Hematopoietic stem cell expansion

Abstract

The present disclosure relates to methods and compositions for expanding human hematopoietic stem cells. The disclosure also relates to therapeutic methods involving the use of expanded HSCs.

Description

[Technical field]

[0001] The present disclosure relates to methods and compositions for expanding human hematopoietic stem cells (HSCs). The present disclosure also relates to therapeutic methods involving the use of expanded HSCs. [Background technology]

[0002] Hematopoietic stem cells (HSCs) have an inherent self-renewal capacity and give rise to all types of mature cells within the blood and immune systems. These characteristics provide broad clinical utility for HSC transplantation, but the primary sources of HSCs (human bone marrow, mobilized peripheral blood, and umbilical cord blood) remain limited in donor supply. These issues are further complicated by the need to find donors well matched to the recipient, thereby increasing the complexity in ensuring a supply of suitable and reliable donor material. Furthermore, patients suffering from diseases caused by genetic mutations would greatly benefit from gene therapy techniques, where autologous material is engineered ex vivo and returned after correction of the corrected genetic defect. Developing effective techniques for ex vivo expansion and genetic manipulation of HSCs in various types of transplantation categories would provide an easy and renewable resource outside of the existing donor infrastructure, and new gene therapy techniques for the treatment of diseases caused by genetic mutations could be established. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO 2004 / 85630 [Patent Document 2] U.S. Patent No. 5,486,359 [Patent Document 3] Patent Publication WO 2007 / 145227 [Patent Document 4] U.S. Patent No. 5,578,475 [Patent Document 5] U.S. Patent No. 6,020,202 [Patent Document 6] U.S. Patent No. 6,051,429 [Patent Document 7] U.S. Patent No. 6,723,561 [Patent Document 8] U.S. Patent No. 6,627,442 [Patent Document 9] International Publication No. 2006 / 108229 [Non-patent literature]

[0004] [Non-Patent Document 1] Kishimoto, Ann. review of 1 mm. 23:1 2005 [Non-Patent Document 2] Smith, MA et al., ACTA Haematologica, 105, 3:143, 2001 [Non-Patent Document 3] Hannum C, Nature 368 (6472): 643~8 [Non-Patent Document 4] Kaushansky K (2006), N. Engl. J. Med. 354 (19): 2034~45 [Non-Patent Document 5] Notta et al. (2011) Science 333: 218-221 [Non-Patent Document 6] Boitano et al. (2010) Science 329: 1345-1348 [Non-Patent Document 7] Fares et al. (2014) Science 345: 1509-1512 Summary of the Invention [Problem to be solved by the invention]

[0005] Unlike the case of embryonic stem cells (ESCs), expansion of HSCs in culture generally comes at the expense of loss of primitive phenotype or "stemness". It is unclear whether exogenous factors can be applied to enhance the expansion of HSC populations without losing "stemness". Thus, there remains a need for methods to generate and expand large numbers of human HSCs to increase the availability of cells for transplantation as a renewable therapeutic resource. [Means for solving the problem]

[0006] Applicants have developed a method for expanding HSCs from a starting population of hematopoietic cells derived from any source, including adult, umbilical cord blood, iPS cells, fetal or embryonic sources. In one embodiment, the method preferentially expands primitive HSCs within the starting population of hematopoietic cells. Primitive HSCs have the phenotype CD34+, CD45RA-, CD90+, CD49f+.

[0007] The ability to expand HSC in this way is advantageous for transplantation and other therapies for hematological and oncological diseases and disorders.As described in the methods herein, HSC number can be significantly increased ex vivo.Methods of increasing stem cell number are useful for autologous donor transplants, which often lack sufficient stem cells.Methods of increasing stem cell number also allow umbilical cord blood to be useful for adult patients, thereby expanding the use of allogeneic transplantation.

[0008] Accordingly, the present disclosure provides a method for expanding hematopoietic stem cells, comprising: The present invention provides a method comprising culturing a population of hematopoietic cells in the presence of mesenchymal progenitor or stem cells (MLPSCs) and at least one histone deacetylase inhibitor (HDACi) such that hematopoietic stem cells having the phenotype CD34+ are expanded.

[0009] In one example, hematopoietic stem cells with the phenotype CD34+, CD90+ are expanded. In another embodiment, hematopoietic stem cells with the phenotype CD34+, CD90+, CD45RA- are expanded. In another embodiment, hematopoietic stem cells with the phenotype CD34+, CD45RA-, CD90+, CD49f+ are expanded. In another embodiment, hematopoietic stem cells that are CD34+, CD45RA-, CD90+, CD49f+ are preferentially expanded compared to hematopoietic stem cells that are CD34+, CD49f-.

[0010] In one embodiment, an increase in CD34+ cell count of at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 5-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold is indicative of HSC expansion.

[0011] In one embodiment, the starting cell population comprises at least 10 5 , 10 6 , 10 7 , 10 8 or 10 9 The cells are cultured for a time sufficient to reach an absolute number of CD34+ cells.

[0012] In one embodiment, the total number of CD34+, CD45RA-, CD90+, CD49f+ hematopoietic stem cells is increased by at least 2-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 44-fold, or at least 50-fold when compared to the starting population of hematopoietic cells.

[0013] In another embodiment, the percentage of CD34+, CD45RA-, CD90+, CD49f+ hematopoietic stem cells in the total cell population after culture is at least 1%, or at least 1.5%, or at least 2%, or at least 5%, or more, compared to the starting population of hematopoietic cells.

[0014] In one embodiment, the starting co-culture population comprises about 300 million, or about 400 million, or about 500 million, or more MLPSCs.

[0015] In one embodiment, the starting co-culture population comprises about 30 million, or about 40 million, or about 50 million, or more CD34+ cells.

[0016] In one embodiment, the starting co-culture population comprises about 1.5 million, or about 2 million, or about 2.5 million, or more, CD34+, CD45RA-, CD90+, CD49f+ cells.

[0017] In one embodiment, the HDACi is selected from the group consisting of valproic acid (VPA), trichostatin (TSA), DLS3, MS275, SAHA, and HDAC6 inhibitor 161.

[0018] In one embodiment, the hematopoietic cells are also cultured in the presence of one or more growth factors selected from the group consisting of s(SCF), GM-SCF, M-CSF, G-CSF, MGDF, EPO, FLT3-ligand, IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-11, TNFα, or thrombopoietin.

[0019] In another embodiment, the hematopoietic cells are also cultured in the presence of one or more stem cell regenerative agents, which can be, for example, SR1 or UM171.

[0020] In another embodiment, the MLPSCs are isolated by immune selection. For example, the MLPSCs can be STRO-1+ mesenchymal progenitor cells or their progeny expanded in culture. In another embodiment, the MLPSCs are mesenchymal progenitor cells or stem cells or their progeny expanded in culture.

[0021] It is understood that the population of hematopoietic cells may be derived from any source, including bone marrow, umbilical cord or cord blood, peripheral blood, liver, thymus, lymph, spleen, or iPS cells.

[0022] In one embodiment, hematopoietic cells are added to an established adherent MLPSC cell culture. The MLPSCs are cultured to confluence, re-implanted, and re-cultured to provide a feeder layer to which the hematopoietic cells are added for co-culture.

[0023] The cells can be co-cultured for about 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or 10 days, or 12 days, or 15 days, or 20 days or more.

[0024] The culture conditions described herein allow contact between the MLPSCs and HSCs that can facilitate the transfer of a genetic payload, such as a heterologous nucleic acid or a CRISPR system, from the MLPSCs to the HSCs.

[0025] Thus, in one embodiment, the MLPSCs comprise a heterologous nucleic acid molecule that is transferred to hematopoietic stem cells having the phenotype CD34+ during culture expansion. In one embodiment, the MLPSCs comprise a heterologous nucleic acid molecule that is transferred to hematopoietic stem cells having the phenotype CD34+, CD45RA-, CD90+, CD49f+ during culture expansion.

[0026] In another embodiment, CD34+, CD45RA-, CD90+, CD49f+ cells are isolated by immune selection following culture expansion to provide an enriched population of CD34+, CD45RA-, CD90+, CD49f+ cells that may be useful for long-term regeneration following administration to a subject.

[0027] In another embodiment, CD34+, CD45RA-, CD90+, CD49f+ cells are removed by immune selection after culture expansion to provide an enriched population of CD34+, CD49f- cells that may be useful for early neutrophil / platelet recovery following administration to a subject.

[0028] In one embodiment, the enriched population of CD34+, CD45RA-, CD90+, CD49f+ cells is subjected to genetic manipulation, for example, by transfection with a heterologous nucleic acid.

[0029] In one embodiment, the enriched population of CD34+, CD49f- cells is subjected to genetic manipulation, for example, by transfection with a heterologous nucleic acid.

[0030] Heterologous nucleic acid can be in the form of expression vector.Suitable expression vectors include, but are not limited to, plasmid, phage, autonomously replicating sequence (ARS), virus, centromere, and artificial chromosome structure.In one example, the expression vector is a viral vector selected from the group consisting of lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV), and recombinant forms thereof.

[0031] In another embodiment, the heterologous nucleic acid encodes a protein selected from the group consisting of a clotting factor, a hormone, or a cytokine.

[0032] In another embodiment, the heterologous nucleic acid comprises a CRISPR system or its components.For example, the CRISPR system can comprise a Cas expression vector and a guide nucleic acid sequence specific to an endogenous gene in HSCs.For example, the CRISPR system can comprise a Cas9 protein complexed with a guide nucleic acid sequence specific to an endogenous gene in HSCs.

[0033] In another embodiment, the expression vector or CRISPR system comprises an inducible promoter.

[0034] In another embodiment, the method comprises exposing the HSCs to an agent that activates the inducible promoter.

[0035] The present disclosure also provides a composition comprising HSCs obtained by a method according to the present disclosure. In one embodiment, the composition obtained by a method according to the present disclosure comprises HSCs having the phenotype CD34+, CD45RA-, CD90+, CD49f+.

[0036] The present disclosure also provides a composition comprising HSCs having the phenotype CD34+, CD45RA-, CD90+, CD49f+, and MLPSCs in respective ratios of at least 1:35, or at least 1:30, or at least 1:20, or at least 1:10, or at least 1:5, or at least 1:4.5, or at least 1:4.

[0037] The present disclosure also provides a composition comprising HSCs having the phenotype CD34+, CD45RA-, CD90+, CD49f+, and MLPSCs, wherein the cells having the phenotype CD34+, CD45RA-, CD90+, CD49f+ constitute at least 10% or at least 20% of the total cell population.

[0038] In one embodiment, the composition further comprises an HDACi.

[0039] The present disclosure also provides a composition comprising a hematopoietic stem cell having the phenotype CD34+, CD45RA-, CD90+, CD49f+, an MLPSC, and an HDACI inhibitor.

[0040] In one embodiment of the composition, the HSCs comprise a heterologous nucleic acid molecule.

[0041] In another embodiment, the heterologous nucleic acid encodes a protein selected from the group consisting of a clotting factor, a hormone, or a cytokine.

[0042] In another embodiment, the heterologous nucleic acid comprises a CRISPR system or its components.For example, the CRISPR system can comprise a Cas expression vector and a guide nucleic acid sequence specific to an endogenous gene in HSCs.For example, the CRISPR system can comprise a Cas9 protein complexed with a guide nucleic acid sequence specific to an endogenous gene in HSCs.

[0043] In one embodiment, HSCs having the phenotype CD34+, CD45RA-, CD90+, CD49f+ constitute at least 5%, or at least 10%, or at least 20%, or at least 30% of the total cell number in the composition.

[0044] In another embodiment, the composition comprises at least 10 5 Cells, at least 10 6 Cells, at least 10 7 Cells, at least 10 8 Cells or at least 10 9 Contains the total amount of cells.

[0045] The present disclosure also provides a method of transfecting HSCs, comprising: Culturing a population of HSCs in the presence of mesenchymal progenitor or stem cells (MLPSCs) and at least one histone deacetylase inhibitor (HDACi). wherein the MLPSC comprises at least one heterologous nucleic acid molecule; The culture conditions allow for the transfer of heterologous nucleic acid molecules from the MLPSCs to the HSCs.

[0046] In one embodiment, the HSCs have the phenotype CD34+. In another embodiment, the HSCs have the phenotype CD34+, CD45RA-, CD90+, CD49f+.

[0047] Heterologous nucleic acid can be in the form of expression vector.Suitable expression vectors include, but are not limited to, plasmid, phage, autonomously replicating sequence (ARS), virus, centromere, and artificial chromosome structure.In one example, the expression vector is a viral vector selected from the group consisting of lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV), and recombinant forms thereof.

[0048] In another embodiment, the heterologous nucleic acid encodes a protein selected from the group consisting of a clotting factor, a hormone, or a cytokine.

[0049] In another embodiment, the heterologous nucleic acid comprises a CRISPR system or its components.For example, the CRISPR system can comprise a Cas expression vector and a guide nucleic acid sequence specific to an endogenous gene in HSCs.For example, the CRISPR system can comprise a Cas9 protein complexed with a guide nucleic acid sequence specific to an endogenous gene in HSCs.

[0050] In another embodiment, the expression vector or CRISPR system comprises an inducible promoter.

[0051] In another embodiment, the method comprises exposing the HSCs to an agent that activates the inducible promoter.

[0052] The present disclosure also provides compositions comprising HSCs transfected according to the above-described methods.

[0053] The present disclosure also provides a method of treating a hematological disorder in a subject in need thereof comprising administering to the subject a composition of the present disclosure.

[0054] As used throughout, subject means an individual. Thus, subject includes, for example, domestic animals such as cats and dogs, livestock (e.g., cows, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. Subjects are optionally mammals, such as primates or humans.

[0055] In one embodiment, the subject is a human. The human can be an adult or pediatric patient.

[0056] It will be understood that the methods and compositions of the present disclosure can be used to treat a range of hematological disorders.

[0057] For example, the methods and compositions of the disclosure can be used to treat disorders of platelet number and / or function, such as thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), or disorders associated with viral infection, drug abuse, or malignancy.

[0058] In another example, the disclosed methods and compositions can be used to treat disorders of red blood cell number and / or function, such as anemia. Examples of anemias that can be treated include aplastic anemia, autoimmune hemolytic anemia, blood loss anemia, Cooley anemia, Diamond-Blackfan anemia, Fanconi anemia, folate (folic acid) deficiency anemia, hemolytic anemia, iron deficiency anemia, pernicious anemia, sickle cell anemia, thalassemia, or polycythemia vera.

[0059] In one example, the methods and compositions of the disclosure are used to treat alpha or beta thalassemia.

[0060] In another example, the disclosed methods and compositions can be used to treat disorders caused by a T cell or B cell deficiency, such as disorders of lymphocyte number and / or function, including AIDS, leukemia, lymphoma, Hodgkin's lymphoma, chronic infections such as miliary tuberculosis, viral infections, rheumatoid arthritis, systemic lupus erythematosus, or genetic disorders such as agammaglobulinemia, DiGeorge anomaly, Wiskott-Aldrich syndrome, or ataxia-telangiectasia.

[0061] In another example, the disclosed methods and compositions can be used to treat disorders of multilineage bone marrow failure, which may be the result of radiation or chemotherapy or malignant replacement, such as myelofibrosis, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), pigmented myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), multiple myeloma (MM), or secondary malignancies disseminated to bone.

[0062] In another example, the methods and compositions of the disclosure are used to treat an inborn error of metabolism, for example, the inborn error of metabolism is selected from the group consisting of mucopolysaccharidoses, Gaucher disease, metachromatic leukodystrophy, and adrenoleukodystrophy.

[0063] The present invention is applicable to a wide range of animals. For example, the subject may be a mammal, such as a human, dog, cat, horse, cow, or sheep. In one embodiment, the subject is a human.

[0064] In another embodiment, the method of the present disclosure further comprises administering an immunosuppressant. The immunosuppressant may be administered for a time sufficient to allow the transplanted hematopoietic cells to be functional. The immunosuppressant may be selected from, but is not limited to, one or more of the following: corticosteroids such as prednisone, budesonide and prednisolone; calcineurin inhibitors such as cyclosporine and tacrolimus; mTOR inhibitors such as sirolimus and everolimus; IMDH inhibitors such as azathioprine, leflunomide and mycophenolate; biologics such as abatacept, adalimumab, etanercept, infliximab or rituximab, and the like.

[0065] In one example, the immunosuppressant is cyclosporine, which may be administered at a dosage of 5 to 40 mg / kg body weight. [Brief description of the drawings]

[0066] [Figure 1] FIG. 1 shows absolute numbers of CD34+ cells per well after 5 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Diagram 2] FIG. 1 shows the percentage of CD34+CD90+ cells per well after 5 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Diagram 3]FIG. 1 shows absolute numbers of CD34+CD90+ cells per well after 5 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Figure 4] FIG. 1 shows the absolute number of cells exhibiting a primitive HSC phenotype (CD34+CD90CD49f+) per well after 5 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Diagram 5] FIG. 1 shows the percentage of CD34+ cells per well after 10 days of culture in the presence or absence of MPC with (i) no HDACi, (ii) TSA or (iii) VPA. [Figure 6] FIG. 1 shows absolute numbers of CD34+ cells per well after 10 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Figure 7] FIG. 1 shows the percentage of CD34+CD45RA-CD90+ cells per well after 10 days of culture in the presence or absence of MPC with (i) no HDACi, (ii) TSA or (iii) VPA. [Figure 8] FIG. 1 shows absolute numbers of CD34+CD45RA-CD90+ cells per well after 10 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Figure 9] FIG. 1 shows the absolute number of cells exhibiting a primitive HSC phenotype (CD34+CD90CD49f+) per well after 10 days of culture in the presence or absence of MPC with (i) no HDACi, (ii) TSA or (iii) VPA. [Figure 10] FIG. 1 shows flow cytometry analysis of cells exhibiting primitive HSC phenotype (CD34+CD90CD49f+) per well after 10 days of culture with (i) no HDACi, (ii) TSA or (iii) VPA, in the presence or absence of MPC. [Figure 11]CD34+CD38-CD45RA-CD90+CD49f+ cells isolated by FACS were cultured in MethoCult™ H4435 enrichment (Stem cell Technologies) and tested for colony-forming efficiency on culture day 14. The overall colony-forming efficiency was 0.42%. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Description of the embodiments General implementation methods and definitions Throughout this specification, unless specifically stated otherwise or the context dictates otherwise, reference to a single step, composition of matter, group of steps or composition of matter is intended to encompass one and more (i.e., one or more) of those steps, compositions of matter, group of steps or compositions of matter.

[0068] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions and compounds referred to or indicated herein, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0069] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0070] Any example disclosed herein shall be construed to apply mutatis mutandis to any other example, unless expressly stated otherwise.

[0071] Unless expressly defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0072] Unless otherwise specified, the stem cell, cell culture, and surgical techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as Perbal, 1984; Sambrook & Green, 2012; Brown, 1991; Glover & Hames, 1995 and 1996; Ausubel., 1987 (including all current revisions), Harlow & Lane, 1988; and Coligan et al., 1991 (including all current revisions).

[0073] As used in this specification and the appended claims, the singular and singular terms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise, for example in some instances.

[0074] The term "subject" as used herein refers to a mammal, including humans and non-human animals. More specifically, the mammal is a human. Terms such as "subject," "patient," or "individual" are terms that may be used interchangeably in this disclosure, where context permits. In certain instances, the subject may be an adult or a child (pediatric) subject.

[0075] "Effective amount" refers to at least an effective amount, at the dosage and time required, to achieve the desired therapeutic or prophylactic result. The effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is used to refer to the amount required to carry out the treatment of a disease or condition as described above. The effective amount can vary depending on the disease or condition being treated, and depending on the weight, age, racial background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, the effective amount falls within a relatively broad range (e.g., "dosage" range) that can be determined by routine trial and experimentation by the practitioner. The effective amount can be administered once or several times in a single dose or repeated doses over the treatment period.

[0076] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of the individual or cells being treated during the course of clinical pathology. The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disease are reduced or eliminated.

[0077] A "hematopoietic stem cell transplant (HSCT)" is a graft that includes multipotent hematopoietic stem cells, which can be derived, for example, from bone marrow or peripheral blood. The transplant can also include some non-stem cells, such as APCs, for example DCs and / or lymphocytes.

[0078] The term "adult," as used herein, means a human subject aged 18 years or older.

[0079] The term "child," as used herein, means a human subject ranging in age from birth to 17 years.

[0080] The term "graft," as used herein, refers to a biological sample selected from bone marrow, blood (e.g., whole blood or peripheral blood mononuclear cells (PBMCs)), blood products, or solid organs in which hematopoietic cells are present.

[0081] The term "allogeneic," as used herein, refers to a graft (e.g., hematopoietic cells) provided by an individual whose genetic characteristics differ from those of the recipient, particularly with respect to major histocompatibility complex (MHC) and minor histocompatibility agents expressed on the surface of the individual's cells.

[0082] The term "autologous" as used herein refers to a graft that uses the subject's own cells (e.g., hematopoietic cells present in bone marrow or peripheral blood). The cells are usually harvested and stored before the subject undergoes treatment (e.g., chemotherapy), and then reinfused into the subject.

[0083] The term "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y" and shall be interpreted as providing explicit support for both meanings or for either meaning.

[0084] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0085] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" are understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0086] hematopoietic stem cells "Hematopoietic stem cell" (HSC), as used herein, refers to an immature blood cell that has the capacity to self-renew and differentiate into more mature blood cells, including granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, thrombocytic megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages).

[0087] It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. It is believed that CD34+ cells comprise a subpopulation of cells with stem cell characteristics as defined above. It is known in the art that HSCs include pluripotent stem cells, multipotent stem cells (e.g., lymphoid stem cells), and / or stem cells committed to specific hematopoietic lineages. Stem cells committed to specific hematopoietic lineages may be T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage, and / or lymphoid tissue-specific macrophage cell lineage.

[0088] Human HSCs capable of long-term renewal and engraftment are considered "primitive" in phenotype and express CD34, CD49f, and CD90. In one embodiment, primitive cells have the phenotype CD34+, CD45RA-, CD90+, CD49f+. In one embodiment, they also lack expression of CD38 and any lineage-restricted antigens. In one embodiment, primitive HSCs are defined as CD34+CD45RA-CD49f+CD90+CD38-Lin- cells (LT-HSCs).

[0089] Mesenchymal progenitor or stem cells As used herein, the term "mesenchymal progenitor or stem cells" refers to undifferentiated multipotent cells that have the ability to self-renew while maintaining pluripotency and to differentiate into a number of cell types, either of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or of non-mesodermal origin, such as hepatocytes, neural cells and epithelial cells.

[0090] The term "mesenchymal precursor or stem cells" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells (MPCs), multipotent stromal cells, mesenchymal stem cells, perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0091] Mesenchymal precursor or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual into whom they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogeneic cells are isolated from genetically identical organisms such as twins, clones, or highly inbred research animal models.

[0092] Mesenchymal progenitor or stem cells reside primarily in bone marrow, but have also been shown to reside in a variety of host tissues including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp.

[0093] Mesenchymal progenitor or stem cells can be isolated from host tissue and enriched by immunoselection. For example, bone marrow aspirate from a subject can be further processed with antibodies against STRO-1 or TNAP to allow for the selection of mesenchymal progenitor or stem cells. In one example, mesenchymal progenitor or stem cells can be enriched by using STRO-1 antibodies as described in Simmons & Torok-Storb, 1991.

[0094] STRO-1+ cells are cells found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germ lineages such as mesoderm and / or endoderm and / or ectoderm. Thus, STRO-1+ cells can differentiate into multiple cell types, including, but not limited to, adipose, bone, cartilage, elastic, muscle, and fibrous connective tissue. The specific lineage-commitment and differentiation pathways that these cells enter depend on a variety of influences from mechanical and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0095] The term "enriched" as used herein describes a population of cells in which the percentage of one particular cell type or the percentage of multiple particular cell types is increased when compared to an untreated population of cells (e.g., cells in their natural environment). In one example, a population enriched for STRO-1+ cells contains at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% STRO-1+ cells. In this regard, the term "population of cells enriched for STRO-1+ cells" is used to expressly support the term "population of cells comprising X% STRO-1+ cells," where X% is a percentage as described herein. STRO-1+ cells can, in some examples, form and have the activity of clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%).

[0096] In one example, the cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but does not necessarily have to be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPCs) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and STRO-1 bright (It may be that the cells are STRO-1+). Thus, the indication that the cells are STRO-1+ does not mean that the cells are selected by STRO-1 expression. In one example, the cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+).

[0097] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells may be selected or isolated or enriched from a wide variety of sources. However, in some instances, these terms support selection from any one or more tissues, including STRO-1+ cells, or vascularized tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or tissues described herein.

[0098] In one example, the mesenchymal progenitor or stem cells of the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90β), CD45+, CD146+, 3G5+.

[0099] "Individually" means that the present disclosure encompasses the described markers or groups of markers separately, and that notwithstanding that individual markers or groups of markers may not be separately recited herein, the appended claims may define such markers or groups of markers separately and separately from one another.

[0100] "Collectively" means that the disclosure encompasses any number or combination of the described markers or groups of markers, and that notwithstanding that such numbers or combinations of markers or groups of markers may not be specifically recited herein, the appended claims may define such combinations or subcombinations separately and in isolation from any other combinations of the markers or groups of markers.

[0101] Cells that are referred to as "positive" for a given marker can express either low (lo or dim or dull), intermediate (median), or high (bright, bri) levels of that marker, depending on the extent to which the marker is present on the cell surface, and these terms refer to the intensity of the fluorescence or other marker used in the cell sorting process or flow cytometric analysis of the cells. The distinction of low (lo or dim or dull), intermediate (median), or high (bright, bri) is understood in the context of the marker used on the particular cell population being sorted or analyzed. A cell that is referred to as "negative" for a given marker does not necessarily mean that the cell is completely absent. This term means that the marker is expressed at a relatively very low level by the cell, producing a very low signal when detectably labeled, or is undetectable above background levels, e.g., levels detected using an isotype control antibody.

[0102] The term "bright" or bri, as used herein, refers to a marker on the cell surface that generates a relatively high signal when detectably labeled. Without wishing to be limited by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by the STRO-1 antibody) than other cells in the sample. For example, STRO-1 bri When cells were labeled with FITC-conjugated STRO-1 antibody, they were classified as non-bright cells (STRO-1 lo / dim / dull / intermediate / median) produces a greater fluorescent signal than STRO-1. In one example, mesenchymal progenitor or stem cells are isolated from bone marrow and enriched by selection for STRO-1+ cells. In this example, the "bright" cells comprise at least about 0.1% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In other examples, the "bright" cells comprise at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In one example, the "bright" cells comprise at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. bright The cells have a 2 log increase in STRO-1 surface expression compared to "background", i.e., cells that are STRO-1-. In contrast, STRO-1 lo / dim / dull and / or STRO-1 intermediate / median The cells exhibit greater expression of STRO-1 surface expression by less than 2 log orders of magnitude, and are typically about 1 log or below "background."

[0103] In one example, STRO-1+ cells are bright In one example, STRO-1 bright STRO-1 cells lo / dim / dull or STRO-1 intermediate / median are preferentially enriched relative to cells.

[0104] In one example, STRO-1 bright The cells may further be one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β) and / or CD146+. For example, the cells may be selected for and / or shown to express one or more of the aforementioned markers. In this regard, cells shown to express a marker need not be specifically tested, rather previously enriched or isolated cells can be tested and then reasonably assumed to also express the same marker.

[0105] In one example, STRO-1 brightThe cells are perivascular mesenchymal precursor cells as defined in WO 2004 / 85630, characterized by the presence of the perivascular marker 3G5.

[0106] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, this term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule that can bind to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0107] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0108] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ lineages. Non-limiting examples of lineages that cells can commit include bone progenitor cells; hepatocyte precursors that are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells that can generate glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; cardiac muscle and cardiac muscle cell precursors, glucose-responsive insulin-secreting pancreatic beta cell lineages. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, as well as the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes and oligodendrocytes progenitors.

[0109] In one example, the mesenchymal progenitor or stem cell is a mesenchymal stem cell (MSC). The MSC can be a homogenous composition or a mixed cell population enriched for MSC. The homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and the MSC can be identified by specific cell surface markers identified with specific monoclonal antibodies. Methods for obtaining cell populations enriched for MSC using plastic adhesion techniques are described, for example, in U.S. Pat. No. 5,486,359. MSC prepared by conventional plastic adhesion isolation relies on the non-specific plastic adhesion properties of CFU-F. Alternative sources of MSC include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0110] The mesenchymal precursor or stem cells can be cryopreserved prior to use.

[0111] Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow cooling methods or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotype, cell surface markers and proliferation rate of the cryopreserved cells compared to non-frozen cells.

[0112] The cryopreservation composition may include a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0113] Cryopreservation solutions can include sterile, non-pyrogenic, isotonic solutions such as PlasmaLyteA™. 100 mL of PlasmaLyteA™ contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C 6 H 11 NaO 7 ), 368 mg of Sodium Acetate Trihydrate, USP(C 2 H 3 NaO 2 3H 2 0); 37 mg of Potassium Chloride, USP (KCl); and 30 mg of Magnesium Chloride, USP (MgCl 2 6H2 It contains no antibacterial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0114] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium.

[0115] To facilitate freezing, cryoprotectants such as dimethyl sulfoxide (DMSO) are usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, non-antigenic, chemically inert, provide high survival rates after thawing, and allow transplantation without washing. However, DMSO, the most commonly used cryoprotectant, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as a substitute or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0116] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0117] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled rate freezer.

[0118] In a preferred embodiment of the invention, the mesenchymal precursor or stem cells are obtained from a master cell bank derived from mesenchymal precursor or stem cells enriched from the bone marrow of healthy volunteers. The use of mesenchymal precursor or stem cells derived from such a source is particularly advantageous for subjects who do not have suitable family members available who can serve as mesenchymal precursor or stem cell donors or who require immediate treatment and are at high risk of relapse, disease-related decline or death during the time required to generate mesenchymal precursor or stem cells.

[0119] The isolated or enriched mesenchymal precursor or stem cells can be expanded ex vivo or in vitro by culture. As will be appreciated by those of skill in the art, the isolated or enriched mesenchymal precursor or stem cells can be cryopreserved, thawed, and then further expanded ex vivo or in vitro by culture.

[0120] Cultured mesenchymal progenitor or stem cells are phenotypically distinct from the cells in vivo, for example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b.

[0121] Cultured mesenchymal progenitor or stem cells are biologically distinct from cells in vivo, having a higher proliferation rate compared to mostly non-cycling (quiescent) cells in vivo.

[0122] In one example, the cell population enriched for mesenchymal progenitor or stem cells is cultured at approximately 6,000-7,000 viable cells / cm in serum-supplemented culture medium, e.g., Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine. 2 and incubated overnight at 37 °C and 20% O 2In one embodiment, the cells are allowed to adhere to the culture vessel at about 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6810, 6820, 6830, 6840, 6850, 6860, 6870, 6880, 6890, 6890, 6900, 6910, 6920, 6930, 6940, 6970, 6980, 6990, or 7000 viable cells / cm. 2 , preferably about 6850-6860 viable cells / cm 2 The culture medium is then replaced and the cells are incubated at 37°C, 5% O for a total of 68-72 h before co-culture with T cells and determining the amount of IL-2Rα expressed by the T cells. 2 and cultured in.

[0123] HSC culture conditions HSCs can be cultured from any cell or cell population that contains HSCs or has the potential to develop into HSCs. In one embodiment, the starting population of cells contains at least 0.1% hematopoietic stem cells. In one embodiment, the HSCs are primary cells. Typically, primary cells are obtained directly from tissue. Methods for obtaining primary cells are well known in the art.

[0124] The starting population of hematopoietic cells can be harvested, for example, from a tissue sample of a subject or from culture. Harvesting is defined as the removal or separation of cells. This can be accomplished using a number of methods, such as enzymatic, non-enzymatic, centrifugation, electrical, or size-based methods, or preferably by washing the cells with culture medium (e.g., the medium in which the cells are incubated) or a buffer solution. The cells are optionally harvested, separated, and further expanded.

[0125] The conditions for culturing the starting cell population for hematopoietic stem cell expansion will vary depending on, for example, the starting cell population, the desired final cell number, and the desired final proportion of HSCs.

[0126] The cells can be co-cultured for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 days, or more. For example, the cells can be co-cultured for at least 2 weeks, or at least 4 weeks.

[0127] In one embodiment, the culture medium is a serum-free medium.

[0128] Expansion of HSCs can be performed in a basal medium supplemented with a mixture of cytokines and growth factors as described above. The basal medium typically contains amino acids, a carbon source, vitamins, serum proteins (e.g., albumin), inorganic salts, divalent cations, buffers, and any other elements suitable for use in the expansion of HSCs. Examples of such basal media suitable for methods of expanding HSCs include, but are not limited to, StemSpan® SFEM-serum-free expansion medium (StemCell Technologies, Vancouver, Canada), StemSpan® H3000-synthetic medium (StemCell Technologies, Vancouver, Canada), CellGro® SCGM (CellGenix, Freiburg Germany), and StemPro®-34 SFM (Invitrogen).

[0129] The culture medium may include an effective amount of one or more additional factors, such as cytokines. Suitable factors include insulin-like growth factors (IGF), IL-1, IL-3, IL-6, IL-11, G-CSF, GM-CSF, SCF, FLT3-L, thrombopoietin (TPO), erythropoietin, and analogs thereof. As used herein, "analog" includes any structural variant of cytokines and growth factors that have biological activity as the naturally occurring form, including, but not limited to, variants with enhanced or decreased biological activity when compared to the naturally occurring form or cytokine receptor agonists, such as agonist antibodies against the TPO receptor (e.g., VB22B sc(Fv)2, detailed in patent publication WO 2007 / 145227, etc.). The combination of cytokines and growth factors is selected to expand HSCs and progenitor cells while limiting the production of terminally differentiated cells. In one specific embodiment, the one or more cytokines and growth factors are selected from the group consisting of SCF, Flt3-L and TPO, hi one specific embodiment, at least TPO is used in serum-free medium under conditions suitable for HSC expansion.

[0130] Human IL6 or interleukin-6, also known as B cell stimulatory factor 2, has been described by (Kishimoto, Ann. review of 1 mm. 23:1 2005) and is commercially available. Human SCF or stem cell factor, also known as c-kit ligand, mast cell growth factor or steel factor, has been described (Smith, MA et al., ACTA Haematologica, 105, 3:143, 2001) and is commercially available. Flt3-L or FLT-3 ligand, also called FL, is a factor that binds to the flt3-receptor. It has been described (Hannum C, Nature 368 (6472): 643-8) and is commercially available. TPO or thrombopoietin, also known as megakaryocyte growth factor (MGDF) or c-Mpl ligand, is described in (Kaushansky K (2006), N. Engl. J. Med. 354 (19): 2034-45) and is commercially available.

[0131] Compositions and Administration Compositions comprising HSCs can be prepared in a pharma- ceutically acceptable carrier. The term "pharma- ceutically acceptable carrier," as used herein, refers to a composition of matter that facilitates storage, administration, and / or maintains the biological activity of mesenchymal progenitor or stem cells.

[0132] In one example, the carrier does not cause significant local or systemic adverse effects in the recipient. Pharmaceutically acceptable carriers can be solid or liquid. Useful examples of pharma- ceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbing agents, dispersion media, coating agents, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, sequestering agents, scaffolds, isotonic and absorption retarding agents that do not affect the viability and activity of mesenchymal progenitor or stem cells. The selection of suitable carriers is within the skill of the art.

[0133] The composition of the present disclosure can be conveniently provided in unit dosage form and can be prepared by any method known in the art.The term "dosage unit form" as used herein refers to a physically discrete unit suitable as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic or prophylactic effect in association with pharmaceutical carrier.The dose of mesenchymal precursor or stem cells can vary according to factors such as disease state, age, sex, and weight of the subject to be treated.

[0134] An exemplary dose is at least about 1×10 6 For example, the dose may be about 1.0×10 6 ~Approx. 1.1×10 10 For example, about 1.1 x 10 6 ~Approx. 1×10 9 Cells, e.g., about 1.2 x 10 6 ~Approx. 1×10 8 Cells, e.g., about 1.3 x 10 6 ~Approx. 1×107 Cells, e.g., about 1.4 x 10 6 ~Approx. 9×10 6 Cells, e.g., about 1.5 x 10 6 ~Approx. 8×10 6 Cells, e.g., about 1.6 x 10 6 ~Approx. 7×10 6 Cells, e.g., about 1.7 x 10 6 ~about 6×10 6 Cells, e.g., about 1.8 x 10 6 ~Approx. 5×10 6 Cells, e.g., about 1.9 x 10 6 ~Approx. 4×10 6 Cells, e.g., about 2 x 10 6 ~Approx. 3×10 6 It may comprise cells.

[0135] In one embodiment, the dose is about 5×10 5 ~2×10 7 Cells, e.g., about 6 x 10 6 Cells ~ approx. 1.8×10 7 The dose may be, for example, about 6×10 6 cells or approximately 1.8 x 10 7 It may be a cell.

[0136] HSCs comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cell population of the composition.

[0137] The compositions of the present disclosure can be administered by a route suitable for the particular disease state to be treated.For example, the compositions of the present disclosure can be administered systemically, i.e., parenterally, intravenously, or by injection.The compositions of the present disclosure can be targeted to a particular tissue or organ.

[0138] Dosage regimen can be adjusted to obtain optimal therapeutic response.For example, a single bolus can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.For ease of administration and uniformity of dosage, parenteral compositions can be advantageously formulated into dosage unit form.

[0139] In some embodiments, it may not be necessary or desirable to immunosuppress the patient prior to the initiation of treatment with the cell composition. This can be accomplished through the use of systemic or local immunosuppressants or by delivering the cells in an encapsulation device. The cells may be encapsulated in a capsule that allows the cells to obtain the nutrients and oxygen they need, as well as immune humoral factors and therapeutic factors that are still impermeable to the cells. Preferably, the encapsulating agent is hypoallergenic, easily and stably located in the target tissue, and provides additional protection to the implanted structure. These and other means for reducing or eliminating the immune response to implanted cells are known in the art. Alternatively, the cells may be genetically modified to reduce their immunogenicity.

[0140] It is understood that the HSCs may be administered with other beneficial drugs or biological molecules (growth factors, trophic factors). When administered with other agents, they may be administered together, in a single pharmaceutical composition, or in separate pharmaceutical compositions, simultaneously with the other agent or sequentially (either before or after administration of the other agent). Bioactive factors that may be co-administered include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs) such as PEMIROLAST™, TRANILAST™, REMICADE™, SIROLIMUS™, and TEPOXALIN™, TOLMETIN™, SUPROFEN™; immunosuppressants / immunomodulators (e.g., calcineurin inhibitors such as cyclosporine, tacrolimus); mTOR inhibitors (e.g., SIROLIMUS™, EVEROLIMUS™); antiproliferatives (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); monoclonal anti-IL-2R alpha receptor antibodies (e.g., basal Antibodies such as anti-T-cell antibodies (e.g., antithymocyte globulin (ATG); antilymphocyte globulin (ALG); monoclonal anti-T-cell antibody OKT3); antithrombotic agents (e.g., heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine proline arginine chloromethyl ketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), and local anesthetics.

[0141] Genetically modified cells In one embodiment, the HSCs or MLPSCs are genetically engineered to express and / or secrete, for example, a protein of interest, eg, a protein that provides a therapeutic and / or prophylactic benefit.

[0142] The term "nucleic acid" as used herein refers to a polymeric form of nucleotides of either ribonucleotide or deoxyribonucleotide length, containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. Polynucleotides of the present embodiments include sequences of deoxyribopolynucleotides (DNA), ribopolynucleotides (RNA), or DNA copies of ribopolynucleotides (cDNA), which may be isolated from natural sources, recombinantly produced, or artificially synthesized. A further example of a polynucleotide is a polyamide polynucleotide (PNA). Polynucleotides and nucleic acids may exist as single-stranded or double-stranded. The backbone of a polynucleotide may include sugar and phosphate groups, as typically found in RNA or DNA, or modified or substituted sugar or phosphate groups. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Nucleic acids, polynucleotides, and polymers made of nucleotides, such as polynucleotides, may also be referred to herein as nucleotide polymers.

[0143] The HSCs or MLPSCs of the present disclosure may be modified to introduce the above referenced nucleic acids. The term "introduced" is used in the context of the present disclosure to refer to the introduction of a nucleic acid into the nucleus or cytoplasm of a mesenchymal progenitor or stem cell according to the present disclosure.

[0144] An HSC or MLPSC is considered to be "modified" if nucleic acid has been transferred into the cell by any suitable means of artificial manipulation, or if the cell is the progeny of an originally altered cell which inherited the nucleic acid.

[0145] The terms "genetically modified," "transfected," "transduced," or "genetically transformed" may also be used interchangeably in the context of this disclosure to refer to modified mesenchymal progenitor or stem cells. HSCs or MLPSCs can be modified in a stable or transient manner.

[0146] In some instances, HSCs or MLPSCs can be modified to introduce a vector that expresses a nucleic acid. Numerous vectors for expression in cells are known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, a sequence encoding a nucleic acid such as an oligonucleotide, an enhancer element, a promoter, and a transcription termination sequence.

[0147] Exemplary expression vectors include plasmids, phages, autonomously replicating sequences (ARS), viruses, centromeres, artificial chromosomes, chromosomes, or other structures capable of expressing a nucleic acid in a mesenchymal progenitor or stem cell according to the present disclosure.

[0148] Suitable vector plasmids for transfecting mesenchymal progenitor or stem cells include the lipid / DNA complexes described in U.S. Patent Nos. 5,578,475, 6,020,202, and 6,051,429. Suitable reagents for making DNA-lipid complexes include Lipofectamine (Gibco / Life Technologies, #11668019) and FuGENE™ 6 (Roche Diagnostics Corp., #1814443); and LipoTAXI™ (Invitrogen Corp., #204110).

[0149] In another example, HSCs or MLPSCs are modified to introduce nucleic acids using viral expression vectors. Exemplary viral expression vectors include lentiviruses, baculoviruses, retroviruses, adenoviruses (AdVs), adeno-associated viruses (AAVs), including recombinant forms such as recombinant adeno-associated viruses (rAAVs), and derivatives thereof, such as self-complementary AAVs (scAAVs) and non-integrating AVs.

[0150] In one example, the viral vector is replication-defective. In this example, the replication gene is deleted or replaced with an expression cassette containing a highly active promoter. For example, in the context of AV, the E1 / E3 genes can be deleted or replaced. In the context of AAV, the E1A and E1B genes can be deleted or replaced. Exemplary highly active promoters include CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, and Ac5.

[0151] In some examples, HSCs or MLPSCs are modified to introduce nucleic acid using AV vectors or recombinant forms thereof. Various AV serotypes may be suitable for modifying cells to introduce nucleic acid. In one example, AV serotype 1 (AV1) is used to modify mesenchymal progenitor or stem cells. In another example, AV2 is used to modify mesenchymal progenitor or stem cells. In another example, AV3, AV4, AV7, AV8, AV9, AV10, AV11, AV12, or AV13 is used to modify HSCs or MLPSCs. In another example, AV5 is used to modify HSCs or MLPSCs. In another example, AV6 is used to modify mesenchymal progenitor or stem cells.

[0152] In one example, HSCs or MLPSCs are modified to introduce the nucleic acid using an AAV vector or a recombinant form thereof. Various AAV serotypes may also be suitable for modifying HSCs or MLPSCs.

[0153] In one example, AAV serotype 1 (AAV1) is used to modify HSCs or MLPSCs. In another example, AAV2 is used to modify HSCs or MLPSCs. In another example, AAV3, AAV4, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 is used to modify HSCs or MLPSCs. In another example, AAV5 is used to modify HSCs or MLPSCs. In another example, AAV6 is used to modify HSCs or MLPSCs.

[0154] The optimal vector can be identified using various techniques known in the art. In one example, mesenchymal progenitor or stem cells can be contacted / transfected with various vectors expressing green fluorescent protein (GFP). In this example, the optimal vector can be identified based on transfection / transduction efficiency, GFP expression level, cytotropism, and / or persistence of GFP expression.

[0155] Viral transduction methods are known in the art (e.g., U.S. Pat. Nos. 6,723,561; 6,627,442). A variety of viral expression vector systems are also available from commercial suppliers such as Miltenyi Biotech (MACSductin), Sigma-Aldrich (ExpressMag), and Thermo Fisher Scientific (ViraPower).

[0156] Modification efficiency is rarely 100%, and it is usually desirable to enrich the population for successfully modified cells. In some instances, modified cells can be enriched by taking advantage of functional characteristics of the new genotype. One exemplary method of enriching modified cells is positive selection using resistance to drugs such as neomycin.

[0157] Delivery to co-cultured HSCs In one example, the disclosure encompasses a method of delivering a nucleic acid to an HSC by contacting the HSC via co-culture with MLPSCs modified to contain a heterologous nucleic acid or a vector expressing same. For the avoidance of doubt, the nucleic acid delivered to the HSC cell is the nucleic acid introduced into the modified mesenchymal progenitor or stem cell.

[0158] The transfer of nucleic acid can occur through direct or indirect contact between MLPSC and HSC. "Direct contact" is used in the context of the present disclosure to refer to physical contact between HSC and modified MLPSC that facilitates the transfer of nucleic acid. For example, the target cell and the modified MLPSC can be directly contacted through a common connexin (i.e., a connexin expressed by both HSC and modified mesenchymal progenitor or stem cell). In this example, the common connexin facilitates the transfer of nucleic acid from MLPSC to HSC via gap junction. In one example, the gap junction is formed by Cx40. In another example, the gap junction is formed by Cx43. In another example, the gap junction is formed by Cx45, Cx32, and / or Cx37.

[0159] "Indirect contact" is used in the context of the present disclosure and refers to the delivery of nucleic acid from MLPSC to HSC without direct contact.For example, the modified MLPSC that is in close proximity to target cell can indirectly contact with target cell.In one example, the modified MLPSC that indirectly contacts with HSC can deliver nucleic acid to target cell via exosome.

[0160] In another example, modified MLPSCs in direct contact with HSCs can deliver nucleic acids to target cells via common connexins and indirectly via exosomes.

[0161] In another example, the HSCs share a connexin in common with the modified MLPSCs. In one example, the HSCs express Cx40. In another example, the HSCs express Cx43. In another example, the target cells express Cx45, Cx32 and / or Cx37.

[0162] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive. EXAMPLES

[0163] Example 1 Immunoselection of mesenchymal progenitor or stem cells (MLPSC) Bone marrow (BM) was collected from healthy adult volunteers (ages 20-35). Briefly, 40 ml of BM was aspirated from the posterior iliac crest into a lithium-heparin anticoagulant-containing tube.

[0164] Bone marrow mononuclear cells (BMMNC) were prepared by density gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo, Norway) as previously described by Zannettino et al., 1998. After centrifugation at 400×g for 30 min at 4° C., the buffy layer was removed with a transfer pipette and washed three times in “HHF” consisting of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0165] STRO-3+ (or TNAP+) cells were isolated by magnetic activated cell sorting as previously described by Gronthos & Simmons, 1995; and Gronthos, 2003. Briefly, approximately 1-3 × 10 8BMMMNCs are incubated for 20 min on ice in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. Cells are incubated for 1 h on ice with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer. Cells are then washed twice in HHF by centrifugation at 400×g. A 1 / 50 dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer is added and cells are incubated for 1 h on ice. Cells are then incubated for 20 min on ice in MACS buffer (Ca2+, supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide) as above. 2+ and Mg 2+ The cells are washed twice in PBS (without HCl) and resuspended in a final volume of 0.9 ml MACS buffer.

[0166] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) are added to the cell suspension and incubated on ice for 15 min. The cell suspension is washed twice, resuspended in 0.5 ml of MACS buffer, and then loaded onto a miniMACS column (MS column, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that do not bind to STRO-3 mAb (Deposited at the American Type Culture Collection (ATCC) on December 19, 2005 under accession number PTA-7282 - see WO 2006 / 108229). After adding another 1 ml of MACS buffer, the column is removed from the magnet and TNAP+ cells are isolated by positive pressure. An aliquot of cells from each fraction can be stained with streptavidin-FITC and purity assessed by flow cytometry.

[0167] Example 2 Co-culture of HSCs and mesenchymal progenitor or stem cells (MLPSCs) Cell::CB CD34+ (Stem Cell Technologies)

[0168] Media: StemSpan SFEM (StemCell Technologies) supplemented with: ● Human low density lipoprotein (Stem Cell Technologies) 10μg / ml Growth Factor ("SFT"): 〇rHu-SCF 100ng / ml 〇rHu-FLT3 ligand 100ng / ml 〇rHu-TPO 50ng / ml (All recombinant cytokines were from R&D Systems.)

[0169] Small molecules: SR1 (500 nM); UM171 (35 nM); trichostatin A (TSA, 50 nM); valproic acid (VPA, 500 μM) (all from Stem Cell Technologies)

[0170] Assay conditions: Day -1 MPCs (MCBCC006) plated at 50,000 / well in 2 x 24 well plates in alpha-MEM / 10% FBS Day 0: 10,000 CD34+ cells per well were seeded into each well of two 24-well plates. MPC-containing wells were first washed to remove FBS medium. The remaining CD34+ cells were cultured at the same concentration in T-25 flasks in StemSpan / SFT / SR-1+UM171. This was to provide bulk cells for the flow cytometry analysis setup and to define electronic compensation settings for multicolor analysis. Day 3 All groups were fed by removing 1.5 mL of medium and replacing it with 2.0 mL of fresh medium plus supplements. Day 5 Flow cytometry analysis: 1.5 mL of culture medium was taken from each well. For the suspension (without MPC) group, this was achieved by first aspirating the medium up and down, completely suspending the CD34+ cells, and then removing 1.5 mL of the suspension. For the +MPC group, the CD34+ cells were resuspended in a way that left the MPC feeder layer intact. After the cells were removed from each well, they were replaced with 2.0 mL of fresh medium + additives. A cell count was performed for all wells. This was necessary to determine not only the incidence of the populations identified by FACS analysis, but also their absolute numbers. Day 8 All groups were fed with 2.0 mL of medium removed and replaced with 2.0 mL of fresh medium and supplements. Day 10 Flow cytometry analysis: The complete contents of each well were harvested. For the suspension (no MPC) group, this was achieved by first aspirating the medium up and down to completely suspend the CD34+ cells, then removing the suspension. For the +MPC group, the CD34+ cells in suspension (as described above for day 5) were first harvested. The CD34+ cells attached to the MPC layer were then detached by brief exposure (5 min) to 0.05% trypsin-EDTA in PBS at 37°C, the trypsin was quenched with 10% FBS, and the detached cells were pooled with the previously harvested suspension fraction to represent the day 10 harvest.

[0171] Flow cytometry analysis: Four-color flow cytometry analysis was performed to allow identification and quantification of CD34+ cells and related subpopulations, including potential hematopoietic stem cells (HSCs), according to the phenotype described for umbilical cord blood: CD34+CD45RA-CD90+CD49f+ (Notta et al. (2011) Science 333: 218-221). All antibody conjugates were used at the concentrations recommended by the manufacturer.

[0172] Bulk cultures of CD34+ cells in StemSpan / SFT / SR-1+UM171 were used at both days 5 and 10 to establish flow cytometry analysis settings and to establish compensation settings, as described above. Staining with the following antibodies / antibody combinations was performed: 1. Cells alone 2. PE-Cy7 / FITC / PE / APC isotype (pool) 3. CD34-PECy7 4. CD45RA-FITC 5. CD90-PE 6. CD49f-APC 7. CD34-PECy7 / CD45RA-FITC / IgG1-PE isotype / CD49f-APC 8. CD34-PECy7 / CD45RA-FITC / CD90-PE / Rat IgG2a isotype-APC 9. CD34-PECy7 / CD45RA-FITC / CD90-PE / CD49f-APC Each group was stained with the four-color panel (9).

[0173] result Cord blood-derived CD34+ HSCs were cultured with and without immunoselected MPCs in the presence of: SFT SFT+VPA SFT+SR-1 SFT+SR-1+UM171 SFT+UM171 SFT+SR-1+TSA SFT+TSA SFT+SR-1+VPA: SFT+UM171+TSA SFT+UM171+VPA SFT+SR-1+UM171+TSA SFT+SR-1+UM171+VPA

[0174] Results showing the numbers of CD34+ cells and various subsets including the candidate HSC phenotype (CD34+CD45RA-CD90+CD49f+) on day 5 of culture are shown in Figures 1-4, and on day 10 of culture are shown in Figures 5-9. Results of flow cytometry analysis on day 10 of culture are shown in Figure 10.

[0175] FIG. 9 shows that the presence of MPC and HDACi (either TSA or VPA) results in a substantially greater expansion of CD34+ cells with the primitive HSC phenotype CD34+CD45RA-CD90+CD49f+ than in the absence of MPC or HDACi.

[0176] FIG. 10 shows that co-culture of CD34+ cells with MPC significantly synergizes with HDACi, enhancing the generation of HSCs with the primitive phenotype CD34+CD45RA-CD90+CD49f+.

[0177] These results show a substantial increase in primitive HSCs, especially during the culture. For example, the starting population of co-cultured cells includes about 100,000 MPCs and about 10,000 CD34+ cells. Of these 10,000 CD34+ cells, about 500 have the primitive phenotype CD34+CD45RA-CD90+CD49f+. After 10 days of co-culture with MPCs, SFV and VP, for example, the number of CD34+ cells increased to about 800,000, and the number of CD34+CD45RA-CD90+CD49f+ cells increased to about 22,000 cells. This represents an approximately 44-fold increase in the number of CD4+CD90+CD49f+ cells over the course of 10 days.

[0178] Over the 10 days, the number of MPCs in culture remained constant (approximately 100,000 cells). Thus, the ratio of CD34+CD45RA-CD90+CD49f+ cells:MPCs increased from 1:200 to 1:4.5 over the 10 days.

[0179] For comparison, prior to this disclosure, the state of the art method for expanding CD34+ cells involved culturing CD34+ cells in the presence of SFT, SR-1 and UM171 and in the absence of MPC (Boitano et al., (2010) Science 329: 1345-1348; Fares et al., (2014) Science 345: 1509-1512). As shown in Figure 9, under these conditions, the number of CD34+CD45RA-CD90+CD49f+ cells present after a 10 day culture period was very low (less than about 800 cells).

[0180] CD34+CD38-CD45RA-CD90+CD49f+ cells were isolated by FACS after 10 days in co-culture with MPC, SFV and VPA. These isolated cells were then cultured in MethoCult™ H4435 Enriched (Stem cell Technologies), a complete methylcellulose-based medium containing IL-3, IL-6, G-CSF, GM-CSF, SCF and EPO, useful for the expansion and enumeration of hematopoietic progenitor cells in colony forming unit (CFU) assays. Colonies were scored on day 14 of culture and tested for colony forming efficiency. As shown in Figure 11, the isolated CD34+CD38-CD45RA-CD90+CD49f+ cells contained clonogenic hematopoietic precursors that gave rise to erythroid progenitors (BFU-E), granulocyte-macrophage progenitors (CFU-GM, CFU-G and CFU-M), and multipotent granulocyte, erythroid, macrophage and megakaryocyte progenitors (CFU-Mix). The overall colony formation efficiency was 0.42%.

[0181] Example 3 Expansion of CD34+ cells from peripheral blood or umbilical cord blood Approximately 25 million MPCs are culture expanded in animal component-free medium for approximately 5 days to obtain a cell population containing approximately 400-500 million MPCs. The MPCs are then washed and placed in co-culture with approximately 50 million CD34+ cells obtained from the peripheral blood or from umbilical cord blood of a subject requiring HSC transplantation. At this stage, there will be approximately 2.5 million primitive HSCs of phenotype CD34+CD45RA-CD90+CD49f+ within the total CD34+ population. The cells are then co-cultured in serum-free medium for approximately 10 days in the presence of HDAC inhibitors, after which the HSCs of phenotype CD34+CD45RA-CD90+CD49f+ are expanded to approximately 100,000,000 cells.

[0182] At this stage, the entire cell population can be used for administration to a subject in need of an HSC transplant.

[0183] Alternatively, CD34+CD45RA-CD90+CD49f+ cells can be isolated by immunoselection, for example, using an antibody that binds to the CD49f antigen, to provide a purified population of these cells that are particularly suitable for long-term regeneration and engraftment. The remaining cell population, depleted of CD34+CD45RA-CD90+CD49f+ cells, is enriched for CD34+CD49- cells, which are particularly useful for early neutrophil / platelet recovery.

[0184] The immunoselected CD34+CD45RA-CD90+CD49f+ cells can be subjected to genetic modification, for example, by transduction with a viral vector containing a genetic sequence encoding a therapeutic protein or by using the CRISPR system.

[0185] References TIFF0007614106000001.tif119170

Claims

1. 1. A method for expanding hematopoietic stem cells, comprising: Culturing the hematopoietic cell population in the presence of a population of mesenchymal progenitor cells isolated by immunoselection and comprising at least 5% TNAP+ cells and at least one histone deacetylase inhibitor (HDACi) so as to expand hematopoietic stem cells having the phenotype CD34+. The method comprising:

2. 2. The method of claim 1, wherein hematopoietic stem cells having the phenotype (i) CD34+, CD90+, or (ii) CD34+, CD45RA-, CD90+, CD49f+ are expanded at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 40-fold.

3. 3. The method of claim 1 or claim 2, wherein the HDACi is selected from the group consisting of valproic acid (VPA), trichostatin (TSA), DLS3, MS275, SAHA, and HDAC6 inhibitor 161.

4. The method of claim 3, wherein the HDACi is VPA or TSA.

5. The method according to any one of claims 1 to 4, wherein the hematopoietic cells are also cultured in the presence of one or more growth factors selected from the group consisting of stem cell factor (SCF), flt3 ligand (FL), TPO, IL3 and IL6.

6. The method of any one of claims 1 to 5, wherein the hematopoietic cells are cultured in the presence of one or more stem cell regenerating agents selected from the group consisting of SR1 and UM171.

7. 7. The method of any one of claims 1 to 6, wherein TNAP+ mesenchymal progenitor cells are isolated by immune selection and culture expanded.

8. 8. The method of any one of claims 1 to 7, wherein the hematopoietic cell population is derived from bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph or spleen.

9. 9. The method of any one of claims 1 to 8, further comprising isolating cells with the phenotype (i) CD34+, CD90+, or (ii) CD34+, CD45RA-, CD90+, CD49f+ after culture expansion to provide an enriched population of cells with the phenotype (i) CD34+, CD90+, or (ii) CD34+, CD45RA-, CD90+, CD49f+.

10. 9. The method of any one of claims 1 to 8, further comprising the step of removing cells with the phenotype CD34+, CD45RA-, CD90+, CD49f+ after culture expansion to provide an enriched population of cells with the phenotype CD34+, CD49f-.

11. 11. The method of claim 9 or 10, further comprising the step of introducing a heterologous nucleic acid into the enriched cells having the phenotype (i) CD34+, CD90+, or (ii) CD34+, CD45RA-, CD90+, CD49f+.

12. The method of claim 11, further comprising the step of introducing a heterologous nucleic acid into the enriched cells having the phenotype CD34+, CD49f-.

13. 9. The method of any one of claims 1 to 8, wherein the TNAP+ mesenchymal progenitor cells comprise a heterologous nucleic acid molecule that is transferred to hematopoietic stem cells having the phenotype CD34+, CD45RA-, CD90+, CD49f+ during culture expansion.

14. 14. The method according to any one of claims 11 to 13, wherein the heterologous nucleic acid is present in the form of an expression vector.

15. 15. The method of claim 14, wherein the expression vector is selected from the group consisting of lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV), and recombinant forms thereof.

16. 16. The method according to any one of claims 11 to 15, wherein the heterologous nucleic acid encodes a protein selected from the group consisting of a clotting factor, a hormone or a cytokine.

17. 14. The method of any one of claims 11 to 13, wherein the heterologous nucleic acid is a CRISPR system.

18. 18. The method of claim 17, wherein the CRISPR system comprises a Cas expression vector and a guide nucleic acid sequence specific for an endogenous gene in the hematopoietic stem cell.

19. 18. The method of claim 17, wherein the CRISPR system comprises a Cas9 protein complexed with a guide nucleic acid sequence specific for an endogenous gene in the HSC.

20. 20. The method of any one of claims 14 to 19, wherein the expression vector or CRISPR system comprises an inducible promoter.

21. 21. The method of claim 20, further comprising exposing the hematopoietic stem cells to an agent that activates the inducible promoter.

22. A composition comprising hematopoietic stem cells having the phenotype (i) CD34+, CD90+, or (ii) CD34+, CD90+, CD45RA-, CD49f+, and a population of mesenchymal progenitor cells isolated by immunoselection and comprising at least 5% TNAP+ cells, in a ratio of at least 1:20, or at least 1:10, or at least 1:5, or at least 1:4.5, or at least 1:4, respectively.

23. 23. The composition of claim 22, further comprising an HDACi.

24. A composition comprising hematopoietic stem cells having the phenotype (i) CD34+, CD90+, or (ii) CD34+, CD45RA-, CD90+, CD49f+, a population of mesenchymal progenitor cells isolated by immune selection and comprising at least 5% TNAP+ cells, and an HDACI inhibitor.

25. 25. The composition of any one of claims 22 to 24, wherein (i) CD34+, CD90+, or (ii) CD34+, CD90+, CD45RA-, CD49f+ cells comprise the heterologous nucleic acid molecule.

26. 26. The composition of claim 25, wherein the heterologous nucleic acid encodes a protein selected from the group consisting of a clotting factor, a hormone or a cytokine.

27. 26. The composition of claim 25, wherein the heterologous nucleic acid comprises a CRISPR system.

28. 28. The composition of any one of claims 22 to 27, wherein hematopoietic stem cells having the phenotype CD34+, CD90+, CD45RA-, CD49f+ constitute at least 2% of the total cells in the composition.

29. The composition comprises at least 10 5 cells, 10 7 cells, 10 8 Cells or 10 9 29. The composition of any one of claims 22 to 28, comprising a whole amount of cells.

30. 30. The composition of any one of claims 22 to 29 for treating a hematological disorder in a subject in need thereof.

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