Urolithins for Improving Stem Cell Function

Abstract

Use of a urolithin to improve stem cell function in a population of hematopoietic stem and / or progenitor cells (HSPCs), wherein stem cell function is improved for at least 40 weeks.

Description

[Technical field]

[0001] The present invention relates to reagents and methods for improving stem cell function in hematopoietic stem and progenitor cells (HSPCs), for example for improving the engraftment and / or improving the self-renewal and differentiation capacity of HSPC populations. In particular, the present invention relates to long-term improvement of stem cell function. [Background technology]

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

[0003] HSCs differ from their lineage-committed progeny in that they rely primarily 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, WM et al. (2013) Cell Stem Cell 12:62-74). This distinct metabolic state is thought to protect HSCs from cellular damage due to reactive oxygen species (ROS) in active mitochondria and maintain 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 tetramethylrhodamine methyl ester (TMRM) fluorescence, has traditionally been used as a surrogate indicator of the metabolic state of cells. It has been demonstrated that phenotypically defined HSCs have a lower mitochondrial membrane potential compared to progenitor cells (Vannini, N. et al. (2016) Nat Commun 7:13125). The same study found that artificially lowering the mitochondrial membrane potential by chemical uncoupling of the mitochondrial electron transport chain maintained HSCs 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 (a precursor of NAD and vitamin B3) to the culture medium resulted in significantly higher levels of engraftment and was able to maintain long-term blood production in both primary and secondary transplanted humanized recipient mice.

[0005] However, there remains a great need for additional approaches to improve stem cell function in HSCs over the long term in vivo and in vitro, in particular approaches that improve the engraftment of HSPC populations (e.g., during hematopoietic stem cell transplant procedures) as well as approaches that improve the self-renewal and differentiation capacity of HSCs. Summary of the Invention

[0006] The inventors have observed that UroA improves hematopoietic stem cell (HSC) function, including by enhancing engraftment and self-renewal.

[0007] Furthermore, the inventors have observed, for example, through serial transplantation studies, that UroA treatment of HSPCs can result in long-term enhancement of stem cell function, in particular, the inventors have found that enhanced stem cell function can be sustained for at least 40 weeks.

[0008] Without wishing to be bound by theory, our studies indicate that relatively short exposure of HSPCs to UroA can result in long-term enhancement of stem cell function through effects on the epigenetic signature of the cells.

[0009] In one aspect, the invention provides for the use of a urolithin to improve stem cell function in a cell population of hematopoietic stem and / or hematopoietic progenitor cells (HSPCs), and to improve stem cell function for at least 40 weeks.

[0010] In some embodiments, the use is an in vitro use. In some embodiments, the use is an ex vivo use.

[0011] In another aspect, the invention provides a method for improving stem cell function in a cell population of hematopoietic stem and / or progenitor cells (HSPCs), comprising contacting the cell population with a urolithin, wherein stem cell function is improved for at least 40 weeks.

[0012] In some embodiments, stem cell function is improved for at least 41 weeks. In some embodiments, stem cell function is improved for at least 42 weeks. In some embodiments, stem cell function is improved for at least 43 weeks.

[0013] In a preferred embodiment, stem cell function is enhanced for at least 44 weeks.

[0014] In some embodiments, the cell population is an isolated HSPC population.

[0015] In some embodiments, the HSPCs have a CD34+ phenotype.

[0016] In some embodiments, the HSPCs have a CD34+CD38- phenotype. In some embodiments, the method comprises: (a) providing a population of HSPCs; (b) optionally isolating a subpopulation of HSPCs characterized by a low mitochondrial membrane potential; (c) contacting the cell population of (a), or the cell subpopulation of (b), with a urolithin; Includes.

[0017] In another aspect, the invention provides a urolithin for use in a method of treatment by improving stem cell function in hematopoietic stem and / or progenitor cells (HSPCs), wherein stem cell function is improved for at least 40 weeks.

[0018] In some embodiments, the urolithin is for use in improving hematopoietic stem cell function in a subject.

[0019] In some embodiments, the methods involve contacting the HSPCs with a urolithin prior to administration of the HSPCs to the subject.

[0020] In some embodiments, the methods include administering a urolithin to a subject.

[0021] In some embodiments, a urolithin is administered to a subject enterally or parenterally, preferably enterally, In preferred embodiments, a urolithin is administered to a subject orally.

[0022] In some embodiments, the method of treatment is the treatment or prevention of (a) anemia, leukopenia and / or thrombocytopenia, (b) infectious disease, and / or (c) cancer.

[0023] In some embodiments, the method of treatment is treatment or prevention of anemia, leukopenia and / or thrombocytopenia. In some embodiments, the method of treatment is treatment or prevention of an infection. In some embodiments, the method of treatment is treatment or prevention of cancer.

[0024] In some embodiments, the cancer is a hematological cancer, hi some embodiments, the cancer is a leukemia, lymphoma, or myeloma.

[0025] In some embodiments, stem cell function includes one or more of the following: engraftment capacity, self-renewal capacity, and blood differentiation and immune cell production capacity.

[0026] In some embodiments, stem cell function includes engraftment, in some embodiments, stem cell function includes self-renewal, in some embodiments, stem cell function includes blood differentiation and immune cell production.

[0027] 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 blood differentiation and immune cell production.

[0028] In some embodiments, improved stem cell function increases blood cell levels in a subject.

[0029] In a preferred embodiment, the urolithin is urolithin A.

[0030] In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for no longer than 7 days.

[0031] In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for 1-3 days. In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for 1-5 days. In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for 1-7 days.

[0032] In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for 3 to 7 days. In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for 5 to 7 days.

[0033] In some embodiments, the population or subpopulation of HSPCs is contacted with the urolithin for 3 to 5 days.

[0034] In some embodiments, the urolithin is in the form of a pharmaceutical or nutritional composition.

[0035] In some embodiments, the urolithin is in the form of a food product, dietary supplement, nutraceutical, food for special medical purposes (FSMP), nutritional supplement, dairy drink, small volume liquid nutritional supplement, or meal replacement drink.

[0036] In some embodiments, the subject has, or is at risk for having, subnormal amounts of hematopoietic cells, eg, red blood cells, white blood cells, and / or platelets.

[0037] In some embodiments, the subject has or is at risk for anemia, leukopenia and / or thrombocytopenia.

[0038] In some embodiments, the subject has undergone an intervention selected from the group consisting of hematopoietic stem cell transplant, bone marrow transplant, myeloablative conditioning, chemotherapy, radiation therapy, and surgery.

[0039] In some embodiments, the subject is an immune-compromised subject.

[0040] In some embodiments, the subject is 3-4 weeks post-intervention.

[0041] In some embodiments, the subject is a human or a non-human mammal, preferably a human, and optionally a human adult, child, or infant.

[0042] In some embodiments, the urolithin is present in a combination formulation for simultaneous, separate, or sequential use with an agent selected from the group consisting of nicotinamide riboside, a G-CSF analog, a TPO receptor analog, SCF, TPO, Flt3-L, FGF-1, IGF1, IGFBP2, IL-3, IL-6, G-CSF, M-CSF, GM-CSF, EPO, and combinations thereof.

[0043] In a preferred embodiment, the urolithin is present in a combined formulation for simultaneous, separate or sequential use with nicotinamide riboside.

[0044] In another aspect, the invention provides a method of expanding an isolated population of hematopoietic stem and / or progenitor cells (HSPCs), comprising contacting the population with a urolithin, whereby stem cell function of the HSPCs is enhanced for at least 40 weeks.

[0045] In some embodiments, the contacting step comprises culturing the cell population in the presence of the urolithin.

[0046] In some embodiments, the method comprises: (a) providing a population of HSPCs; (b) optionally culturing the HSPC population, preferably in an HSPC expansion or maintenance medium; (c) optionally isolating a subpopulation of HSPCs characterized by a low mitochondrial membrane potential; (d) contacting the cell population of (a) or (b), or the cell subpopulation of (c), with a urolithin; Includes.

[0047] In some embodiments, the cell population provided in step (a) is obtained from bone marrow, mobilized peripheral blood, or umbilical cord blood.

[0048] In some embodiments, the product of step (d) is enriched for cells with long-term multilineage blood reconstituting potential.

[0049] In another aspect, the present invention provides a cell population of hematopoietic stem and / or progenitor cells (HSPCs) obtainable by the method of the present invention.

[0050] In another aspect, the invention provides pharmaceutical compositions comprising the hematopoietic stem and / or hematopoietic progenitor cell (HSPC) populations of the invention.

[0051] In another aspect, the invention provides a method of engrafting hematopoietic stem and / or progenitor cells (HSPCs) in a subject comprising contacting an isolated population of HSPCs with a urolithin and administering the population of HSPCs to a subject in need thereof, wherein stem cell function of the HSPCs is enhanced for at least 40 weeks.

[0052] In another aspect, the invention provides a method of improving hematopoietic stem cell function comprising contacting a population of hematopoietic stem and / or hematopoietic progenitor cells (HSPCs) with a urolithin, wherein stem cell function is improved for at least 40 weeks.

[0053] In another aspect, the invention provides a method of improving hematopoietic stem cell function in a subject, the method comprising contacting a population of hematopoietic stem and / or hematopoietic progenitor cells (HSPCs) with a urolithin and administering the HSPC population to a subject in need thereof, wherein stem cell function is improved for at least 40 weeks.

[0054] In another aspect, the invention provides a method of improving engraftment by a population of hematopoietic stem and / or progenitor cells (HSPCs), comprising contacting the HSPC population with a urolithin, whereby engraftment and blood reconstitution capacity are improved for at least 40 weeks. In another aspect, the invention provides a method of improving hematopoietic stem cell self-renewal, comprising contacting a population of hematopoietic stem and / or progenitor cells (HSPCs) with a urolithin, whereby hematopoietic stem cell self-renewal is improved for at least 40 weeks. In another aspect, the invention provides a method of improving hematopoietic stem cell differentiation, comprising contacting a population of hematopoietic stem and / or progenitor cells (HSPCs) with a urolithin, whereby stem cell differentiation is improved for at least 40 weeks. In some embodiments, engraftment, self-renewal and / or differentiation are improved in a subject, and the method further comprises administering the HSPC population to a subject in need of the cell population.

[0055] In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vivo method.

[0056] In some embodiments, the cell population is an isolated HSPC population.

[0057] In another aspect, the invention provides a method of improving hematopoietic stem cell function comprising administering a urolithin to a subject in need thereof, wherein stem cell function is improved for at least 40 weeks.

[0058] In another aspect, the invention provides a method of improving hematopoietic stem cell engraftment, comprising administering a urolithin to a subject in need of a urolithin, whereby hematopoietic stem cell engraftment is improved for at least 40 weeks.In another aspect, the invention provides a method of improving hematopoietic stem cell self-renewal, comprising administering a urolithin to a subject in need of a urolithin, whereby stem cell self-renewal is improved for at least 40 weeks.In another aspect, the invention provides a method of improving hematopoietic stem cell differentiation, comprising administering a urolithin to a subject in need of a urolithin, whereby hematopoietic stem cell differentiation is improved for at least 40 weeks. [Brief description of the drawings]

[0059] [Figure 1A-1] Figure 1 shows that UroA leads to a decrease in mitochondrial membrane potential. Bone marrow-derived mouse HSCs cultured in basal medium (control) supplemented with various concentrations of UroA. The percentage of cells in the TMRM low gate increases and the MFI TMRM decreases in a dose-dependent manner. Mitochondrial mass (measured by Mitotracker) decreases with increasing concentrations of UroA in the culture medium. [Figure 1A-2] Figure 1 shows that UroA leads to a decrease in mitochondrial membrane potential. Bone marrow-derived mouse HSCs cultured in basal medium (control) supplemented with various concentrations of UroA. The percentage of cells in the TMRM low gate increases and the MFI TMRM decreases in a dose-dependent manner. Mitochondrial mass (measured by Mitotracker) decreases with increasing concentrations of UroA in the culture medium. [Figure 1B-1]Figure 1 shows that UroA leads to a decrease in mitochondrial membrane potential. Human umbilical cord blood-derived HSPCs cultured for 7 days in basal medium (control) containing various concentrations of UroA. FACS analysis shows a decrease in TMRM signal at all three time points [day 3 (top), day 5 (middle), and day 7 (bottom)]. The percentage of cells in the CD34+TMRM low gate increases, while the MFI TMRM decreases in a dose-dependent manner. [Figure 1B-2] Figure 1 shows that UroA leads to a decrease in mitochondrial membrane potential. Human umbilical cord blood-derived HSPCs cultured for 7 days in basal medium (control) containing various concentrations of UroA. FACS analysis shows a decrease in TMRM signal at all three time points [day 3 (top), day 5 (middle), and day 7 (bottom)]. The percentage of cells in the CD34+TMRM low gate increases, while the MFI TMRM decreases in a dose-dependent manner. [Figure 1B-3] Figure 1 shows that UroA leads to a decrease in mitochondrial membrane potential. Human umbilical cord blood-derived HSPCs cultured for 7 days in basal medium (control) containing various concentrations of UroA. FACS analysis shows a decrease in TMRM signal at all three time points [day 3 (top), day 5 (middle), and day 7 (bottom)]. The percentage of cells in the CD34+TMRM low gate increases, while the MFI TMRM decreases in a dose-dependent manner. [Figure 2A] We show that in vitro UroA treatment enhances the in vivo function of mHSCs and hHSPCs. HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated recipient mice. Mice injected with cells cultured with UroA show higher blood reconstitution over 24 weeks. This increase is also seen in myeloid and lymphoid lineages. [Figure 2B]We show that in vitro UroA treatment enhances the in vivo function of mHSCs and hHSPCs. Human umbilical cord blood-derived HSPCs were cultured in basal medium with or without 50 μM UroA. Two functional assays were performed. After 5 days of culture, cells were injected into irradiated NSG-SGM3 neonates. After 7 days of culture, cells were seeded on methylcellulose plates and their colony-forming ability was estimated (CFU assay). [Figure 2C] We show that in vitro UroA treatment enhances the in vivo function of mHSCs and hHSPCs. UroA-treated cells produced significantly more colonies than control (Ctrl) after 15 days of methylcellulose culture. [Figure 2D] We show that in vitro UroA treatment enhances mHSC and hHSPC function in vivo. Mice transplanted with UroA-treated cells show a significant increase in human cell chimerism in the peripheral blood. [Figure 2E] We show that in vitro UroA treatment enhances the in vivo function of mHSCs and hHSPCs. UroA treatment increases blood cell numbers primarily of human lymphoid lineage (T and B cells). [Diagram 3] Figure 1 shows that UroA drives metabolic gene expression in mHSCs. A) QPCR analysis performed on bone marrow-derived mHSCs cultured in basal medium with or without 20 μM UroA. Higher expression of mitophagy / autophagy genes, glycolysis genes, and genes involved in protection from ROS was observed in cells treated with UroA. [Figure 4A] We show that UroA treatment improves survival of recipient mice after transplantation. A) Human umbilical cord blood-derived HSPCs were cultured in basal medium with or without 50 μM UroA. After 3 days of culture, cells were counted and a limiting dose (40,000 cells) was injected into each irradiated recipient adult NSG mouse and survival was monitored over several months. Twelve mice were transplanted for each of the control and UroA conditions. B) Mice transplanted with UroA-treated cells had significantly improved survival over a period of 8 months. [Figure 4B] We show that UroA treatment improves survival of recipient mice after transplantation. A) Human umbilical cord blood-derived HSPCs were cultured in basal medium with or without 50 μM UroA. After 3 days of culture, cells were counted and a limiting dose (40,000 cells) was injected into each irradiated recipient adult NSG mouse and survival was monitored over several months. Twelve mice were transplanted for each of the control and UroA conditions. B) Mice transplanted with UroA-treated cells had significantly improved survival over a period of 8 months. [Figure 5A] Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 5B]Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 5C] Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 5D]Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 5E] Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 5F]Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 5G] Analysis of serial transplants demonstrates that in vitro UroA treatment enhances HSC function in vivo over the long term. (Figure 5A) HSCs were isolated from mouse bone marrow and cultured in basal medium with or without 20 μM UroA. At the end of the culture period, cells were injected intravenously into lethally irradiated first recipient mice. Blood analysis was performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples. Bone marrow from the first mice was transplanted into lethally irradiated second recipient mice by intravenous tail vein injection. Blood analysis was performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5E) samples. UroA-cultured cells show higher blood reconstitution over the entire period of at least 44 weeks. This increase is also expressed in myeloid and lymphoid lineages. [Figure 6A] Gene expression analysis of UroA-treated mouse HSCs. RNA sequencing analysis was performed on HSCs after brief ex vivo UroA treatment. [Figure 6B-1] Gene expression analysis of UroA-treated mouse HSCs. Gel electrophoresis and fragment analyzer analysis. [Figure 6B-2] Gene expression analysis of UroA-treated mouse HSCs. Gel electrophoresis and fragment analyzer analysis. [Figure 6C] Gene expression analysis of UroA-treated mouse HSCs. Multidimensional scaling (MDS) plots of RNA sequencing data and differential expression analysis. [Figure 6D-1] Gene expression analysis of UroA-treated mouse HSCs. Analysis of biological pathways altered by UroA treatment. [Figure 6D-2] Gene expression analysis of UroA-treated mouse HSCs. Analysis of biological pathways altered by UroA treatment. [Figure 6D-3] Gene expression analysis of UroA-treated mouse HSCs. Analysis of biological pathways altered by UroA treatment. [Figure 6D-4] Gene expression analysis of UroA-treated mouse HSCs. Analysis of biological pathways altered by UroA treatment. [Figure 6E] Gene expression analysis of UroA-treated mouse HSCs. Differential expression analysis of mitochondrial genes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes" or "containing" or "contains" and are inclusive, i.e., open-ended, and do not exclude additional, unrecited components, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0061] 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 the ability to self-renew by mitosis, as well as the ability to differentiate into a variety of specialized cell types. The two main types of mammalian stem cells are embryonic stem cells, which are isolated from the inner cell mass of the blastocyst, and adult stem cells, which are found in adult tissues. In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In the adult organism, stem and progenitor cells act as the body's repair system, replenishing specialized cells as well as maintaining the normal turnover of regenerative organs such as blood, skin or intestinal tissues.

[0062] Hematopoietic stem cells (HSCs) are multipotent stem cells that can be found, for example, in peripheral blood, bone marrow, and umbilical cord blood. HSCs have the capacity for self-renewal and differentiation into any blood lineage. HSCs have the capacity to repopulate the erythroid and myeloid lineages of the entire immune system, as well as all hematopoietic tissues, including bone marrow, spleen, and thymus. HSCs are life-long producers of hematopoietic cells of all lineages.

[0063] Hematopoietic progenitor cells have the capacity to differentiate into specific cell types. However, in contrast to stem cells, hematopoietic progenitor cells are already quite specific and differentiate into the cells they are "targeted" to. The 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. Hematopoietic progenitor cells can only be strictly distinguished from HSCs by in vivo functional assays (i.e., demonstration of their ability to engraft and give rise to all blood lineages over the long term).

[0064] Differentiated cells are more specialized cells compared to stem cells or progenitor cells. Differentiation occurs during the development of a multicellular organism, as the organism changes from a single zygote to a complex system of multiple tissues and cell types. Differentiation is also a common process in adults: adult stem cells divide to produce fully differentiated daughter cells during tissue repair and normal cell turnover. Differentiation dramatically changes cell size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are primarily due to highly controlled regulation in gene expression. In other words, differentiated cells are cells that have specific structures and exhibit specific functions due to developmental processes that involve the activation and inactivation of specific genes. Here, differentiated cells include differentiated cells of the hematopoietic lineage, such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, T cells, B cells, and NK cells. For example, differentiated cells of the hematopoietic lineage can be distinguished from stem and progenitor cells by detection of cell surface molecules that are not expressed or are expressed to a lesser extent on undifferentiated cells. Examples of suitable human lineage markers include CD33, CD13, CD14, CD15 (myeloid), CD19, CD20, CD22, CD79a (B), CD36, CD71, CD235a (erythroid), CD2, CD3, CD4, CD8 (T), CD56 (NK).

[0065] HSC source In some embodiments, the hematopoietic stem cells are obtained from a tissue sample.

[0066] For example, HSCs can be obtained from adult and fetal peripheral blood, umbilical cord blood, bone marrow, liver, or spleen. HSCs are obtained after mobilization of cells in vivo by treatment with growth factors.

[0067] Mobilization may be performed, for example, using G-CSF, plerixafor, or a combination thereof. Other agents such as NSAIDs, CXCR2 ligands (Grobeta), and dipeptidyl peptidase inhibitors may also be useful as mobilizing agents.

[0068] With the availability of the stem cell growth factors GM-CSF and G-CSF, most hematopoietic stem cell transplant procedures are now performed using stem cells harvested from peripheral blood rather than bone marrow. Harvesting peripheral blood stem cells can result in larger grafts and does not require the donor to undergo general anesthesia to harvest the graft, potentially resulting in shorter engraftment times and lower long-term relapse rates.

[0069] Bone marrow can be harvested by standard aspiration techniques (either steady state or after mobilization) or by using next generation harvesting tools (eg, the Marrow Miner).

[0070] In addition, HSCs may also be derived from induced pluripotent stem cells.

[0071] Characteristics of HSC HSCs typically have low forward and side scatter profiles by flow cytometry. Some are metabolically quiescent, as demonstrated by rhodamine labeling, which allows for the determination of mitochondrial activity. 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 CD38 and CD45RA cell surface markers. However, 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," do not accept Hoechst 33342 dye, which is detected by flow cytometry. Thus, HSCs have descriptive characteristics that allow for their identification and isolation.

[0072] Negative markers CD38 is the most established and useful single negative marker for human HSCs.

[0073] Human HSCs may also be negative for lineage 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 enrichment of HSCs.

[0074] By "negative markers" it is understood that human HSCs do not express these markers.

[0075] Positive markers CD34 and CD133 are the most useful positive markers for HSCs.

[0076] Some HSCs are also positive for lineage markers such as CD90, CD49f, and CD93, however, these markers may need to be used in combination to enrich for HSCs.

[0077] By "positive markers" it is understood that human HSCs express these markers.

[0078] In some embodiments, the HSCs have a CD34+ phenotype.

[0079] In some embodiments, the HSCs have a CD34+CD38- phenotype.

[0080] Further separation may be performed, for example, to obtain CD34+CD38-CD45RA-CD90+CD49f+ cells.

[0081] stem cell function As used herein, the term "stem cell function" refers to characteristics typically associated with stem cells, such as the ability to engraft, the ability to differentiate into specific cell lineages, and / or the ability to self-renew.

[0082] As used herein, the term "engraftment" refers to the ability of hematopoietic stem cells and / or hematopoietic progenitor cells to establish and survive in a subject following transplantation, i.e., short-term and / or long-term after transplantation. For example, engraftment can refer to the number and / or percentage of hematopoietic cells (e.g., graft-derived cells) derived from transplanted hematopoietic stem cells and / or hematopoietic progenitor cells detected at about 1 day to 24 weeks, 1 day to 10 weeks, or 1 day to 30 days, or 10 days to 30 days after transplantation. In some embodiments, engraftment is assessed at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days after transplantation. In other embodiments, engraftment is assessed at about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, or about 24 weeks after transplantation. In other embodiments, engraftment is assessed at about 16 to 24 weeks, preferably 20 weeks, after transplantation.

[0083] Engraftment can be easily analyzed by one of skill in the art. For example, the transplanted hematopoietic stem and / or progenitor cells can be engineered to contain a marker (e.g., a reporter protein such as a fluorescent protein), which can be used to quantitate graft-derived cells. Samples for analysis can be extracted from the relevant tissue and analyzed ex vivo (e.g., using flow cytometry).

[0084] As used herein, the term "self-renewal" refers to the ability of a cell to undergo multiple cycles of cell division while maintaining an undifferentiated state.

[0085] The number and / or percentage of cells in a state (e.g., live, dead, or apoptotic cells) can be quantified using any of a number of methods known in the art, such as the use of a hemocytometer, an automated cell counter, a flow cytometer, and a fluorescence-activated cell sorter. These techniques may allow for the differentiation of live, dead, and / or apoptotic cells. Additionally or alternatively, apoptotic cells can 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, which binds to exposed PS; apoptotic cells can be quantified by the use of fluorescently labeled Annexin V), and such assays may be used to complement other techniques.

[0086] Hematopoietic stem and / or progenitor cells, as well as cells differentiated therefrom, can be identified and / or quantified using the characteristics and / or markers disclosed herein (eg, CD34 and CD38).

[0087] "Improved stem cell function" may refer to improved stem cell function, e.g., engraftment, self-renewal, and / or differentiation, compared to stem cell function in the absence of a urolithin. One of skill in the art can readily analyze stem cell function, for example, using the methods disclosed herein (e.g., disclosed in the Examples).

[0088] Stem cell function (e.g., self-renewal and / or differentiation) can also be determined using a colony forming unit (CFU) assay as disclosed in the Examples herein. For example, prior to performing a CFU assay on each of the populations of HSPCs, an experiment can be performed in which the HSPC populations are cultured in the presence or absence of urolithin, but under otherwise substantially identical conditions. The level of stem cell function can be determined by analyzing the number of colonies in each CFU assay.

[0089] Stem cell function (e.g., engraftment, self-renewal and / or differentiation) may be determined using in vivo transplantation assays as disclosed in the Examples herein. For example, experiments may be performed in which a population of human HSPCs is cultured in the presence or absence of urolithins, but under otherwise substantially identical conditions, prior to transplantation of the population into irradiated mice. Engraftment may be determined, for example, by analyzing the number of human cells in the mice, as disclosed herein. Self-renewal and / or differentiation may be determined, for example, by analyzing blood reconstitution levels, particularly blood reconstitution levels over time, as disclosed herein. Blood may be further analyzed for levels of specific blood cell lineages.

[0090] Improved stem cell function (e.g., engraftment, self-renewal and / or differentiation) can be at least about a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500% improvement in stem cell function compared to stem cell function in the absence of urolithin. Improved stem cell function can be at least about a 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold improvement in stem cell function compared to stem cell function in the absence of urolithin.

[0091] The improvement of stem cell function over a period of time (e.g., at least 40 weeks, 41 weeks, 42 weeks, 43 weeks, preferably at least 44 weeks) can be determined by analyzing stem cell function by blood chimerism analysis over that period. For example, an in vivo transplantation assay can be performed in which stem cell function as disclosed herein is analyzed over a relevant period of time. The in vivo transplantation assay can be a primary transplantation assay, e.g., a HSPC population can be transplanted into a mouse and then analyzed as disclosed herein. To perform analysis over a longer period of time, the in vivo transplantation assay can be a serial transplantation assay, e.g., a HSPC population can be transplanted into a first mouse and then analyzed over a first period of time. Then, a HSPC population is extracted from the first mouse, and the extracted HSPC population is transplanted into a second mouse and then analyzed over a second period of time. The sum of the first period and the second period of time can result in an analysis of stem cell function over a longer period of time than may be achievable using, for example, a primary transplantation assay alone.

[0092] Isolation and enrichment of cell populations Disclosed herein are cell populations, such as hematopoietic stem and / or progenitor cells (HSPCs). In some embodiments, the cell population is an isolated cell population.

[0093] As used herein, the term "isolated cell population" refers to a population of cells that is not contained within the body. The isolated cell population may have been previously removed from a subject. The isolated cell population may 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 originally isolated, or a different subject.

[0094] From a cell population, cells exhibiting a specific phenotype or characteristic can be selectively purified from other cells that do not exhibit, or exhibit to a lesser extent, that phenotype or characteristic. For example, a cell population expressing a particular marker (such as CD34) can be purified from the starting cell population. Alternatively, or in addition, a cell population that does not express another marker (such as CD38) can be purified.

[0095] As used herein, the term "enrichment" refers to increasing the concentration of one cell type within a population. The concentrations of other cell types may be concomitantly decreased.

[0096] Purification or enrichment may result in a substantially pure cell population of the other cell type.

[0097] Purification or enrichment of a cell population expressing a specific marker (eg, CD38 or CD34) can be achieved by using a reagent that binds to that marker, preferably a reagent that is substantially specific for that marker.

[0098] The reagent that binds to a cell marker may be an antibody, such as an anti-CD34 antibody or an anti-CD38 antibody.

[0099] As used herein, the term "antibody" refers to intact antibodies or antibody fragments capable of binding to a selected target, including Fv, ScFv, F(ab') and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies, such as chimeric, CDR-grafted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative technologies.

[0100] In addition, alternatives to typical antibodies, such as "avibodies", "avimers", "anticalins", "nanobodies" and "DARPins" may also be used in the present invention.

[0101] The reagent that binds to the specific marker may be identifiably labeled using any of a number of techniques known in the art. The reagent may be inherently labeled or may be modified by conjugating a label to the reagent. By "conjugate" it is understood that the reagent and the label are operably linked. This means that the reagent and the label are linked so that both can perform their functions (e.g., bind to the marker, allow identification by fluorescence, or be separated when placed in a magnetic field) without substantial hindrance. Suitable methods of conjugation are well known in the art and can be easily identified by those skilled in the art.

[0102] The label allows, for example, for the labeled reagent and any cells to which it is linked to be purified from their environment (e.g., the agent may be labeled with a magnetic bead or an affinity tag such as avidin), detected, or both. Detectable markers that are suitable 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 tag, Myc tag, FLAG tag, and HA tag).

[0103] Numerous techniques are known in the art for isolating cell populations expressing specific markers, including magnetic bead-based separation techniques (e.g., closed-loop magnetic bead separation), flow cytometry, fluorescence activated cell sorting (FACS), affinity tag purification (e.g., affinity columns or beads, e.g., biotin columns for separating avidin-labeled reagents), and microscopy-based techniques.

[0104] Separation can also be performed using a combination of different techniques, for example a magnetic bead-based separation step followed by flow cytometric sorting of the resulting cell population for one or more additional (positive or negative) markers.

[0105] 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.

[0106] It is also envisioned that dye exclusion properties (eg, side population or rhodamine labeling) or enzymatic activity (eg, ALDH activity) can be used to enrich for HSCs.

[0107] Urolithin Urolithins are metabolic products of food-derived ellagic acid derivatives such as ellagitannins and are produced in the human intestine by gut bacteria.

[0108] Ellagitannins are a class of antioxidant polyphenols present in several fruits, especially pomegranates, strawberries, raspberries, and walnuts. Ellagitannins are very poorly absorbed, but are rapidly metabolized to urolithins by the gut microbiota in the large intestine.

[0109] Due to their superior absorption, urolithins are believed to be the bioactive molecules that mediate the effects of ellagitannins. For example, urolithins have previously been shown to have antioxidant and anti-inflammatory properties.

[0110] Examples of urolithins include urolithin A (3,8-dihydroxyurolithin), urolithin B (3-hydroxyurolithin), and urolithin D (3,4,8,9-tetrahydroxyurolithin), urolithin A glucuronide, and urolithin B glucuronide.

[0111] Urolithin A (UroA) has the following structure: [ka]

[0112] In some embodiments, HSPCs are contacted with urolithin at a urolithin concentration 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, HSPCs are contacted with urolithin at a urolithin concentration 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, HSPCs are contacted with urolithin at a urolithin concentration 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, HSPCs are contacted with urolithin at a urolithin concentration 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.

[0113] In a preferred embodiment, HSPCs are contacted with a urolithin at a urolithin concentration of between 20 μM and 50 μM.

[0114] The urolithins of the invention may exist as salts or esters, particularly pharma- ceutically acceptable salts or esters.

[0115] Pharmaceutically acceptable salts of the agents of the present invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts can be found in Berge et al. (1977) J Pharm Sci 66:1-19.

[0116] The present invention also includes, where appropriate, all enantiomers and tautomers of the reagents. Those skilled in the art will recognize compounds that have optical properties (e.g., one or more chiral carbon atoms) or tautomeric properties. The corresponding enantiomers and / or tautomers can be isolated / prepared by methods known in the art.

[0117] Pharmaceutical and nutritional compositions In some embodiments, the urolithin is in the form of a pharmaceutical composition.

[0118] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, diluent, or excipient.

[0119] In some embodiments, the hematopoietic stem and / or progenitor cells (HSPCs) are in the form of a pharmaceutical composition.

[0120] The cells of the invention can be formulated for administration to a subject with a pharma- ceutically acceptable carrier, diluent, or excipient. Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline, optionally containing human serum albumin.

[0121] Handling of cell therapy products is preferably performed in accordance with the FACT-JACIE International Standard for Cell Therapy.

[0122] In some embodiments, the urolithin is in the form of a nutritional composition.

[0123] In some embodiments, the urolithin is in the form of a food product, dietary supplement, nutraceutical, food for special medical purposes (FSMP), nutritional supplement, dairy drink, small volume liquid nutritional supplement, or meal replacement drink. In some embodiments, the composition is an infant formula.

[0124] In some embodiments, the urolithin is in the form of a food additive or pharmaceutical.

[0125] The food additive or medicine may be in the form of, for example, a tablet, capsule, lozenge, or liquid. The food additive or medicine is preferably provided as a sustained release formulation, allowing for a constant supply of urolithin or its precursors over an extended period of time.

[0126] The composition may be selected from the group consisting of: milk powder based products; instant beverages; ready-to-drink formulations; nutritional powders; nutritional liquids; milk based products, in particular yoghurt or ice cream; cereal products; beverages; water; coffee; cappuccino; malt beverages; chocolate flavoured beverages; cooked products; soups; tablets; and / or syrups.

[0127] The compositions may further contain protective hydrocolloids (gums, proteins, modified starches, etc.), binders, film formers, encapsulants / encapsulants, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavoring agents, bulking agents, jellifying agents, gel forming agents, antioxidants, and antimicrobial agents.

[0128] Additionally, the compositions may contain vitamins, minerals, trace elements and other micronutrients as recommended by government agencies such as the USRDA, in addition to organic or inorganic carrier materials suitable for oral or enteral administration.

[0129] The compositions of the present invention may contain a protein source, a carbohydrate source and / or a lipid source.

[0130] Any suitable food-derived protein may be used, such as animal proteins (such as milk proteins, meat proteins, and egg proteins); vegetable proteins (such as soybean proteins, wheat proteins, rice proteins, and pea proteins); mixtures of free amino acids; or combinations thereof. Milk proteins such as casein and whey proteins, and soybean proteins are particularly preferred.

[0131] If the composition includes a fat source, the fat source preferably provides 5% to 40% of the energy of the formula; 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.

[0132] The carbohydrate source more preferably provides 40% to 80% of the energy of the composition. Any suitable carbohydrate may be used, such as sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, and mixtures thereof.

[0133] hematopoietic stem cell transplantation The invention provides cell populations of hematopoietic stem and / or hematopoietic progenitor cells prepared according to the methods of the invention for use in methods of treatment.

[0134] The use may be part of a hematopoietic stem cell transplantation procedure.

[0135] Hematopoietic stem cell transplantation (HSCT) is the transplantation of blood stem cells derived from bone marrow (in this case known as bone marrow transplant) or blood. Stem cell transplantation is a medical procedure in the fields of hematology and oncology, and is most often performed in people with blood or bone marrow disorders or certain types of cancer.

[0136] Many recipients of HSCT are patients with multiple myeloma or leukemia who would not benefit from long-term treatment with chemotherapy or who are already resistant to chemotherapy. Candidates for HSCT include pediatric cases in which the patient has a congenital defect, such as severe combined immunodeficiency or congenital neutropenia with stem cell deficiency, 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, desmoplastic small round cell tumor, and Hodgkin's disease. More recently, non-myeloablative so-called "mini-transplant" procedures have been developed that require lower doses of preoperative chemotherapy and radiation. This development has allowed HSCT to be performed in patients, such as the elderly, who were considered too frail to tolerate traditional treatment regimens.

[0137] In some embodiments, the hematopoietic stem and / or progenitor cells are administered as part of an autologous stem cell transplant procedure.

[0138] In other embodiments, the hematopoietic stem and / or progenitor cells are administered as part of an allogeneic stem cell transplant procedure.

[0139] In an "autologous stem cell transplant procedure," it is understood that the cell population from which the manipulation begins (i.e., prior to contact with the reagents of the invention) is obtained from the same subject as the subject to whom the final cell population will be administered. Autologous transplant procedures are advantageous because they avoid problems associated with immune incompatibility and can be utilized for subjects regardless of the possibility of finding a genetically compatible donor.

[0140] In an "allogeneic stem cell transplant procedure," it is understood that the cell population from which the procedure begins (i.e., prior to contact with the reagents of the invention) is obtained from a subject that is different from the subject to which the final cell population will be administered. Preferably, the donor will be selected to be genetically compatible with the subject to which the cells will be administered, to minimize the risk of immune incompatibility.

[0141] Treatment method It will be understood that all references made herein to "treatment" include curative, palliative and prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatment are within the scope of the present invention.

[0142] Administration The agents used in the present invention may be administered alone but, particularly in human therapy, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent.

[0143] In some embodiments, the urolithin is present in a combined formulation for simultaneous, separate, or sequential use with an agent selected from the group consisting of nicotinamide riboside, a G-CSF analog, a TPO receptor analog, and combinations thereof.

[0144] As used herein, the term "combination" or the terms "in combination," "used in combination with," or "combined preparation" refers to the simultaneous, sequential, or separate combined administration of two or more agents.

[0145] As used herein, the term "concurrently" means that the reagents are administered simultaneously, i.e., at the same time.

[0146] As used herein, the term "sequential" means that one agent is administered after the other.

[0147] As used herein, the term "separately" means that the reagents are administered independently of each other, but within a time interval that allows the reagents to exhibit a combined, preferably synergistic, effect. Thus, "separately" administration can allow for one reagent to be administered, for example, within 1 minute, 5 minutes, or 10 minutes after the other.

[0148] Dosage Those skilled in the art can easily determine the appropriate dosage of one of the reagents of the present invention to be administered to a subject without undue experimentation.Typically, a physician will determine the actual dosage that is most suitable for an individual patient based on a variety of factors, including the activity of the specific reagent used, metabolic stability and duration of action of the reagent, age, body weight, general health, sex, dietary requirements, method and time of administration, excretion rate, drug combination, severity of the particular condition, and the individual being treated.Of course, there may be individual cases where higher or lower dosage ranges are superior, and such cases are within the scope of the present invention.

[0149] subject In some embodiments, the subject is a human or a non-human animal.

[0150] Examples of non-human animals include vertebrates, e.g., mammals such as non-human primates (especially higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs, and cats. The non-human animal may be a companion animal.

[0151] Preferably, the subject is a human.

[0152] The present invention may be useful, for example, to increase the production of blood cells in a subject.

[0153] The present invention may be useful, for example, to increase blood cell levels in a subject.

[0154] In some embodiments, the subject has, or is at risk for having, subnormal amounts of hematopoietic cells, eg, red blood cells, white blood cells, and / or platelets.

[0155] The normal range for white blood cells in humans is 4500 / μL to 10000 / μL. The normal range for red blood cells in men is 5 million / μL to 6 million / μL, and for women is 4 million / μL to 5 million / μL. The normal range for platelets is 140000 to 450000 per μL. Blood cell levels, sometimes referred to as blood counts, can also be readily measured by one of skill in the art using any of a number of techniques known in the art, such as hemocytometers and automated blood analyzers.

[0156] In some embodiments, the subject has or is at risk for anemia, leukopenia and / or thrombocytopenia.

[0157] In some embodiments, subnormal amounts of hematopoietic cells are secondary to a primary or autoimmune disease of the hematopoietic system, such as congenital bone marrow failure syndromes, idiopathic thrombocytopenia, aplastic anemia, and myelodysplastic syndromes.

[0158] Subjects at risk of developing reduced blood cell levels include patients suffering from anemia or myelodysplastic syndrome, patients undergoing chemotherapy, bone marrow transplantation or radiation therapy, and patients suffering from autoimmune cytopenias, including, but not limited to, immune thrombocytopenic purpura, pure red cell aplasia, and autoimmune neutropenia.

[0159] Subjects at risk for developing post-transplant complications include those who have been hematopoietic cell-depleted and have undergone autologous or allogeneic hematopoietic stem cell or progenitor cell transplantation with primary HSPCs or in vitro manipulated HSPCs.

[0160] In some embodiments, the subject may have undergone myeloablative conditioning, chemotherapy, radiation therapy, and / or surgery, which may result in less than normal amounts of hematopoietic cells.

[0161] Subjects having or at risk of developing subnormal amounts of hematopoietic cells include subjects suffering from blood cancers (e.g., leukemia, lymphoma, and myeloma), blood diseases (e.g., hereditary anemias, inborn errors of metabolism, aplastic anemia, beta-thalassemia, Blackfan-Diamond syndrome, globoid cell leukodystrophy, sickle cell anemia, severe combined immunodeficiency, X-linked lymphoproliferative syndrome, Wiskott-Aldrich syndrome, Hunter syndrome, Hurler syndrome, Lesch-Nyhan syndrome, osteopetrosis), and subjects undergoing chemotherapy for immune system and other diseases (e.g., autoimmune diseases, diabetes, rheumatoid arthritis, systemic lupus erythematosus). Further subjects having or at risk of developing subnormal amounts of hematopoietic cells include subjects exhibiting severe neutropenia and / or severe thrombocytopenia and / or severe anemia, such as post-transplant subjects or subjects undergoing ablative chemotherapy for solid tumors, patients suffering from toxic, drug-induced or infectious hematopoietic disorders (i.e., benzene derivatives, chloramphenicol, B19 parvovirus, etc.), as well as patients suffering from myelodysplastic syndromes, patients suffering from severe immune disorders, or patients suffering from congenital blood disorders, whether of central (i.e., Fanconi anemia) or peripheral origin (i.e., G6PDH deficiency).

[0162] The present invention may be useful, for example, in the treatment or prevention of anemia, leukopenia and / or thrombocytopenia, infectious diseases (e.g., non-viral or viral infections), and / or cancer, such as a blood cancer (e.g., leukemia, lymphoma, or myeloma).

[0163] The reagents, compositions, and cell populations of the present invention may be useful in the treatment of diseases listed in WO 1998 / 005635, a partial list of which is provided here for ease of reference: cancer, inflammation or inflammatory diseases, dermatological disorders, fever, cardiovascular effects, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infections, HIV infection, shock states, graft versus host reaction, autoimmune diseases, reperfusion injury, meningitis, migraine and aspirin-dependent antithrombosis; tumor growth, invasion and spread, angiogenesis, metastasis, malignant tumors, ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease, osteoarthritis, joint inflammation, osteoporosis ... 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.

[0164] Additionally, or alternatively, the reagents, compositions and cell populations of the invention may be useful in the treatment of the diseases listed in WO 1998 / 007859. For ease of reference, a partial list is provided here: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity (e.g., for treating immune deficiencies including human immunodeficiency virus infection; regulating lymphocyte proliferation; treating cancer and many autoimmune diseases, and preventing transplant rejection or inducing tumor immunity); regulating hematopoiesis, e.g., treating myeloid or lymphoid diseases; promoting the growth of bone, cartilage, tendon, ligament and nerve tissue, e.g., for wound healing, burns, ulcers and the treatment of periodontal disease and neurodegeneration; inhibiting or activating follicle-stimulating hormone (regulating fertility); chemotactic / chemokinetic activity (e.g., to recruit specific cell types to sites of injury or infection); 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., as a metabolism or behavior modulator; as an analgesic; treating certain deficiencies; in human or veterinary medicine, e.g., treating psoriasis.

[0165] Additionally or alternatively, the reagents, compositions and cell populations of the invention may be useful in the treatment of diseases listed in WO 1998 / 009985, a partial list of which is provided here for ease of reference: macrophage inhibitory activity and / or T cell inhibitory activity, and thus anti-inflammatory activity; anti-immune activity, i.e., inhibitory effects on cellular and / or humoral immune responses, including responses without inflammation; inhibition of the adhesive 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, Inflammation associated with systemic lupus erythematosus, collagen disease and other autoimmune diseases, atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, inflammatory vascular disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcers, ulcerative colitis and other gastrointestinal diseases, liver fibrosis, liver cirrhosis or other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urinary diseases, otitis or other ear, nose and throat diseases, dermatitis or other skin diseases, periodontal disease or other dental diseases, orchitis / epididymo-orchitis, infertility, testicular trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and / or inflammatory related gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation (e.g. retinitis or cystoid macular oedema), sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fundus diseases, inflammatory components of ocular injuries, infections Ophthalmitis due to glaucoma, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring (e.g., following glaucoma filtration surgery), immune and / or inflammatory responses to ocular implants, other immune and inflammation-related eye diseases, inflammation associated with autoimmune diseases or conditions or disorders, both in the central nervous system (CNS) or other organs, where suppression of immunity and / or inflammation is beneficial, Parkinson's disease, complications of Parkinson's disease and / or side effects associated with treatment, AIDS-related dementia complex HIV-related encephalopathyencephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, Inflammatory component of Stokes' disease, Post-polio syndrome, Immune and inflammatory components of psychiatric illness, Myelitis, Encephalitis, Encephalomyelitis, Acute neuropathies, Subacute neuropathies, Chronic neuropathies, Guillain-Barre syndrome, Sydenham's chorea suppressing or inhibiting humoral and / or cellular immune responses by inhibiting unwanted immune responses and inflammation including those caused by chronic myelopathy, myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, the inflammatory component of CNS compression or CNS trauma or CNS infection, the inflammatory component of muscular atrophy and muscular dystrophies, and immune- and inflammatory-related diseases, conditions or disorders of the central and peripheral nervous system, post-traumatic inflammation, septic shock, infections, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplant complications and / or side effects, inflammatory and / or immune complications and side effects of gene therapy (e.g., due to infection with viral carriers, inflammation associated with AIDS, etc.); reducing the amount of monocytes or lymphocytes to treat or ameliorate monocyte or leukocyte proliferative disorders, e.g., leukemia; preventing and / or treating graft rejection in the case of transplantation of natural or artificial cells, tissues or organs, such as corneas, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

[0166] Amplification method and culture medium In another aspect, the invention provides a method of expanding an isolated population of hematopoietic stem and / or progenitor cells (HSPCs), comprising contacting the population with a urolithin, whereby stem cell function of the HSPCs is enhanced for at least 40 weeks.

[0167] In some embodiments, the contacting step comprises culturing the cell population in the presence of the urolithin.

[0168] In some embodiments, the method comprises: (a) providing a population of HSPCs; (b) optionally culturing the HSPC population, preferably in an HSPC expansion or maintenance medium; (c) optionally isolating a subpopulation of HSPCs characterized by a low mitochondrial membrane potential; (d) contacting the cell population of (a) or (b), or the cell subpopulation of (c), with a urolithin; Includes.

[0169] In some embodiments, the cell population provided in step (a) is obtained from bone marrow, mobilized peripheral blood, or umbilical cord blood.

[0170] In some embodiments, the product of step (d) is enriched for cells with long-term multilineage blood reconstituting potential.

[0171] 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 medium described in the Examples herein below or the medium described in Boitano et al. (2010) Science 329:1345-1348.

[0172] In another aspect, the invention provides a cell culture medium comprising a urolithin.

[0173] In some embodiments, the medium comprises cytokines and growth factors, which 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, angiopoietins and angiopoietin-like family (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).

[0174] Membrane potential, particularly mitochondrial membrane potential, in the HSC compartment 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).

[0175] kit In another aspect, the invention provides kits comprising the reagents and / or cell populations of the invention.

[0176] The cell population may be provided in a suitable container.

[0177] The kit may also include instructions for use.

[0178] It is understood that a person skilled in the art can combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0179] Preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0180] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology which are within the capabilities of those skilled in the art and are explained 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; 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

[0181] Example 1 Results and Discussion UroA leads to a decrease in mitochondrial membrane potential.

[0182] We first tested the effect of UroA on bone marrow-derived mouse HSCs (mHSCs) (Figure 1A). Freshly isolated mHSCs (LKS CD150+CD48-) were cultured in basal medium (Stemline+SCF+FLT3L+Penicillin / Streptomycin) supplemented with different concentrations of UroA. Cells were harvested on day 3, stained using tetramethylrhodamine methyl ester (TMRM; to measure mitochondrial membrane potential) and Mitotracker (to measure mitochondrial mass), and analyzed by flow cytometry.

[0183] TMRM with increasing doses of UroA low A stepwise increase in the percentage of cells within the gate was found, resulting in a significant decrease in TMRM fluorescence intensity (mean fluorescence intensity, MFI) (Figure 1A, top). Mitotracker staining showed that mitochondrial mass was reduced at all concentrations of UroA. A significant decrease occurred at 20 μM (Figure 1A, bottom). Next, the effect of UroA on human umbilical cord blood-derived hematopoietic stem and progenitor cells (hHSPCs) was examined (Figure 1B). Cryopreserved hHSPCs (CD34+) were thawed and cultured in basal medium (Stemspan+SCF+FLT3L+TPO+LDLP+penicillin / streptomycin) supplemented with different concentrations of UroA. Aliquots of cells were harvested on days 3, 5, and 7, followed by staining for CD34 and TMRM and analysis by flow cytometry. At all three time points, the increase in TMRM mass with increasing doses of UroA was significantly greater than that with increasing doses of UroA. low It was confirmed that the proportion of cells within the gate increased, and at the same time, the TMRM signal (median fluorescence intensity, MFI) decreased (Figure 1B).

[0184] In vitro UroA treatment enhances the in vivo function of mHSCs and hHSPCs.

[0185] Since a reduction in mitochondrial membrane potential has previously been shown to enhance HSC function (Vannini, N. et al. (2016) Nat Commun 7:13125), we investigated whether UroA treatment would improve the in vivo reconstitution potential of HSCs. To this end, freshly isolated mHSCs were cultured in basal medium with or without UroA (20 μM). At the end of the culture period (3 days), cells were counted and injected into lethally irradiated recipient mice (Figure 2A). Recipient blood analysis showed higher reconstitution levels in mice injected with UroA-treated cells (Figure 2A). This trend was evident in both myeloid and lymphoid lineages of blood (Figure 2A).

[0186] Next, we cultured cord blood-derived human HSPCs with or without UroA (50 μM) and performed two functional assays: colony-forming unit (CFU) assay—after 7 days of culture, and NSG-SGM3 neonatal in vivo transplantation assay—after 5 days of culture (Figure 2B). UroA-treated cells formed significantly higher numbers of colonies in methylcellulose CFU assay plates (Figure 2C), indicating improved stem cell and progenitor function of hHSCs exposed to UroA. In a second assay, blood analysis of NSG-SGM3 mice transplanted with cultured cells showed increased human graft engraftment (both percentage and absolute numbers) in UroA-treated conditions (Figure 2D). Furthermore, we analyzed different human blood lineages and found greater human cell numbers under UroA conditions, mainly in lymphoid lineages (T and B cells) (Figure 2E). These data demonstrate that treatment with UroA enhances HSC function.

[0187] UroA drives the expression of metabolic genes in mHSCs.

[0188] To analyze the molecular mechanism by which UroA enhances HSC function, we performed gene expression analysis of mHSCs cultured in basal medium with or without UroA (20 μM). Fold change (ΔΔCT) analysis showed that the expression of autophagy genes (ATG5, PARK2), glycolysis genes (HK2, Glut1), and genes involved in protection from ROS (Fox1, SOD2) was increased under UroA treatment (Figure 3). This suggests that autophagy and protection from ROS are key drivers of HSC self-renewal (Takubo, K. et al. (2013) Cell Stem Cell 12:49-61; Vannini, N. et al. (2016) Nat Commun 7:13125; Ito, K. et al. (2006) Nat Med 12:446-51; Warr, MR et al. (2013) Nature 494:323-327; Ito, K. et al. (2016) Science 354:1156-1160) as well as upregulated glycolysis, a key metabolic pathway that maintains HSC stemness (Takubo, K. et al. (2013) Cell Stem Cell 12:49-61; Yu, WM et al. (2013) Cell Stem Cell 12:62-74), which is consistent with the inventors' previous studies and various literature.

[0189] In summary, our findings demonstrated the ability of UroA to restore HSC function through regulating mitochondrial membrane potential by inducing mitophagy, which leads to the application of UroA in HSC transplantation for the treatment of hematological malignancies.

[0190] Materials and Methods Flow cytometry Flow cytometry analysis was performed on freshly isolated bone marrow (BM) from C57Bl6 mice. BM was extracted from crushed femurs and tibiae. The cell suspension was filtered through a 70 μm cell strainer and red blood cells were removed by incubation with red blood cell lysis buffer (eBioscences). Isolation and staining were performed in ice-cold 1 mM EDTA / PBS. Lineage positive cells were then removed using magnetic lineage depletion (BD biosciences). The cell suspension was then stained with specific antibodies against the stem cell compartment and sorted by FACS (BD FACS Aria III) into 1.5 mL Eppendorf tubes.

[0191] antibody The following antibodies were used in this study: rat mAbs against cKit (2B8), Sca1 (D7), CD150 (TC-15-12F12.2), CD48 (HM48-1), CD45.2 (104), CD45.1 (A20), Gr1 (RB6-8C5), F4 / 80 (BM8), CD19 (6D5), CD3 (17A2), CD16 / CD32 (2.4G2). 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 lineage markers ("lineage cocktail") and was purchased from BD. Human specific antibodies were obtained from either eBioscience or BD: hCD56 (NCAM16.2), hCD16 (3G8), hCD45 (HI30), hCD19 (HIB19), hCD4 (RPA-T4), hCD3 (SK7), hCD14 (M5E2), hCD8b (SIDI8BEE), hCD34 (8G12), hCD38 (HB-7). DAPI or propidium iodide (PI) staining was used to distinguish live / dead cells.

[0192] Culture of mHSCs and hHSPCs 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). Different concentrations of UroA (dissolved in DMSO) were added as indicated, and an equal volume of DMSO was added to control wells.

[0193] CD34+ cells isolated and cryopreserved from fetal liver / cord blood were thawed and cultured in vitro in StemSpan (Stem cell tech) medium supplemented with hSCF (100ng / mL), hFLT3L (100ng / mL), hTPO (50ng / mL), hLDLP (10μg / mL), and different concentrations of UroA (dissolved in DMSO). Equivalent amounts of DMSO were added to control wells. For longer culture periods, half of the medium was replenished every 2 or 3 days.

[0194] Analysis of mitochondrial activity Already cultured mouse HSCs were incubated with 200 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen) and 100 nM Mitotracker green for 1 hour at 37° C. Cells were then washed with FACS buffer and analyzed by flow cytometry on a BD LSR II.

[0195] Precultured human HSCs were incubated with 200 nM TMRM (Invitrogen) for 1 hour at 37° C. Cells were then washed with FACS buffer and subsequently stained with CD34 antibody for 1 hour at 4° C. Cells were washed with FACS buffer and analyzed by flow cytometry on a BD LSR II.

[0196] Mouse and humanized transplants C57Bl / 6 Ly5.2 mice were lethally irradiated with a total dose of 8 Gy in a gamma radiator 24 hours before transplantation. Mice were injected with 200 cultured donor cells from C57Bl / 6 Ly5.1 mice and 200,000 competitor cells from C57Bl / 6 Ly5.1 / 5.2 mice via tail vein injection. Peripheral blood was collected every few weeks and the percentage of chimerism was calculated by FACS analysis.

[0197] NSG mice were purchased from Jackson Laboratory and bred and maintained in a pathogen-free room in the research facility. For transplantation, 1-day-old NSG pups were irradiated with 1 Gy (RS-2000, RAD source) and several hours later, intrahepatically injected with in vitro expanded HSCs. Each pup was injected with a cell mass derived after in vitro culture of an initial 50,000 CD34+ cells. Mice were bled at 12 weeks to estimate the level of human reconstitution (% of human CD45+ cells) in the peripheral blood. A combination of antibodies was used to further estimate human B cells, T cells, monocytes, neutrophils, and NK cells.

[0198] CFU assay CFU assays were performed using H4434 (Stem cell tech) according to the manufacturer's instructions. 1000 cells were seeded in duplicate from each well. 15 days after seeding, colonies were counted using Stem Vision (Stem cell tech).

[0199] QPCR RNA was extracted from cultured HSCs using ZR RNA MicroPrep (Zymo Research). RNA extraction was performed according to the manufacturer's instructions. st RNA was reverse transcribed into cDNA using a 3-strand cDNA kit (TAKARA) according to the manufacturer's instructions.

[0200] For qPCR, 0.5 μL of cDNA, 5 μL of Power Syber Green master mix (Applied Biosystem) and 500 nM primers were used in a final volume of 10 μL for each reaction. Reactions were performed on a 7900HT system (Applied Biosystem).

[0201] The mouse primer sequences are as follows:

[0202] [Table 1]

[0203] Example 2 A limited transplantation experiment was designed to determine whether a brief in vitro treatment with UroA could improve survival after transplantation into irradiated recipients. Human umbilical cord blood-derived HPSCs were cultured for 3 days in the absence or presence of UroA. After culture, cells were counted and 40,000 were injected into each recipient mouse (irradiated adult NSG mice). These mice were followed up for several months to determine post-transplant survival. The group of mice transplanted with UroA-treated cells showed a significant improvement in survival, especially in the early stages of post-transplant recovery.

[0204] Example 3 Serial transplantation analyses demonstrate that in vitro UroA treatment enhances HSC function in vivo over the long term.

[0205] HSCs were isolated from mouse bone marrow and cultured in the presence or absence of UroA (Figure 5A). At the end of the culture period, cells were injected intravenously into lethally irradiated primary recipient mice.

[0206] Blood chimerism analysis of the first mouse was then performed over a 24-week period (Figure 5B), followed by analysis of spleen (Figure 5D) and bone marrow (Figure 5E) samples from the first mouse.

[0207] Bone marrow cells were then extracted from the bone marrow of the first mouse and transplanted into a lethally irradiated second recipient mouse via tail vein injection.

[0208] Blood chimerism analysis of the second mouse was then performed over a 20-week period (Figure 5C), followed by analysis of spleen (Figure 5F) and bone marrow (Figure 5G) samples from the second mouse.

[0209] UroA cultured cells show higher blood reconstitution over the entire period of at least 44 weeks, with the increase also being expressed in myeloid and lymphoid lineages.

[0210] Example 4 Gene expression analysis of HSCs from UroA-treated mice.

[0211] To investigate the mechanism by which UroA mediates its effects, we performed RNA sequencing analysis on HSCs after brief ex vivo UroA treatment (Figure 6A). After culture, we first isolated RNA from six control samples (D1-6) and six UroA-treated samples (U1-U6). Due to the limited number of cells, the amount of isolated RNA was found to be very low. However, gel electrophoresis and fragment analyzer analysis confirmed that the quality of the RNA was suitable for RNA sequencing (Figure 6B). One of the control samples (D3) had a large peak at the end of the chromatogram, but we concluded that this peak was an artifact of the fragment analyzer. Furthermore, a multidimensional scaling (MDS) plot of the RNA sequencing data revealed that the UroA samples (U1-6) clustered together, while the control samples appeared to be more dispersed (Figure 6C). Differential expression analysis revealed several candidate genes that were differentially expressed upon UroA treatment ( Fig. 6C , Volcano plot).

[0212] Next, we investigated various biological pathways that were altered by UroA treatment. We found that the response to topologically incorrect proteins and unfolded proteins was significantly upregulated in UroA conditions (Figure 6D, top left). In addition, the response to endoplasmic reticulum unfolded proteins was also significantly upregulated (Figure 6D, top left). Interestingly, we previously showed that the response to unfolded proteins is one of the important pathways that regulate HSC function. Reactome analysis revealed that the activation of mitochondrial biogenesis was downregulated upon UroA treatment (Figure 6D, bottom right). This was consistent with the observed decrease in mitochondrial mass upon UroA treatment.

[0213] Molecular function and cellular component analyses revealed that several candidates involved in epigenetic modifications, such as histone methyltransferase, histone acetyltransferase complex, and histone deacetylase complex, were significantly downregulated (Figure 6D), suggesting that important epigenetic changes occur in HSCs upon UroA exposure.

[0214] Differential expression analysis of mitochondrial genes revealed altered gene expression of several candidates ( Fig. 6E ).

[0215] All publications mentioned in the above specification are incorporated herein by reference.Various modifications and alterations of the compositions, uses and methods disclosed in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.Although the present invention has been disclosed in connection with certain preferred embodiments, it should be understood that the invention claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the disclosed modes for carrying out the invention will be apparent to those skilled in the art and are intended to be within the scope of the following claims.

Claims

1. 1. An in vitro method for improving stem cell function in a population of hematopoietic stem and / or progenitor cells (HSPCs), comprising contacting the population in vitro with a urolithin; the stem cell function comprises one or more selected from the group consisting of immune cell differentiation, blood cell differentiation, engraftment and self-renewal; The method, wherein said stem cell function is enhanced for at least 40 weeks.

2. The following steps: (a) providing a population of HSPCs; (b) optionally isolating a subpopulation of HSPCs characterized by low mitochondrial membrane potential; and (c) contacting the cell population of (a), or the cell subpopulation of (b), with a urolithin.

3. The method of claim 1, wherein the improved stem cell function increases blood cell levels in the subject.

4. The method of claim 1, wherein the urolithin is urolithin A.

5. The method of claim 1, wherein the population or subpopulation of HSPCs is contacted with the urolithin for a period of not more than 7 days.

6. Hematopoietic stem and / or progenitor cells (HSPCs) obtained by the method according to any one of claims 1 to 5.

7. The hematopoietic stem and / or progenitor cells (HSPCs) of claim 6 for use in the treatment or prevention of (a) anemia, leukopenia, and / or thrombocytopenia, (b) infectious disease, and / or (c) cancer in a subject.

8. The hematopoietic stem and / or progenitor cells (HSPCs) described in claim 7 for treating the subject for at least 40 weeks.

9. The hematopoietic stem and / or progenitor cells (HSPCs) of claim 7, wherein the subject has or is at risk of having less than normal amounts of hematopoietic cells, and optionally the hematopoietic cells are red blood cells, white blood cells, and / or platelets.

10. The hematopoietic stem and / or progenitor cells (HSPCs) of claim 7, wherein the subject has or is at risk of having anemia, leukopenia and / or thrombocytopenia.

11. The hematopoietic stem and / or progenitor cells (HSPCs) of claim 7, wherein the subject is undergoing an intervention selected from the group consisting of hematopoietic stem cell transplantation, bone marrow transplantation, myeloablative conditioning, chemotherapy, radiation therapy, and surgery.

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