Method for mobilizing stem cells

Abstract

The present invention has found that soluble P-selectin (sP-sel) can interfere with the interaction between stem cells and the niche and, thus, mobilize stem cells from the bone marrow. Accordingly, mobilizing stem cells with sP-sel can treat an individual in need of one or more of tissue preservation, repair, or regeneration or neovascularization in an individual.

Description

Technical Field

[0001] The present invention generally relates to methods for mobilizing stem cells by administering soluble P-selectin. Background Art

[0002] Stem cells (SCs) are defined as cells having the unique ability to self-renew throughout the life cycle of an organism and differentiate into the various cell types of the body. Two well-known types of stem cells are embryonic stem cells and adult stem cells. Because stem cells are able to differentiate into a wide variety of cell types, they play an important role in the healing and regeneration processes of various tissues and organs. Some stem cells, such as bone marrow stem cells and hematopoietic stem cells, are released from their source tissues and circulate in the circulation or immune system of an individual to migrate into various organs and tissues to become mature terminally differentiated cells. Thus, enhancing stem cell trafficking (i.e., release, circulation, homing, and / or migration) can amplify these physiological processes and provide potential therapies for various pathologies.

[0003] Currently, stem cells are mobilized from the bone marrow to peripheral blood prior to collection for allogeneic stem cell transplantation in a clinical setting rather than bone marrow. The recently approved CXCR4 inhibitor AMD3100 for stem cell mobilization induces more specific mobilization of cells into the circulation compared to G-CSF by disrupting the CXCR4-SDF1 interaction between bone marrow cells and their microenvironment. For example, US 20180142211 uses a CXCR4 antagonist peptide to mobilize mesenchymal stem cells to peripheral blood and then harvests the cells.

[0004] However, there remains a need to develop reagents for stem cell mobilization. Summary of the Invention

[0005] The present invention provides a method for mobilizing circulating stem cells in an individual, which comprises administering to the individual an amount of soluble P-selectin (sP-sel) effective to mobilize a population of hematopoietic stem cells or progenitors. In one embodiment, the soluble P-selectin can interfere with the interaction between stem cells and the niche.

[0006] The present invention also provides a method for treating an individual in need of one or more of tissue preservation, repair or regeneration or vascular reformation in an individual, which comprises administering to the individual an amount of sP-sel effective to mobilize a population of hematopoietic stem cells or progenitors.

[0007] The present invention also provides a method for performing allogeneic hematopoietic stem cell transplantation in a patient in need thereof, the method comprising infusing into the patient a therapeutically effective amount of allogeneic hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from the bone marrow of a human donor to the peripheral blood of the human donor by a method comprising administering an effective amount of sP-sel to the donor.

[0008] In one embodiment, the stem cells are hematopoietic cells, progenitor cells, or bone marrow stem cells.

[0009] In one embodiment, the method further comprises administering a second agent before, after, or simultaneously with the administration of sP-sel. In another embodiment, the second agent is selected from the group consisting of: G-CSF, GM-CSF, IL-3, GM-CSF / IL-3 fusion protein, FLK-2 / FLT-3 ligand, stem cell factor, IL-6, IL-11, TPO, VEGF, AMD3100, and combinations thereof.

[0010] In one embodiment, the amount of sP-sel is from about 10 -5 μg to about 1.5 mg per kilogram of body weight per administration.

[0011] In one embodiment, the sP-sel mobilized circulating stem cells (PselMSC) can produce stem cell-derived extracellular vesicles.

[0012] In one embodiment, PselMSC can improve tissue or organ injury, increase repair, improve glucose tolerance, and / or reduce inflammation. In another embodiment, the tissue injury is liver injury.

[0013] In one embodiment, PselMSC can repopulate the bone marrow or hematopoietic stem cell population.

[0014] In one embodiment, PselMSC can repopulate the bone marrow or hematopoietic stem cell population and rescue tissue injury, proliferative disorders, inflammatory diseases, immunodeficiency diseases, autoimmune disorders, and / or metabolic diseases.

[0015] In one embodiment, sP-sel is a naturally occurring sP-sel or recombinant sP-sel.

[0016] In one embodiment, sP-sel can be further conjugated to a vesicle or liposome.

[0017] The present invention also provides a method for cell therapy in an individual, comprising administering to the individual an amount of sP-sel effective to mobilize stem cells and an amount of stem cells effective for cell therapy. In one embodiment, sP-sel and the stem cells are administered simultaneously, separately, or intermittently.

[0018] Brief Description of the Drawings

[0019] Figure 1 . Soluble P-selectin but not G-CSF treatment induces mobilization of CD34+ stem cells in mice. Recombinant murine soluble P-selectin and G-CSF (Both at 0.1 mg / kg body weight) were intravenously injected twice into 8-week-old male C57Bl / 6J experimental mice at 24-h intervals, and blood samples were collected after another 24 h (A, overview). The content of circulating CD34+ monocytes was measured using flow cytometry (FC) (A), and the vehicle (saline) control group was normalized to 100% (B).

[0020] Figure 2 . Soluble P-selectin rather than G-CSF treatment improves thioacetamide (TAA)-induced thrombocytopenia and liver injury in mice. Recombinant murine soluble P-selectin and G-CSF (Both at 0.1 mg / kg body weight) were intravenously injected twice into 8-week-old male C57Bl / 6J experimental mice at 24-h intervals; and the hepatotoxic drug thrombocytopenia (TAA) was additionally administered for 24 h (A, overview). Platelet (PLT) counts and the content of the circulating liver-specific enzyme aspartate transaminase (AST) were analyzed 48 h after TAA treatment according to the previously described method 1 (B).

[0021] Figure 3 . Soluble P-selectin-mobilized CD34+ cells can rescue thioacetamide (TAA)-induced liver injury. After the aforementioned method, soluble P-selectin-mobilized CD34+ cells and peripheral blood mononuclear cells (PBMCs; monocytes) (both injected at 5×106 cells / mouse) were co-injected with TAA challenge (n = 4; results were statistically significant, P < 0.05 for P-sel vs. "no cell transfer" and "monocyte" groups).

[0022] Figure 4 . The level of circulating Lin-Sca-1+c-Kit+ (LSK) stem cells after soluble P-selectin stimulation. LSK hematopoietic stem cells are the stem cell lineage capable of re-populating bone marrow stem cells after lethal γ-irradiation. The experimental overview is illustrated (A). Soluble P-selectin (0.1 mg / kg) was intravenously injected into C57BL / 6J mice twice daily (n = 5). Peripheral blood (PB) was collected and analyzed using flow cytometry (FC) at once before the first injection and 24 h after the first injection. The absolute number of LSK cells in peripheral blood was quantified (B). Data are reported as mean ± SD. *P < 0.05, compared with the pre-experiment group.

[0023] Figure 5. LSK stem cells mobilized by P-selectin rescue the mortality of γ-irradiated mice, indicating the reconstitution of bone marrow hematopoietic stem cells. C57BL / 6J mice were subjected to lethal-dose γ-irradiation (which is commonly used in bone marrow transplantation) or not. These γ-irradiated mice were further treated with vehicle, LSK stem cells obtained through mobilization mediated by P-selectin or G-CSF treatment. Since both G-CSF and P-selectin-mobilized LSK stem cells can rescue the mortality of γ-irradiated mice, these results indicate the reconstitution of bone marrow hematopoietic stem cells using two methods for preparing LSK stem cells.

[0024] Figure 6 . Improvement of thioacetamide (TAA)-mediated liver injury by P-selectin-mobilized CD34+ stem cells (PselMSC), P-selectin-mobilized CD34+ stem cell-derived microvesicles (PselSCMV), soluble P-selectin, and soluble P-selectin-conjugated liposomes. Treatment with PselMSC and PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes can rescue TAA-mediated liver injury, as indicated by circulating ALT, a hepatocyte-specific enzyme content. **P < 0.01, compared with the normal / vehicle group; #P < 0.05, ##P < 0.01, compared with the TAA group. n = 6.

[0025] Figure 7 . Improvement of glucose tolerance in high-fat diet (HFD)-induced diabetic mice by P-selectin-mobilized CD34+ stem cells (PselMSC), P-selectin-mobilized CD34+ stem cell-derived microvesicles (PselSCMV), soluble P-selectin, and soluble P-selectin-conjugated liposomes. A glucose tolerance test (OGTT) experiment was performed, and plasma glucose levels were measured in different groups of experimental mice.

[0026] *P < 0.05; **P < 0.01, compared with the respective HFD group. n = 6.

[0027] Figure 8 . Anti-inflammatory effect of P-selectin-mobilized CD34+ stem cells (PselMSC), P-selectin-mobilized CD34+ stem cell-derived microvesicles (PselSCMV), soluble P-selectin, and soluble P-selectin-conjugated liposomes on TAA-induced hepatitis in mice. The degree of TAA-induced inflammation was indicated by the circulating TNF-α content in mice; the average TNF-α content in the normal / vehicle group was normalized to 100%. **P < 0.01, compared with the normal / vehicle group; *P < 0.05, **P < 0.01, compared with the TAA group. n = 6. Detailed Description

[0028] Unless otherwise specified, when the definition of a term deviates from its ordinary meaning, the applicant intends to use the definitions provided hereinbelow.

[0029] Unless the context clearly indicates otherwise, the singular forms "a / an" and "the" as used in this specification and the appended claims include plural referents.

[0030] As used herein, unless otherwise stated, the use of "or" means "and / or". In the case of multiple dependent claims, the use of "or" merely re-refers to more than one of the foregoing independent or dependent claims in an alternative manner.

[0031] As used herein, the term "one or more" is readily understood by those skilled in the art, particularly when read in the context in which it is used.

[0032] As used interchangeably herein, the terms "individual", "subject", "host", and "patient" refer to a mammal, including but not limited to murine (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, sheep, suids, goats), and the like.

[0033] As used herein, the term "soluble P-selectin" refers to the naturally occurring soluble form of P-selectin and its recombinant forms, or its polymorphs or allelic variants or other isoforms. The term also encompasses soluble P-selectin, whether modified or unmodified, such as glycosylated or unglycosylated forms.

[0034] As used herein, the terms "mobilize" and "mobilization" refer to the process of release of a population of hematopoietic stem cells or progenitor cells from the stem cell niche.

[0035] As used herein, the term "niche" refers to the in vivo or in vitro cellular and molecular microenvironment that regulates stem cell function along with the stem cell's intrinsic mechanisms. This includes controlling the balance between quiescence, self-renewal, and differentiation, as well as specific programs involved in response to stress.

[0036] As used herein, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that is capable of differentiating into myeloid lineages (i.e., monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, and some dendritic cells) and lymphoid lineages (i.e., T cells, B cells, NK cells, and some dendritic cells).

[0037] As used herein, the term "subject" refers to any animal, including mammals, birds, reptiles, and amphibians, and in a preferred embodiment refers to mammals, including humans, companion animals, food production animals, and wild animals.

[0038] As used herein, the term "donor" refers to a subject from whom one or more cells are isolated prior to administration of the cells or their progeny to a recipient.

[0039] As used herein, the term "effective amount" refers to an amount of one or more agents, such as an amount of soluble P-selectin and / or a second agent described herein, that mobilizes a population of hematopoietic stem or progenitor cells upon administration to a subject.

[0040] Stem cells can generate new cells to repair damage to tissues and thus have great potential for regenerative medicine. However, stem cells are present in tissues, and particularly in peripheral blood, in small numbers, making it difficult to harvest stem cells or to use stem cells clinically. Mobilization of stem cells is a means of collecting stem cells from the bone marrow into the blood. The present invention has carefully discovered that soluble P-selectin (sP-sel) can interfere with the interaction between stem cells and the niche and thus mobilize stem cells from the bone marrow. Accordingly, mobilization of stem cells with sP-sel can treat a subject in need of one or more of tissue preservation, repair or regeneration, or angiogenesis in a subject.

[0041] P-selectin is a member of the selectin family located in the membranes of the α-granules of platelets and in the Weibel-Palade bodies (WP bodies) of endothelial cells. P-selectin exists in two different forms; one is the "cell surface" form and the other is the "soluble" form. The former is expressed on activated platelets or endothelial cells involved in leukocyte inflammation and HSC homing. The latter (i.e., sP-sel) is expressed almost exclusively in the plasma of animals / humans under stress (e.g., hypoxia) (Chang, H.H. and Sun, D.S. Methods of reducing hypoxic stress in a mammal by adminstring soluble P-selectin. U.S. Patent US 8377887 B1 (2012)). Soluble P-selectin molecules present as monomers in the blood are 3 kDa smaller than P-selectin molecules present as oligomers in the membrane. Soluble P-selectin in healthy individuals is derived from alternative splicing forms present in endothelial cells and platelets. The present invention unexpectedly found that treatment with soluble P-selectin can interfere with the interaction between stem cells and the niche, and thus mobilize stem cells. Hematopoietic stem cells and progenitors mobilized in this way can then be withdrawn from the donor and administered to the patient, where these cells can home to the hematopoietic stem cell niche and reconstitute the damaged or insufficient cell population in the patient.

[0042] Any form of sP-sel suitable for stem cell mobilization can be used in the present invention. Examples of sP-sel of the present invention include, but are not limited to, naturally occurring sP-sel and recombinant sP-sel. sP-sel can be readily obtained by conventional techniques, such as isolation from natural sources, purchase from commercial sources, or synthesis by molecular biotechnology.

[0043] sP-sel can be used in combination with a second reagent to mobilize stem cells. Examples of the second reagent include, but are not limited to, G-CSF, GM-CSF, IL-3, GM-CSF / IL-3 fusion protein, FLK-2 / FLT-3 ligand, stem cell factor, IL-6, IL-11, TPO, VEGF, AMD3100, and combinations thereof. Preferably, the second reagent is G-CSF. sP-sel and the second reagent can be used simultaneously or sequentially.

[0044] sP-sel can be used in combination with stem cells. sP-sel and stem cells are administered simultaneously, separately, or intermittently.

[0045] A hematopoietic stem cell transplantation therapy can be administered to an individual in need of treatment in order to repopulate or re-populate one or more blood cell types, such as blood cell lineages that are deficient or lacking in a patient with a stem cell disorder. Hematopoietic stem cells and progenitors exhibit multi-potency and can therefore differentiate into multiple different blood lineages. Hematopoietic stem cells give rise to different types of blood cells, in lineages known as myeloid and lymphoid. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, red blood cells, and megakaryocytes to platelets. Lymphocytes include T cells, B cells, natural killer cells, and innate lymphocytes.

[0046] Hematopoietic stem cells or progenitors mobilized to the peripheral blood of an individual can be drawn (e.g., harvested or collected) from the individual by any suitable technique. For example, hematopoietic stem cells or progenitors can be drawn by venipuncture. In some embodiments, apheresis can be used to harvest (i.e., collect) hematopoietic stem cells or progenitors mobilized to the peripheral blood of an individual as covered herein. In some embodiments, apheresis can be used to enrich the donor's blood with mobilized hematopoietic stem cells or progenitors.

[0047] The sP-sel disclosed herein can be administered according to various routes, typically by injection, such as local or systemic injection. However, other administration routes can also be used, such as intramuscular, intravenous, intradermal, subcutaneous, etc. For administration, sP-sel is typically combined with one or more adjuvants suitable for the indicated administration route. In addition, repeated injections can be performed if needed. sP-sel is administered in the range of about 10 -5 μg to 1.5 mg per kilogram of body weight.

[0048] Circulating stem cells mobilized by sP-sel (PselMSC) can improve tissue or organ injury, increase repair, improve glucose tolerance and / or reduce inflammation. PselMSC can also repopulate the bone marrow or hematopoietic stem cell population and can rescue tissue injury, proliferative disorders, inflammatory diseases, immunodeficiency diseases, genetic disorders, degenerative disorders, autoimmune disorders and / or metabolic diseases. Examples of proliferative disorders include, but are not limited to, blood cancers and myeloproliferative diseases. Examples of immunodeficiency diseases include, but are not limited to, congenital immunodeficiency diseases and acquired immunodeficiency diseases. Examples of autoimmune disorders include, but are not limited to, juvenile arthritis, ulcerative colitis, Type 1 diabetes mellitus / Type 1 diabetes, multiple sclerosis (MS), inflammatory bowel disease (IBD), psoriasis, psoriatic arthritis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS), and lymphocytic colitis. Examples of metabolic diseases include, but are not limited to, glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurler's Disease, sphingolipid storage diseases, and metachromatic leukodystrophy.

[0049] The recovered hematopoietic stem cells or progenitor cells can be reinfused into a patient such that the cells then return to the hematopoietic tissue and establish productive hematopoiesis, thereby repopulating or replenishing a cell line that is lacking or deficient in the patient.

[0050] Although the present invention has been described with reference to preferred embodiments and examples of the invention, the scope of the invention is not limited solely to the embodiments described herein. As will be apparent to those skilled in the art, modifications and variations can be made to the present invention above without departing from the spirit and scope of the invention as defined and limited by the appended claims. The following examples are provided for the purpose of illustrating the embodiments and advantages of the invention and are not intended to limit its scope.

[0051] Examples

[0052] Example 1 Mobilization of Soluble P-Selectin-Mediated CD34+ Cells

[0053] According to a previous report (Tajima, F., Sato, T., Laver, J. H., & Ogawa, M. CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor. Blood 96, 1989 - 1993 (2000)), mobilization of murine CD34+ cells by G-CSF requires 5 injection doses. Here we show that on day 3 (after 2 G-CSF injection doses), induction of circulating CD34+ cells has not been elicited in mice.

[0054] At each treatment, recombinant murine P-selectin (rmP-sel) and granulocyte-colony stimulating factor (G-CSF; ) were intravenously injected into mice (0.1 mg / kg body weight). Compared to not inducing circulating CD34+ stem cells, rmP-sel treatment elicited a large number of CD34+ cells by only 2 injections (see Figure 1 Overview of experiment A, 1B). These results demonstrate that P-selectin is much more effective than G-CSF in mobilizing circulating CD34+ cells.

[0055] Example 2 Soluble P-selectin ameliorates thioacetamide (TAA)-induced thrombocytopenia and liver injury

[0056] C57BL / 6J mice were injected with TAA to induce acute liver injury and were then rescued with or without rm-Psel ( Figure 2 A, schedule). We found that pretreatment with twice the amount of rmP-sel rescued the decreased platelet count in peripheral blood to normal levels ( Figure 2 B). Aspartate transaminase (AST) enzyme activity is a standard marker for measuring liver injury. The data also revealed that rmP-sel ameliorated liver injury ( Figure 2 C). Our data indicate that rmP-sel treatment exerted a tissue-protective effect to reduce TAA-induced injury (n = 4; results were statistically significant, P < 0.05 for rmP-sel vs. saline).

[0057] It has been previously shown that stem cell treatment may have a beneficial effect on the amelioration of liver injury. To characterize whether the CD34+ cells elicited by rmP-Sel have a tissue-protective effect, we performed adoptive transfer experiments. Our data show that adoptive transfer of P-selectin-mobilized CD34+ stem cells but not peripheral blood mononuclear cells (PBMC) can rescue TAA-induced hepatitis in mice ( Figure 3 ).

[0058] In humans, G-CSF treatment mobilizes CD34+ stem cells, which are capable of re-populating γ-irradiated bone marrow. LSK hematopoietic stem cells in mice are a cell lineage equivalent to CD34+ stem cells in humans. To study the potential role of P-selectin-mobilized LSK cells in bone marrow re-population, lethally γ-irradiated C57Bl / 6 recipient mice were transplanted with 1×105 LSK cells that were mobilized with soluble P-selectin ( Figure 4 ) and G-CSF ( Figure 5 ). As shown by the rescue of 100% lethal γ-irradiation in mice, the grafts were successfully transplanted ( Figure 5 ; 100% lethality in the non-transplanted group versus ~83% survival in both the soluble P-selectin-mobilized LSK and G-CSF-mobilized LSK groups).

[0059] The protective effects of PselMSC and PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes were demonstrated using a TAA hepatitis mouse model. Treatment with PselMSC and PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes significantly rescued the elevated circulating alanine transaminase (ALT) levels induced by TAA in mice, indicating that these agents have ameliorative effects on TAA-induced liver injury.

[0060] Previously, it was shown that adipose-derived mesenchymal stem cells improve glucose homeostasis in high-fat diet-induced obese mice2,3. However, whether PselMSC, PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes are still difficult to achieve. The results of glucose tolerance (OGTT) analysis revealed that PselMSC, PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes all improved high-fat diet (HFD)-induced glucose resistance, thereby reducing the blood glucose levels in HFD-fed mice ( Figure 7 ).

[0061] Circulating TNF-α levels, which reflect the degree of inflammation, were analyzed in TAA-induced hepatitis mice. The data showed that PselMSC, PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes all possess anti-inflammatory properties, as treatment with these agents significantly reduced the induction of plasma TNF-α levels in TAA-induced mice ( Figure 8 ).

Claims

1. Use of a soluble P-selectin (sP-sel) in the preparation of a medicament for improving liver injury, improving diabetes induced by a high-fat diet (HFD), or hepatitis caused by thioacetamide TAA.

2. The use according to claim 1, wherein the soluble P-selectin can interfere with the interaction between stem cells and a niche.

3. The use according to claim 2, wherein these stem cells are hematopoietic cells, progenitor cells or bone marrow stem cells.

4. Use according to claim 1, wherein the dosage of sP-sel administered is in the range of 10 -5 μg to 1.5 mg per kilogram of body weight per administration.

5. The use according to claim 2, wherein these stem cells can repopulate the bone marrow or hematopoietic stem cell population.

6. The use according to claim 1, wherein the sP-sel is a naturally-occurring sP-sel or a recombinant sP-sel.

7. The use according to claim 1, wherein the sP-sel can be further conjugated to vesicles and liposomes.

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