject in need thereof. [Brief explanation of the drawing]

[0067] [Figure 1] The combination of UroA, NR, and vitamin B12 induces a decrease in mitochondrial membrane potential. A) Mouse HSCs cultured in basic medium (control) supplemented with NR, Vit B12 (MC), NR+MC, and NR+UroA+MC (concentrations: NR: 500uM, UroA: 20uM, Vit B12 (MC): 100uM). NR was supplemented every 24 hours. The percentage of cells in the TMRM low gate increased, and MFI TMRM decreased under NR conditions and MC conditions only (left and center panels). Furthermore, the NR+MC combination was significantly different from NR alone or MC alone. The triple combination of UroA+MC+NR showed the strongest effect and was significantly different from the double combination. Mitochondrial mass (measured by Mitotracker) (right panel) was significantly reduced in the NR+MC and UroA+MC+NR combinations compared to culture in basic medium or NR alone. [Modes for carrying out the invention]

[0068] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” “containing,” or “contains,” and are open-ended and do not exclude additional, unlisted components, elements, or processes that may include others. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0069] hematopoietic stem cells Stem cells can differentiate into many cell types. Cells capable of differentiating into all cell types are known as totipotent. In mammals, only zygotes and early embryonic cells are totipotent. Stem cells are found in most, if not all, multicellular organisms. Stem cells are characterized by their ability to self-renew through mitosis and to differentiate into a variety of specialized cell types. The two main types of mammalian stem cells are embryonic stem cells isolated from the inner cell mass of the blastocyst and adult stem cells found in adult tissues. In developing embryos, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells function as the body's repair system, not only replenishing specialized cells but also maintaining the normal metabolic turnover of regenerative organs such as blood, skin, or intestinal tissue.

[0070] Hematopoietic stem cells (HSCs) are pluripotent stem cells that can be found, for example, in peripheral blood, bone marrow, and umbilical cord blood. HSCs have the ability to self-renew and differentiate into any blood cell lineage. HSCs have the ability to re-colonize the entire immune system, as well as the erythrocytes and myeloid lineages of all hematopoietic tissues (such as bone marrow, spleen, and thymus). HSCs produce hematopoietic cells of all lineages for life-long use.

[0071] Hematopoietic progenitor cells have the ability to differentiate into specific cell types. However, in contrast to stem cells, hematopoietic progenitor cells are already quite specific, and they differentiate into cells whose differentiation fate is "determined (target)." The difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, while progenitor cells can divide only a limited number of times. Hematopoietic progenitor cells can only be strictly distinguished from hematopoietic stem cells (HSCs) by in vivo functional assays (i.e., demonstration of whether they can produce transplants and entire bloodlines over the long term).

[0072] Differentiated cells are more specialized cells compared to stem cells or progenitor cells. Differentiation occurs during the development of multicellular organisms, in which the organism changes from a single zygote into a complex system consisting of multiple tissues and multiple cell types. Differentiation is also a common process in adults: adult stem cells divide during tissue repair and normal cell turnover to produce fully differentiated daughter cells. Differentiation dramatically alters the size, shape, membrane potential, metabolic activity, and responsiveness to signaling of cells. These changes are primarily due to highly controlled regulation of gene expression. In other words, differentiated cells are cells that have specific structures and exhibit specific functions through developmental processes involving the activation and inactivation of specific genes. Here, differentiated cells include hematopoietic differentiated cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, T cells, B cells, and NK cells. For example, hematopoietic differentiated cells can be distinguished from stem cells and progenitor cells by detecting cell surface molecules that are not expressed or are expressed at lower levels in undifferentiated cells. Examples of suitable human lineage markers include CD33, CD13, CD14, CD15 (bone marrow), CD19, CD20, CD22, CD79a(B), CD36, CD71, CD235a (erythrocytes), CD2, CD3, CD4, CD8(T), and CD56(NK).

[0073] HSC source In some embodiments, hematopoietic stem cells are obtained from tissue samples.

[0074] For example, HSCs can be obtained from adult and fetal peripheral blood, umbilical cord blood, bone marrow, liver, or spleen. HSCs are obtained after recruiting cells in vivo by treatment with growth factors.

[0075] Mobilization may be carried out using, for example, G-CSF, prelixafor, or a combination thereof. Other drugs such as NSAIDs, CXCR2 ligand (Grobeta), and dipeptidyl peptidase inhibitors may also be useful as mobilizers.

[0076] Because stem cell growth factors GM-CSF and G-CSF are available, most hematopoietic stem cell transplants are now performed using stem cells collected from peripheral blood rather than bone marrow. Collecting peripheral blood stem cells allows for larger grafts, eliminates the need for general anesthesia in the donor to collect the graft, and consequently shortens the graft survival time and can reduce the long-term relapse rate.

[0077] Bone marrow can be collected by standard aspiration (either in a steady state or after mobilization) or by using a next-generation collection tool (e.g., Marrow Miner).

[0078] In addition, HSCs may also be derived from induced pluripotent stem cells.

[0079] HSC characteristics Human stromal cells (HSCs) typically exhibit low forward and side scattering profiles by flow cytometry. Some are metabolically dormant, as demonstrated by rhodamine labeling, which allows for the determination of mitochondrial activity. Human HSCs may contain specific cell surface markers such as CD34, CD45, CD133, CD90, and CD49f. HSCs can also be defined as cells lacking expression of the CD38 and CD45RA cell surface markers. However, the expression of some of these markers depends on the developmental stage and tissue specificity of the HSC. Some HSCs, referred to as "side population cells," reject the Hoechst 33342 dye, which is detected by flow cytometry. Therefore, HSCs possess descriptive features that enable their identification and isolation.

[0080] Negative markers CD38 is the most established and useful single negative marker for human herpes simplex sclerosis (HSC).

[0081] Human HSCs may also be negative for series markers such as CD2, CD3, CD14, CD16, CD19, CD20, CD24, CD36, CD56, CD66b, CD271, and CD45RA. However, these markers may need to be used in combination for HSC enrichment.

[0082] Please understand that "negative markers" indicate that human HSCs do not express these markers.

[0083] Positive marker CD34 and CD133 are the most useful positive markers for HSC.

[0084] Some HSCs are also positive for series markers such as CD90, CD49f, and CD93. However, these markers may need to be used in combination for HSC enrichment.

[0085] The term "positive markers" should help you understand that human HSCs express these markers.

[0086] In some embodiments, the HSC has the CD34+ phenotype.

[0087] In some embodiments, the HSC has a CD34+CD38- phenotype.

[0088] Further separation may be performed, for example, to obtain CD34+CD38-CD45RA-CD90+CD49f+ cells.

[0089] stem cell function As used herein, the term “stem cell function” refers to characteristics typically associated with stem cells, such as the ability to differentiate into specific cell lineages and / or the ability to self-renew.

[0090] In one embodiment, stem cell function includes engraftment, self-renewal, and / or differentiation.

[0091] In some embodiments, stem cell function includes engraftment. In some embodiments, stem cell function includes self-renewal. In some embodiments, stem cell function includes differentiation.

[0092] In one embodiment, stem cell functions include engraftment, self-renewal, and / or differentiation.

[0093] In some embodiments, the stem cell function is engraftment. In some embodiments, the stem cell function is self-renewal. In some embodiments, the stem cell function is differentiation.

[0094] As used herein, the term “engraftment” refers to the ability of hematopoietic stem cells and / or hematopoietic progenitor cells to establish themselves and survive in a subject after transplantation, i.e., for a short and / or long period after transplantation. For example, engraftment may refer to the number and / or proportion of hematopoietic cells (e.g., graft-derived cells) derived from the transplanted hematopoietic stem cells and / or hematopoietic progenitor cells, detected at approximately 1 to 24 weeks, 1 to 10 weeks, or 1 to 30 or 10 to 30 days after transplantation. In some embodiments, engraftment is assessed at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after transplantation. In other embodiments, graft survival is evaluated at approximately 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, or 24 weeks after transplantation. In other embodiments, graft survival is evaluated at approximately 16 to 24 weeks, preferably 20 weeks, after transplantation.

[0095] Engraftment can be easily analyzed by those skilled in the art. For example, the transplanted hematopoietic stem cells and / or hematopoietic progenitor cells may be manipulated to contain a marker (e.g., a reporter protein such as a fluorescent protein), which can then be used to quantify graft-derived cells. Samples for analysis may be extracted from the relevant tissue and analyzed ex vivo (e.g., using flow cytometry).

[0096] As used herein, the term “self-renewal” refers to the ability of a cell to undergo multiple cycles of cell division while remaining undifferentiated.

[0097] The number and / or percentage of cells in a given state (e.g., living cells, dead cells, or apoptotic cells) can be quantified using any of the many methods known in the art, such as the use of hemocytometers, automated cell counters, flow cytometers, and fluorescence-activated cell sorting devices. These techniques may enable the distinction between living cells, dead cells, and / or apoptotic cells. Additionally or alternatively, apoptotic cells may be detected using readily available apoptosis assays (e.g., assays based on the detection of phosphatidylserine (PS) on the cell membrane surface, such as those using annexin V bound to exposed PS; apoptotic cells can be quantified using fluorescently labeled annexin V), which may complement other techniques.

[0098] Isolation and enrichment of cell populations Cell populations such as hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) are disclosed herein. In some embodiments, the cell population is an isolated cell population.

[0099] As used herein, the term “isolated cell population” refers to a population of cells not present in the body. The isolated cell population may have been previously removed from a subject. The isolated cell population can be cultured and manipulated ex vivo or in vitro using standard techniques known in the art. The isolated cell population may later be reintroduced into a subject. The subject may be the same subject from which the cells were initially isolated, or a different subject.

[0100] From a cell population, cells exhibiting a specific phenotype or characteristics can be selectively purified from other cells that do not exhibit that phenotype or characteristic, or exhibit it to a low degree. For example, a cell population expressing a specific marker (such as CD34) can be purified from the cell population used to start the process. Alternatively, or in addition, a cell population that does not express another marker (such as CD38) can also be purified.

[0101] As used herein, the term “concentration” refers to increasing the concentration of a particular cell type within a population. The concentrations of other cell types may be consequently reduced.

[0102] Purification or concentration may result in a substantially pure cell population of other cell types.

[0103] The purification or enrichment of cell populations expressing a specific marker (e.g., CD38 or CD34) can be achieved by using a drug that binds to the marker, preferably a drug that is substantially specific to the marker.

[0104] The drug that binds to the cell marker may be an antibody, such as an anti-CD34 antibody or an anti-CD38 antibody.

[0105] As used herein, the term “antibody” means a complete antibody or antibody fragment capable of binding to a selected target, including Fv, ScFv, F(ab') and F(ab')2, monoclonal antibodies and polyclonal antibodies, modified antibodies, such as chimeric antibodies, CDR-grafted antibodies and humanized antibodies, and artificially selected antibodies produced using phage display or alternative technologies.

[0106] In addition, the present invention may also utilize substitutes for typical antibodies, such as "Avibody," "Avimer," "Antichalin," "Nanobody," and "DARPin."

[0107] Agents that bind to specific markers may be labeled in an identifiable manner using one of many techniques known in the art. The agent may be intrinsically labeled or modified by conjugating a label onto the agent. “Conjugation” should be understood as the agent and the label being operably linked. This means that the agent and the label are linked so that both can perform their functions (e.g., binding to a marker, enabling fluorescence identification, or being separable in a magnetic field) with substantially no hindrance. Preferred methods of conjugation are well known in the art and readily identifiable to those skilled in the art.

[0108] The labeling allows, for example, the purification of the labeling agent to which it is linked and any cells from their environment (e.g., the agent may be labeled with magnetic beads or affinity tags such as avidin), detection, or both. Suitable detectable markers for use as labels include fluorescent molecules (e.g., green fluorescent protein, cherry fluorescent protein, cyan fluorescent protein, and orange fluorescent protein) and peptide tags (e.g., His tags, Myc tags, FLAG tags, and HA tags).

[0109] Numerous techniques for isolating cell populations expressing specific markers are known in the art. These techniques include magnetic bead-based separation techniques (e.g., separation using closed-circuit magnetic beads), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g., affinity columns or beads, such as biotin columns for separating avidin-labeled agents), and microscopy-based techniques.

[0110] Separation can also be performed using a combination of different techniques, for example, a magnetic bead-based separation step followed by a step of sorting the resulting cell population by flow cytometry for one or more additional (positive or negative) markers.

[0111] Clinical-grade separation can be performed, for example, using the CliniMACS® system (Miltenyi), which is an example of a closed-circuit magnetic bead-based separation technology.

[0112] It is also conceivable that HSCs can be concentrated using dye exclusion properties (e.g., side population or rhodamine labeling) or enzymatic activity (e.g., ALDH activity).

[0113] composition When used in contact with in vitro T cell cultures, the composition of the present invention may be used in any form suitable for in vitro cell culture (e.g., a non-toxic form). When administered to a subject, the composition of the present invention may be used in any form suitable for ingestion by animals, preferably humans (e.g., a non-toxic form).

[0114] The composition may be used in any appropriate amount in a composition, such as a nutritional composition. Those skilled in the art will be able to determine an appropriate amount depending on the desired dose of the agent. The dose may vary depending on factors such as the age, size, and health status, lifestyle, and genetic heritage of the recipient. The dose may be in line with the Recommended Daily Allowance (RDA) developed by organizations such as the Food and Nutrition Board of the National Academy of Sciences.

[0115] NAD+ precursor NAD of the present invention +The precursors are selected from the group consisting of nicotinic acid (niacin), nicotinamide (niacinamide), nicotinamide riboside (NR), reduced nicotinamide riboside (NRH), β-nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid riboside, food extracts concentrated with at least one of these compounds, for example, food extracts concentrated with nicotinamide adenine dinucleotide (NAD), and mixtures thereof. As used herein, "nicotinamide riboside" includes L-valine and L-phenylalanine esters of nicotinamide riboside.

[0116] In preferred embodiments, the NAD+ precursor is nicotinamide riboside.

[0117] Nicotinamide riboside Nicotinamide riboside (NR) is the pyridine-nucleoside form of vitamin B3, which is a precursor to nicotinamide adenine dinucleotide (NAD).

[0118] Nicotinamide riboside has the following structure.

[0119] [ka]

[0120] In some embodiments, T cells are exposed to NR at concentrations of 1–10 mM, 1–5 mM, 1–2.5 mM, or 1–2 mM. In other embodiments, T cells are exposed to NR at concentrations of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM, preferably 2 mM.

[0121] Nicotinamide riboside or NAD+ precursor can be administered in amounts ranging from approximately 0.001 mg / day to approximately 2000 mg / day, preferably approximately 0.001 mg / day to approximately 1000 mg / day, more preferably approximately 0.001 mg / day to approximately 750 mg / day, even more preferably approximately 0.001 mg / day to approximately 500 mg / day, most preferably approximately 0.001 mg / day to approximately 250 mg / day, for example, approximately 0.001 mg / day to approximately 100 mg / day, approximately 0.001 mg / day to approximately 75 mg / day, approximately 0.001 mg / day to approximately 50 mg / day, approximately 0.001 mg / day to approximately 25 mg / day, approximately 0.001 mg / day to approximately 10 mg / day, or approximately 0.001 mg / day to approximately 1 mg / day. Naturally, the daily dose can be divided and administered at various times throughout the day. However, in any case, the amount of compound administered will vary depending on factors such as the solubility of the active ingredient, the formulation used, the condition of the subject (e.g., body weight), and / or the route of administration. For example, the daily dose of nicotinamide riboside disclosed herein is not limiting and may differ in some embodiments, in particular the compositions disclosed herein which can be used as foods for acute special medical purposes (FSMPs) and contain up to about 2.0 mg / day of nicotinamide riboside.

[0122] Vitamin B12 Vitamin B12 (also known as cobalamin) is a cobalt-containing, water-soluble vitamin that cannot be synthesized in the human body and therefore must be obtained from food or through synthesis by the gut microbiota.

[0123] The vitamin B12 pool in the human body consists of several forms, including cyanocobalamin, which is inactive and requires conversion to become active, as well as methylcobalamin and adenosylcobalamin, which are the metabolically active forms of vitamin B12.

[0124] Two enzymes, methionine synthase and methylmalonyl-CoA mutase, are known to utilize vitamin B12 as a cofactor. Methionine synthase is an intracellular enzyme that uses methylcobalamin to convert homocysteine ​​to methionine. This enzyme plays an important role in preventing the accumulation of homocysteine ​​toxicity by providing S-adenosylmethionine (SAM) as a methylation donor through this conversion. Low SAM levels and high homocysteine ​​levels observed in severe vitamin B12 deficiency impair myelin formation in peripheral nerves and the spinal cord. Methionine synthase also catalyzes the activation of 5-methyltetrahydrofolate to the bioactive tetrahydrofolate, a catalytic effect required for 1-carbon metabolism and DNA synthesis, and consequently contributing to an increase in red blood cells. Methylmalonyl-CoA mutase is a mitochondrial enzyme that utilizes adenosylcobalamin to convert methylmalonyl-CoA to succinyl-CoA, which is then incorporated into the TCA cycle. This process is characterized by the breakdown of branched-chain amino acids and odd-chain fatty acids, and is essential for regulating neural development during the embryonic stage, but not essential in adulthood.

[0125] The vitamin B12 of the present invention may be, for example, vitamin B12 itself, or the semi-synthetic derivatives cyanocobalamin, hydroxocobalamin, methylcobalamin, and / or adenosylcobalamin. Methylcobalamin may be particularly effective.

[0126] In some embodiments, T cells are in contact with vitamin B12 at concentrations of 10–100 μM, 10–75 μM, or 10–50 μM. In other embodiments, T cells are in contact with vitamin B12 at concentrations of 25–100 μM, 25–75 μM, or 25–50 μM. In other embodiments, T cells are in contact with vitamin B12 at concentrations of 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or 100 μM, preferably 50 μM.

[0127] In some embodiments, vitamin B12 is administered to subjects at a dose of 0.1 to 40 times the recommended daily allowance (RDA) of vitamin B12 per day, for example, 1 to 10 times the recommended daily allowance (RDA) of vitamin B12 per day.

[0128] Therefore, vitamin B12 can be administered in a daily dose of approximately 10, 20, 30, or 40 times the RDA of vitamin B12 per day. Preferably, the daily dose provides 10 to 40 times, more preferably 10 to 30 times, or even more preferably 10 to 25 times, and most preferably about 12 to 21 times, the RDA of vitamin B12 per day.

[0129] In the United States, the RDA for vitamin B12 is 2.4 μg per day for individuals aged 14 years and older. Therefore, individuals in this category may be administered a daily dose that provides approximately 0.002 mg to approximately 0.4 mg of vitamin B12 per day, preferably 0.02 mg to 0.07 mg of vitamin B12 per day, and more preferably 0.03 mg to 0.05 mg of vitamin B12 per day.

[0130] Uroricin Uroritin is a metabolite of ellagic acid derivatives such as ellagitannins derived from food, and is produced in the human gut by intestinal bacteria.

[0131] Ellagitannins are a type of antioxidant polyphenol found in several fruits, particularly pomegranates, strawberries, raspberries, and walnuts. Although ellagitannins are absorbed very poorly, they are rapidly metabolized into urolithin by the gut microbiota in the large intestine.

[0132] Due to its excellent absorption, urolithin is considered a bioactive molecule that mediates the effects of ellagitannins. For example, urolithin has been shown to possess antioxidant and anti-inflammatory properties.

[0133] Examples of uroritins include uroritin A (3,8-dihydroxyuroritin), uroritin B (3-hydroxyuroritin), and uroritin D (3,4,8,9-tetrahydroxyuroritin), as well as uroritin A glucuronide and uroritin B glucuronide.

[0134] Urolithin A (UroA) has the following structure:

[0135] [ka]

[0136] In some embodiments, HSPC is contacted with urorithin at urorithin concentrations of 5 μM to 250 μM, 5 μM to 200 μM, 5 μM to 150 μM, 5 μM to 100 μM, or 5 μM to 50 μM. In other embodiments, HSPC is contacted with urorithin at urorithin concentrations of 10 μM to 250 μM, 10 μM to 200 μM, 10 μM to 150 μM, 10 μM to 100 μM, or 10 μM to 50 μM. In other embodiments, HSPC is contacted with urorithin at urorithin concentrations of 20 μM to 250 μM, 20 μM to 200 μM, 20 μM to 150 μM, 20 μM to 100 μM, or 20 μM to 50 μM. In other embodiments, HSPC is brought into contact with urorithin at urorithin concentrations of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 225 μM, or 250 μM.

[0137] In a preferred embodiment, HSPC is brought into contact with urolithin at a urolithin concentration of 20 μM to 50 μM.

[0138] Therefore, urolithin may be administered in a total amount of approximately 0.2 to 150 milligrams (mg) of urolithin per kilogram (kg) of body weight of the subject. Preferably, at least one type of urolithin is administered in a daily dose equivalent to or corresponding to 2 to 120 mg of urolithin per kg of body weight of the subject, more preferably 4 to 90 mg of urolithin per kg of body weight of the subject, and most preferably 8 to 30 mg of urolithin per kg of body weight of the subject. Any given dose may be given as a single dose or as a divided dose.

[0139] The urolithin of the present invention may exist as a salt or ester, particularly as a pharmaceutically acceptable salt or ester.

[0140] Suitable pharmaceutically acceptable salts of the drug of the present invention include its acid addition salts or base salts. A review of suitable pharmaceutically acceptable salts can be found in Berge et al. (1977) J Pharm Sci 66:1-19.

[0141] The present invention also includes, as appropriate, all enantiomers and tautomers of the activator. Those skilled in the art will recognize compounds having optical properties (e.g., one or more chiral carbon atoms) or tautomer properties. The corresponding enantiomers and / or tautomers can be isolated / prepared by methods known in the art.

[0142] Pharmaceutical compositions and nutritional compositions In some embodiments, a composition comprising urolithin, nicotinamide riboside, and vitamin B12 is in the form of a pharmaceutical composition.

[0143] The pharmaceutical composition may further contain pharmaceutically acceptable carriers, diluents, or additives.

[0144] In some embodiments, hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) are in the form of a pharmaceutical composition.

[0145] The cells of the present invention can be formulated for administration to a subject together with a pharmaceutically acceptable carrier, diluent, or additive. Suitable carriers and diluents include isotonic saline, such as phosphate-buffered saline, and optionally contain human serum albumin.

[0146] The handling of cell therapy products should preferably be carried out in accordance with the FACT-JACIE international standard for cell therapy.

[0147] In some embodiments, a composition comprising urolithin, nicotinamide riboside, and vitamin B12 is in the form of a nutritional composition.

[0148] In some embodiments, compositions comprising urolithin, nicotinamide riboside, and vitamin B12 are in the form of food products, dietary supplements, nutraceuticals, foods for specific medical purposes (FSMPs), nutritional supplements, milk beverages, small-volume liquid nutritional supplements, or meal replacement beverages. In some embodiments, the composition is an infant formula.

[0149] In some embodiments, compositions comprising urolithin, nicotinamide riboside, and vitamin B12 are in the form of food additives or pharmaceuticals.

[0150] Food additives or pharmaceuticals may be in the form of, for example, tablets, capsules, lozenges, or liquids. Food additives or pharmaceuticals are preferably provided as sustained-release formulations to allow for a continuous supply of urolithin or its precursor over a long period of time.

[0151] The composition may be selected from the group consisting of: powdered milk-based products; instant beverages; ready-to-drink formulations; nutritional powders; nutritional liquids; milk-based products, especially yogurt or ice cream; cereal products; beverages; water; coffee; cappuccino; malt beverages; chocolate-flavored beverages; cooked products; soups; tablets; and / or syrups.

[0152] The composition may further contain protective hydrophilic colloids (such as gum, protein, or modified starch), binders, film-forming agents, encapsulants / encapsulating materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (such as oils, fats, waxes, or lecithin), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), fluidizers, flavoring agents, bulking agents, gelling agents, gel-forming agents, antioxidants, and antibacterial agents.

[0153] Furthermore, in addition to organic or inorganic carrier materials suitable for oral or enteral administration, the composition may also contain vitamins, minerals, trace elements, and other micronutrients, in accordance with the recommendations of government agencies such as the USRDA.

[0154] The composition of the present invention may contain a protein source, a carbohydrate source, and / or a lipid source.

[0155] Examples include proteins of any suitable food origin, such as animal proteins (milk proteins, meat proteins, and egg proteins, etc.); plant proteins (soybean proteins, wheat proteins, rice proteins, and pea proteins, etc.); mixtures of free amino acids; or combinations thereof. Milk proteins such as casein and whey proteins, and soybean proteins are particularly preferred.

[0156] If the composition contains a fat source, the fat source preferably accounts for 5% to 40% of the formula's energy; for example, 20% to 30% of the energy. DHA may be added. A suitable fat profile can be obtained using a blend of canola oil, corn oil, and high-oleic sunflower oil.

[0157] The carbohydrate source more preferably accounts for 40% to 80% of the energy of the composition. Any suitable carbohydrates, such as sucrose, lactose, glucose, fructose, solid corn syrup, maltodextrin, and mixtures thereof, can be used.

[0158] hematopoietic stem cell transplantation The present invention provides a cell population of hematopoietic stem cells and / or hematopoietic progenitor cells, prepared according to the method of the present invention, for use in therapeutic purposes.

[0159] It may be used as part of a hematopoietic stem cell transplantation procedure.

[0160] Hematopoietic stem cell transplantation (HSCT) is the transplantation of hematopoietic stem cells derived from bone marrow (in this case, known as bone marrow transplantation) or blood. Stem cell transplantation is a medical procedure in the fields of hematology and oncology, most commonly performed on people with blood or bone marrow disorders, or certain types of cancer.

[0161] Many recipients of HSCT are patients with multiple myeloma or leukemia who are not expected to respond to long-term chemotherapy or who have already developed resistance to chemotherapy. Candidates for HSCT include pediatric cases in which patients have congenital defects such as severe combined immunodeficiency with stem cell defects or congenital neutropenia, and children or adults who have lost stem cells after birth and have aplastic anemia. Other conditions treated with stem cell transplantation include sickle cell disease, myelodysplastic syndrome, neuroblastoma, lymphoma, Ewing's sarcoma, fibrinogenic round cell neoplasm, and Hodgkin's disease. More recently, non-myeloablative, so-called "mini-transplant" procedures have been developed that require less neoadjuvant chemotherapy and radiation. This development has made HSCT possible in elderly patients and others who were considered constitutionally weak and unable to withstand conventional treatment plans.

[0162] In some embodiments, hematopoietic stem cells and / or hematopoietic progenitor cells are administered as part of an autologous stem cell transplantation procedure.

[0163] In other embodiments, hematopoietic stem cells and / or hematopoietic progenitor cells are administered as part of an allogeneic stem cell transplantation procedure.

[0164] In "autologous stem cell transplantation," it should be understood that the cell population from which the procedure begins (i.e., before contact with the drug of the present invention) is obtained from the same subject to which the final cell population will be administered. Autologous transplantation is advantageous because it avoids problems associated with immunological incompatibility and can be used on subjects regardless of the likelihood of finding a genetically compatible donor.

[0165] In "allogeneic stem cell transplantation," it should be understood that the cell population at the start of the procedure (i.e., before contact with the drug of the present invention) is obtained from a different subject than the subject to which the final cell population will be administered. Preferably, to minimize the risk of immunological incompatibility, the donor will be selected to be genetically matched to the subject to which the cells will be administered.

[0166] Treatment method All references to “treatment” in this specification are understood to include curative, palliative, and prophylactic treatments. Treatment of mammals, particularly humans, is preferred. Both human and animal treatments are within the scope of this invention.

[0167] Administration The compositions used in this invention may be administered alone, but in particular in human therapeutics, they will generally be administered in mixtures with pharmaceutical carriers, additives, or diluents.

[0168] In some embodiments, compositions comprising urolithin, an NAD+ precursor, preferably nicotinamide riboside, and vitamin B12 are present in combination preparations for simultaneous, separate, or sequential use, preferably for simultaneous use.

[0169] In some embodiments, compositions comprising urolithin, an NAD+ precursor, and vitamin B12 are present in combination formulations for simultaneous, separate, or sequential use with agents selected from the group consisting of G-CSF analogs, TPO receptor analogs, and combinations thereof.

[0170] As used herein, the terms “combination,” “in combination,” “used in combination with,” or “combined preparation” refer to the administration of two or more agents / agents simultaneously, sequentially, or separately.

[0171] As used herein, the term "simultaneous" means that the activators / agents are administered at the same time, i.e., at the same time.

[0172] As used herein, the term "sequential" means that one agent / drug is administered after the other.

[0173] As used herein, the term “separately” means that the agonists / agents are administered independently of each other, but within a time interval that allows for a combined, preferably synergistic, effect of the agonists / agents. Therefore, “separately” administration may allow for one agonist / agent to be administered, for example, within 1 minute, 5 minutes, or 10 minutes after the other.

[0174] Dosage Those skilled in the art can easily determine an appropriate dose for administering one of the agents of the present invention to a target without excessive experimentation. Typically, a physician determines the actual dose best suited to an individual patient based on various factors, including the activity, metabolic stability, and duration of action of the specific agent used, age, weight, overall health, sex, prescribed diet, method and timing of administration, excretion rate, drug combination, severity of the particular condition, and the individual being treated. Naturally, there may be individual cases where a higher or lower dose range is preferable, and such cases are also within the scope of the present invention.

[0175] subject In some embodiments, the subject is a human or a non-human animal.

[0176] Examples of non-human animals include vertebrates, such as non-human primates (especially higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), and mammals such as pigs and cats. Non-human animals may also be companion animals.

[0177] Preferably, the subject is a human being.

[0178] The present invention may be useful, for example, for increasing the production of blood cells in a subject.

[0179] The present invention may be useful, for example, for increasing blood cell levels in a subject.

[0180] In some embodiments, the subject has, or is at risk of having, a normal amount of hematopoietic cells, such as red blood cells, white blood cells, and / or platelets.

[0181] The normal range for white blood cells in humans is 4,500 to 10,000 cells / μL. The normal range for red blood cells in men is 5 million to 6 million cells / μL, and in women it is 4 million to 5 million cells / μL. The normal range for platelets is 140,000 to 450,000 per μL. Blood cell levels, sometimes called blood cell counts, can be easily measured by those skilled in the art using any of the many techniques known in the art, such as hemocytometers and automated blood analyzers.

[0182] In some embodiments, the subjects have or are at risk of having anemia, leukopenia, and / or thrombocytopenia.

[0183] In some embodiments, hematopoietic cells below normal levels are secondary to primary or autoimmune diseases of the hematopoietic system, such as congenital bone marrow failure syndrome, idiopathic thrombocytopenia, aplastic anemia, and myelodysplastic syndrome.

[0184] Patients at risk of developing a decrease in blood cell levels include those with anemia or myelodysplastic syndrome, those undergoing chemotherapy, bone marrow transplantation, or radiotherapy, and those with autoimmune cytopenia, including but not limited to immune thrombocytopenic purpura, pure red cell atrophy, and autoimmune neutropenia.

[0185] Patients at risk of developing post-transplant complications include those with depleted hematopoietic cells who have undergone autologous or allogeneic hematopoietic stem cell transplantation or progenitor cell transplantation using primary HSPCs or HSPCs manipulated in vitro.

[0186] In some embodiments, subjects may have undergone myeloablative conditioning, chemotherapy, radiotherapy, and / or surgery. Myeloablative conditioning, chemotherapy, radiotherapy, and / or surgery may result in sub-normal levels of hematopoietic cells.

[0187] Subjects with or at risk of developing hematopoietic cells below a normal level include those suffering from blood cancers (e.g., leukemia, lymphoma, and myeloma), blood disorders (e.g., hereditary anemia, congenital metabolic disorders, aplastic anemia, β-thalassemia, Blackfan-Diamond syndrome, globoid cell leukodystrophy, sickle cell anemia, severe combined immunodeficiency, X-linked lymphoproliferative syndrome, Wiscott-Aldrich syndrome, Hunter syndrome, Hurler syndrome, Lesch-Nyhan syndrome, osteopetrosis), and those undergoing chemotherapy for immune system and other diseases (e.g., autoimmune diseases, diabetes, rheumatoid arthritis, systemic lupus erythematosus). Furthermore, subjects with or at risk of developing hematopoietic cells below normal levels include those exhibiting severe neutropenia and / or severe thrombocytopenia and / or severe anemia, such as post-transplant subjects or subjects undergoing decongestive chemotherapy for solid tumors; patients suffering from hematopoietic disorders due to toxicity, drug-induced disorders or infections (i.e., benzene derivatives, chloramphenicol, B19 parvovirus, etc.); patients suffering from myelodysplastic syndromes; patients suffering from severe immune disorders; or patients suffering from congenital hematological disorders, whether of central origin (i.e., Fanconi anemia) or peripheral origin (i.e., G6PDH deficiency).

[0188] The present invention may be useful, for example, in the treatment or prevention of anemia, leukopenia and / or thrombocytopenia; infections (e.g., non-viral or viral infections); and / or cancers such as blood cancers (e.g., leukemia, lymphoma, or myeloma).

[0189] The combinations, compositions, and cell populations of the present invention may be useful in the treatment of diseases listed in International Publication No. 1998 / 005635. For ease of reference, a portion of that list is provided hereby: cancer, inflammation or inflammatory diseases, dermatological disorders, fever, cardiovascular effects, bleeding, coagulation and acute responses, cachexia, anorexia, acute infections, HIV infection, shock, graft-versus-host reactions, autoimmune diseases, reperfusion injury, meningitis, migraines and aspirin-dependent antithrombosis; tumor growth, invasion and spread, angiogenesis, metastasis, malignant tumors, ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease, osteoarthritis, arthritis Rheumatism, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease, and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulcers, retinopathy, and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis, or endosclerosis.

[0190] In addition, or alternatively, the combinations, compositions, and cell populations of the present invention may be useful in treating the diseases listed in International Publication No. 1998 / 007859. For ease of reference, a portion of the list is shown here: cytokine and cell proliferation / differentiation activity; immunosuppressive or immunostimulatory activity (e.g., to treat immunodeficiency including human immunodeficiency virus infection; regulation of lymphocyte proliferation; for the treatment of cancer and many autoimmune diseases, and for the prevention of transplant rejection or induction of tumor immunity); regulation of hematopoiesis, e.g., for the treatment of myeloid or lymphoid diseases; promotion of bone, cartilage, tendon, ligament and nerve tissue growth, e.g., for the treatment of wound healing, burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (regulation of fertilization capacity); chemotaxis / chemokinetic activity (e.g., to recruit specific cell types to injury or infection sites); hemostatic and thrombolytic activity (e.g., to treat hemophilia and stroke); anti-inflammatory activity (e.g., to treat septic shock or Crohn's disease); as an antimicrobial agent; e.g., a modulator of metabolism or behavior; as an analgesic; for the treatment of certain deficiencies; in humans or veterinary medicine, e.g., for the treatment of psoriasis.

[0191] In addition, or alternatively, the combinations, compositions, and cell populations of the present invention may be useful in the treatment of diseases listed in International Publication No. 1998 / 009985. For ease of reference, a portion of that list is shown here: macrophage inhibitory activity and / or T cell inhibitory activity, and therefore anti-inflammatory activity; anti-immune activity, i.e., inhibitory effects on cellular immune responses and / or humoral immune responses, including non-inflammatory responses; inhibition of the adhesion ability of macrophages and T cells to extracellular matrix components and fibronectin, and inhibition of upregulated Fas receptor expression in T cells; arthritis, including rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic Unwanted immune responses and inflammation such as inflammation associated with autoimmune diseases like lupus erythematosus and collagen diseases, inflammation associated with arteriosclerosis and arteriosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory diseases, cardiopulmonary diseases such as respiratory distress syndrome, inflammation associated with gastrointestinal diseases such as peptic ulcers and ulcerative colitis, liver diseases such as hepatic fibrosis and cirrhosis, glandular diseases such as thyroiditis, kidney and urinary tract diseases such as glomerulonephritis, ear, nose, and throat diseases, skin diseases such as dermatitis, dental diseases such as periodontitis, orchitis and upper respiratory tract infections, infertility, and immune-related testicular diseases such as testicular trauma.Placental dysfunction, placental insufficiency, recurrent miscarriage, eclampsia, pre-eclampsia and other immunological and / or inflammatory gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation (e.g., retinitis or cystic macular edema), retinitis or cystic macular hydrops, sympathetic ophthalmopathy, scleritis, retinitis pigmentosa, immunological and inflammatory components of degenerative eye diseases, inflammatory components of ocular trauma, ocular inflammation due to infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring (e.g., after glaucoma filtration surgery), ocular use Immune and / or inflammatory responses to plant, other immune and inflammation-related ophthalmic diseases, inflammation associated with autoimmune diseases, conditions or disorders in which suppression of immunity and / or inflammation is beneficial in both the central nervous system (CNS) and other organs, Parkinson's disease, AIDS-related dementia complex HIV-associated encephalopathy, Davis's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of Stork's disease, post-polio syndrome, immune and inflammatory components of mental disorders, complications and / or side effects of treatment for myelitis, encephalitis, subacute Sclerosing panencephalitis, encephalomyelitis, acute neurological disorders, subacute neurological disorders, chronic neurological disorders, Gilliam-Barré syndrome, Sydenham chorea, myasthenia gravis, pseudocerebellum, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or CNS infection, inflammatory components of muscular dystrophy and dystophy, and immune and inflammation-related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infections, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplant complications and / or side effects, To inhibit inflammatory and / or immune complications and side effects of gene therapy (e.g., those caused by infection of viral carriers, inflammation associated with AIDS, etc.), to treat or improve monocyte or leukocyte proliferative disorders, such as leukemia, by reducing the amount of monocytes or lymphocytes in order to suppress or inhibit humoral and / or cellular immune responses, and to prevent and / or treat graft rejection in transplants of natural or artificial cells, tissues, or organs, such as corneas, bone marrow, organs, lenses, pacemakers, and natural or artificial skin tissue.

[0192] Amplification method and culture medium In another embodiment, the present invention provides a method for growing a population of isolated hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs), comprising the step of contacting the population with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0193] In some embodiments, contact involves culturing the population in the presence of a composition comprising urolithin, nicotinamide riboside, and vitamin B12.

[0194] In some embodiments, the method is (a) A step to prepare a population of HSPC cells, (b) Optionally, a step of culturing a population of HSPC cells, preferably in HSPC growth medium or maintenance medium; (c) A step of optionally isolating a cell subpopulation of HSPCs characterized by a low mitochondrial membrane potential, (d) The method involves contacting the group of (a) or (b), or the subgroup of (c), with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

[0195] In some embodiments, the population prepared in step (a) is obtained from bone marrow, mobilized peripheral blood, or umbilical cord blood.

[0196] In some embodiments, the product of step (d) is enriched with cells that have long-term multiseries blood reconstitution ability.

[0197] As used herein, the terms “growth medium” and “maintenance medium” refer to any standard stem cell culture medium suitable for the growth and maintenance of stem cells, such as the media described in the following examples herein or the media described in Boitano et al. (2010) Science 329:1345-1348.

[0198] In another embodiment, the present invention provides a cell culture medium comprising a composition containing urolithin, an NAD+ precursor, preferably NR, and vitamin B12.

[0199] In some embodiments, the culture medium includes cytokines and growth factors. Cytokines and growth factors may be used with or without stromal cell feeder or mesenchymal cell support and may include SCF, TPO, Flt3-L, FGF-1, IGF1, IGFBP2, IL-3, IL-6, G-CSF, M-CSF, GM-CSF, EPO, oncostatin-M, EGF, PDGF-AB, angiopoietin, and angiopoietin-like families (including Angl5), prostaglandins and eicosanoids including PGE2, aryl hydrocarbon (AhR) receptor inhibitors, such as StemRegeninI (SRI) and LGC006 (Boitano et al. (2010) Science 329:1345-1348).

[0200] The membrane potential in the HSC compartment, particularly the mitochondrial membrane potential, can be measured by methods known to those skilled in the art, such as those described in the examples herein, in particular by flow cytometry of cells stained with tetramethylrhodamine methyl ester (TMRM).

[0201] kit In another embodiment, the present invention provides a kit comprising the combination and / or cell population of the present invention.

[0202] The cell population may be provided in a suitable container.

[0203] The kit may also include instructions for use.

[0204] Those skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the invention as disclosed.

[0205] Preferred features and embodiments of the present invention are described below by non-limiting examples.

[0206] In carrying out the present invention, unless otherwise specified, the prior art of chemistry, biochemistry, molecular biology, microbiology, and immunology, which is within the scope of the skills of those skilled in the art, will be used. Such art is described in the literature. For example, Sambrook, J., Fritsch, EFand Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FMet al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch.9,13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JMand McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press and Lilley, DMand. Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures. See Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. [Examples]

[0207] Example 1 Materials and methods Flow cytometry Flow cytometry analysis was performed on bone marrow (BM) newly isolated from C57Bl6 mice. BM was extracted from fragmented femurs and tibias. The cell suspension was filtered through a 70 μm cell filter, and erythrocytes were removed by incubation with erythrocyte lysis buffer (eBioscences). Isolation and staining were performed in ice-cold 1 mM EDTA / PBS solution. Lineage-positive cells were then removed using a magnetic lineage depletion kit (BD Biosciences). The cell suspension was then stained with a specific antibody against the stem cell compartment and sorted into 1.5 mL Eppendorf tubes by FACS (BD FACS Aria III).

[0208] antibody The following antibodies were used in this study: rat mAbs against cKit(2B8), Sca1(D7), CD150(TC-15-12F12.2), and CD48(HM48-1). Antibodies were purchased from Biolegend, eBiosciences, and BD. A mixture of biotinylated mAbs against CD3, CD11b, CD45R / B220, Ly-6G, Ly-6C, and TER-119 was used as a series marker ("series cocktail") and purchased from BD. DAPI or propidium iodide (PI) staining was used to distinguish between live and dead cells.

[0209] mHSC culture Mouse HSCs were sorted into 1.5 mL Eppendorf tubes and cultured in Stemline II (SIGMA) supplemented with 100 ng / mL SCF (R&D) and 2 ng / mL Flt3 (R&D). This was used as the baseline condition. Different concentrations of UroA, NR, and Vit B12 (MC) were added to specific wells as described. NR was added every 24 hours.

[0210] Analysis of mitochondrial activity Mouse HSCs already in culture were incubated with 200 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen) and 100 nM Mitotracker green at 37°C for 1 hour. The cells were then washed with FACS buffer and analyzed by flow cytometry using BD LSR II.

[0211] Results and Discussion The combination of UroA, NR, and vitamin B12 induces a decrease in mitochondrial membrane potential. Newly selected mHSCs (LKS CD150+CD48-) by FACS were cultured in specific wells (as shown) in basal medium (Stemline+SCF+FLT3L) supplemented with UroA, NR, and MC. Cells were harvested on day 3, stained with tetramethylrhodamine methyl ester (TMRM, to measure mitochondrial membrane potential) and Mitotracker (to measure mitochondrial mass), and analyzed by flow cytometry.

[0212] The inventors have found that under NR and MC conditions, TMRM is superior compared to the reference condition. low We found that the proportion of cells in the gate significantly increased, and TMRM fluorescence intensity (mean fluorescence intensity, MFI) significantly decreased. The dual combination of NR+MC showed a significant increase compared to NR alone or MC alone (TMRM low The percentage of cells in the gate decreased (MFI TMRM). Interestingly, the triple combination of UroA, NR, and MC was better than the double combination in TMRM. low The proportion of cells in the gate was significantly higher, and MFI TMRM was significantly lower, showing the strongest effect. Mitochondrial mass (measured by Mitotracker) (right panel) was significantly reduced in the NR+MC and UroA+MC+NR combinations compared to culture in basic medium or NR alone.

[0213] In summary, our findings demonstrate the ability of the combination of UroA, nicotinamide riboside, and vitamin B12 to synergistically restore HSC function, possibly through mitophagy-induced regulation of mitochondrial membrane potential. This ability suggests the application of this combination in HSC transplantation for the treatment of hematological malignancies.

[0214] All publications referenced in the above specification are incorporated herein by reference. Various modifications and changes to the compositions, uses and methods disclosed herein will be obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the invention is disclosed in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the form disclosed for carrying out the invention will be obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

1. Use of a composition comprising urolithin, NAD+ precursor, and vitamin B12 to improve stem cell function in a population of hematopoietic stem cells and / or progenitor cells (HSPCs).

2. The use according to claim 1, wherein the uroritin is uroritin A.

3. The use according to claim 1 or 2, wherein the NAD+ precursor is selected from the group consisting of nicotinic acid, nicotinamide, nicotinamide riboside (NR), reduced nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid riboside, and mixtures thereof.

4. The use according to claim 1 or 2, wherein the combination is urolithin, nicotinamide riboside, and vitamin B12.

5. Urolithin, NAD+ precursor, and vitamin B12 for use in improving hematopoietic stem cell function.

6. The combination for use according to claim 5, wherein the hematopoietic stem cell function includes one or more of engraftment, self-renewal, and differentiation.

7. The combination of uses according to claim 5 or 6, wherein the use increases blood cell levels in the subject.

8. A composition comprising urolithin, an NAD+ precursor, and vitamin B12 for use in the treatment or prevention of (a) anemia, leukopenia and / or thrombocytopenia, (b) infections, and / or (c) cancer.

9. The composition for use according to any one of claims 5 to 8, wherein the urolithin is urolithin A.

10. The composition for use according to any one of claims 5 to 9, wherein the NAD+ precursor is selected from the group consisting of nicotinic acid, nicotinamide, nicotinamide riboside (NR), reduced nicotinamide riboside (NRH), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid riboside, and mixtures thereof.

11. The composition for use according to any one of claims 5 to 10, wherein the combination is urolithin, nicotinamide riboside, and vitamin B12.

12. The composition for use according to any one of claims 5 to 11, wherein the composition is in the form of a pharmaceutical composition or a nutritional composition, and optionally in the form of a food product, a nutritional supplement, a nutraceutical, a food for specific medical purposes (FSMP), a nutritional supplement, a milk beverage, a small-volume liquid nutritional supplement, or a meal replacement beverage.

13. A composition for use according to any one of claims 5 to 12, wherein the subject has or is at risk of having a normal amount of hematopoietic cells, and the hematopoietic cells are, in some cases, red blood cells, white blood cells, and / or platelets.

14. A composition for use according to any one of claims 5 to 13, wherein the subject has or is at risk of having anemia, leukopenia, and / or thrombocytopenia.

15. A composition for use according to any one of claims 5 to 14, wherein the subject has previously undergone an intervention selected from the group consisting of hematopoietic stem cell transplantation, bone marrow transplantation, myeloablative conditioning, chemotherapy, radiotherapy, and surgery.

16. The composition for use according to any one of claims 5 to 15, wherein the urolithin, NAD+ precursor, and vitamin B12 are present in a combination formulation for simultaneous use, separate use, or sequential use with a drug selected from the group consisting of a G-CSF analog, a TPO receptor analog, and combinations thereof.

17. A method for growing an isolated population of hematopoietic stem cells and / or progenitor cells (HSPCs), comprising contacting the population with a composition comprising a combination of urolithin, an NAD+ precursor, and vitamin B12.

18. The method according to claim 17, (a) A step of preparing a population of HSPC cells, (b) Optionally, a step of culturing a population of HSPC cells, preferably in HSPC growth medium or maintenance medium; (c) A step of isolating a cell subpopulation of HSPCs characterized by low mitochondrial membrane potential, A method comprising the step of contacting a group of (a) or (b), or a subgroup of (c), with a composition comprising urolithin, an NAD+ precursor, preferably a combination of nicotinamide riboside and vitamin B12.

19. A cell culture medium comprising a composition containing urolithin, an NAD+ precursor, preferably nicotinamide riboside, and vitamin B12.

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