method

Abstract

The present invention provides methods for expanding CD34+ hematopoietic stem cells (HSCs), related uses, cells produced by such methods, and medical treatments.

Description

[Technical field]

[0001] The present invention provides methods for expanding CD34+ hematopoietic stem cells (HSCs), uses thereof, cells produced thereby, and medical treatments. [Background technology]

[0002] Transplantation of HSCs holds great potential for the medical treatment of a variety of diseases, including various hematological, oncological, and immunological disorders. However, the practical application of the procedure is limited by the low availability of HSCs and, in many cases, the lack of availability of HSC donors that match potential patients. The low amount of HSCs available for transplantation leads to slow engraftment and treatment failure.

[0003] To overcome the challenge of low availability of HSCs, ex vivo expansion of HSCs, especially from umbilical cord blood, has shown great promise, as the availability of these cells in clinical quantities would increase the chances of therapeutic success.

[0004] Several approaches have been developed for ex vivo expansion of HSCs using molecules such as hematopoietic growth factors, cytokines, and differentiation inhibitors (e.g., Notch ligands, copper chelators, nicotinamide, and aryl hydrocarbon receptor antagonists), but expansion methods need to be further improved.

[0005] For example, [Non-Patent Document 1] (Liedtke et. al., 2019, Stem Cell Translational Medicine 8: S20-S20, doi:10.1002 / sctm.12563) describes a method for expanding hematopoietic stem / progenitor cells. In a specific example, hematopoietic stem / progenitor cells are thawed, concentrated, and cultured for 3 to 4 days in a serum-free medium supplemented with 100 ng / mL interleukin-3 (IL-3), 100 ng / mL interleukin-6 (IL-6), 100 ng / mL thrombopoietin (TPO), 100 ng / mL Flt-3 ligand (Flt-3L), and 100 ng / mL stem cell factor or KIT ligand or steel factor (SCF).

[0006] Another example of a method for expanding hematopoietic stem / progenitor cells is described in [Non-Patent Document 2] (Rogers et. al., 2008, biology of Blood and Marrow transplantation, 14:927-937, doi:10.1016 / j.bbmt.2008.06.00). In a specific example, hematopoietic stem / progenitor cells are cultured in a serum-free medium containing IMDM, 1% bovine serum albumin, 10 mg / ml L bovine pancreatic insulin, 200 mg / mL human transferrin, 10-4 M β-mercaptoethanol, 2 mM L-glutamic acid, 40 mg / mL low density lipoprotein and growth factors, 25 ng / mL FGF-4, 25 ng / mL IL-3, 25 ng / mL SCF, 25 ng / mL FLT3 ligand and 25 ng / mL heparin. Cells are cultured for 4-28 days, with medium and growth factors changed three times a week. All cells are cultured in tissue culture-treated 6-, 12-, or 24-well plates.

[0007] However, neither of the above methods of Liedtke et. al., 2019 or Rogers et. al., 2008 provide as high quality quantities of HSCs as the method of the present invention.

[0008] Although estrogens have been used as media supplements for the growth of lineage-committed progenitor cells and the differentiation of stem cells, the use of estrogens has not been considered or identified as a molecule that can be used to expand HSCs. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Liedtke et. al., 2019, Stem Cell Translational Medicine 8: S20-S20, doi:10.1002 / sctm.12563 [Non-Patent Document 2] Rogers et. al., 2008, biology of Blood and Marrow transplantation, 14:927-937, doi:10.1016 / j.bbmt.2008.06.002 Summary of the Invention [Problem to be solved by the invention]

[0010] Against this background, the present inventors have now for the first time identified the use of estrogen receptor agonists (including estrogen) to expand medically highly relevant HSCs expressing the CD34 marker (e.g., CD34+HSCs) as well as subpopulations of CD34+HSCs expressing the CD133 marker (e.g., CD34+CD133+HSCs).Surprisingly, the method of expanding CD34+HSCs and CD34+CD133+HSCs using estrogen receptor agonists has been shown to allow the expansion of high quality cell populations, which are highly beneficial for medical treatment. [Means for solving the problem]

[0011] In a first aspect, the present invention provides a method for expanding CD34+ hematopoietic stem cells, comprising the steps of: i) providing a sample comprising one or more CD34+ hematopoietic stem cells; ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonists to expand the CD34+ hematopoietic stem cells.

[0012] In a second aspect, the present invention provides the use of an estrogen receptor agonist for expanding one or more CD34+ haematopoietic stem cells. The use of this second aspect of the invention may comprise the steps described herein in relation to the method of the first aspect of the invention. [Brief description of the drawings]

[0013] [Figure 1] Fold expansion of UCB-HSPCs by 0, 1 and 10 nM E2 treatment (p<0.05). [Diagram 2] Flow cytometric analysis of expanded cells. No changes in surface phenotype were observed in the CD34+ and CD34+CD133+ cell populations. [Diagram 3] Fold expansion of CD34+ cells by treatment with 0, 1 and 10 nM E2 (p<0.05). [Figure 4] Flow cytometry analysis of expanded UCB-HSCs. Cells maintained their phenotype and stemness in control and all treatment conditions. [Diagram 5] (Figure 5a,b) Expansion of CD34+CD38- HSCs with various combinations of growth factors and test compounds. a) Cell numbers (in millions) and b) cell proliferation rate (compared to control) after 14 days (left) and 21 days (right) of expansion (p<0.05). [Figure 6] (Figure 6a, b) Phenotype of cells expanded from CD34+CD38- HSCs with various combinations of growth factors and test compounds. a) Proliferation rate of CD34+ (HSPCs) and b) percentage of CD34+ cells (p<0.05) after 14 days (left) and 21 days (right) of expansion. [Figure 7]Proliferation rate of CD34+CD133+ cells (LT-HSCs) expanded from CD34+CD38- HSCs with various combinations of growth factors and test compounds for 14 days (left) and 21 days (right) (p<0.05). [Figure 8] Assessment of the hematopoietic multilineage potential of in vitro expanded UCB-HSCs after 21 days of culture with various growth factor and test compound combinations. [Figure 9] (Figure 9a,b) Expansion of CD34+CD38- selected HSCs in various serum-containing (FBS) and serum-free media supplemented with combinations of growth factors and estrogen ligands. a) Cell counts (in millions) on days 14 (left) and 21 (right) and b) fold expansion of cells after days 14 (left) and 21 (right) of culture (p<0.05). [Figure 10] (Figure 10a,b) Phenotype of cells expanded from CD34+CD38-selected HSCs in various serum-containing (FBS) and serum-free media supplemented with combinations of growth factors and estrogen ligands. a) Fold expansion of CD34+ (HSPCs) on days 14 (left) and 21 (right) and b) Fold expansion of CD34+CD38- (LT-ICHSCs) on days 14 (left) and 21 (right) (p<0.05). [Figure 11] Assessment of the hematopoietic multilineage potential of ex vivo expanded CD34+ HSPCs after 21 days of culture in various serum (FBS) and serum-free media supplemented with combinations of growth factors and estrogen ligands. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hematopoietic stem cells (HSCs) are a type of pluripotent stem cell that have the ability to self-renew and differentiate into progenitor cells such as myeloid progenitor cells and lymphoid progenitor cells, and can ultimately differentiate into any blood cell (e.g., red blood cells) or immune system cells (e.g., T lymphocytes and B lymphocytes). HSCs are found in the bone marrow and umbilical cord blood in the body.

[0015] HSC expressing cell surface marker CD34 is known as CD34+HSC. CD34 is a transmembrane phosphorylated glycoprotein, and the nucleic acid sequence of CD34 is known and described, for example, in [Non-Patent Document 3], and the amino acid sequence of CD34 is described in [Non-Patent Document 4]. [Non-Patent Document 3] Satterthwaite AB et al., Structure of the gene encoding CD34, a human hematopoietic stem cell antigen, Genomics, 1992 Apr;12(4):788-794 [Non-Patent Document 4] Sutherland DR et al., Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34, Leukemia, 1988 Dec;2(12):793-803

[0016] CD34 can be used to identify and isolate CD34+ HSCs using methods such as flow cytometry (e.g., fluorescence-activated cell sorting (FACS)), immunomagnetic cell separation and / or immunopanning. The concepts of HSCs and CD34+ HSCs and methods for their identification and isolation are known to medical and hematology experts. For example, standard procedures for identifying HSCs as CD34+ include immunohistochemistry, flow cytometry and gene expression.

[0017] Thus, "one or more CD34+ hematopoietic stem cells" includes one or more hematopoietic stem cells that comprise the CD34 marker and / or express the CD34 gene.

[0018] "One or more" CD34+ hematopoietic stem cells include about two or more CD34+ hematopoietic stem cells, also referred to as a population of CD34+ hematopoietic stem cells. In one embodiment of the present invention, about two or more CD34+ hematopoietic stem cells and / or a population of CD34+ hematopoietic stem cells refers to about 10 or more CD34+ hematopoietic stem cells, for example, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 100 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 600 or more, about 700 or more, about 800 or more, and more. 900 or more, 1,000 or more, 1,500 or more, 2,000 or more, 2,500 or more, 3,000 or more, 3,500 or more, 4,000 or more, About 4,500 or more, about 5,000 or more, about 5,500 or more, about 6,000 or more, about 6,500 or more, about 7,000 or more, about 7,500 or more, about 8,000 or more, about 8,500 or more, about 9,000 or more, about 9,500 or more, about 10,000 or more, about 11,000 or more, about 12,000 or more, about 13,000 or more, about 14,000 or more, about 15,000 or more, about 16,0 00 or more, about 17,000 or more, about 8,000 or more, about 19,000 or more, about 20,000 or more, about 50,000 or more, about 100,000 or more, about 200,000 or more, about 300,0 "CD34+ hematopoietic stem cells" refers to about 1,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more, about 1,000,000 or more, about 1,500,000 or more, about 2,000,000 or more, about 2,500,000 or more, or about 3,000,000 or more CD34+ hematopoietic stem cells.

[0019] In some embodiments, the sample comprises about 100 CD34+ hematopoietic stem cells. In some other embodiments, the sample comprises more than about 100 CD34+ hematopoietic stem cells. In some preferred embodiments, the sample comprises about 1,000 CD34+ hematopoietic stem cells. In some other preferred embodiments, the sample comprises more than about 1,000 CD34+ hematopoietic stem cells. In some preferred embodiments, the sample comprises about 3,000,000 CD34+ hematopoietic stem cells. In some other preferred embodiments, the sample comprises more than about 3,000,000 CD34+ hematopoietic stem cells.

[0020] In some other embodiments, the sample comprises up to about 3,000,000 CD34+ hematopoietic stem cells. For example, the "one or more" populations of CD34+ hematopoietic stem cells and / or CD34+ hematopoietic stem cells are about 1,000,000 or more CD34+ hematopoietic stem cells, e.g., about 1,500,000 or more, about 2,000,000 or more, about 2,500,000 or more, or about 3,000,000 or more. In some preferred embodiments, the sample comprises about 3,000,000 CD34+ hematopoietic stem cells. In some embodiments, the sample comprises about 100 to about 3,000,000 CD34+ hematopoietic stem cells.

[0021] The number of CD34+ hematopoietic stem cells in the sample of step (i) depends on how long the culturing step of step (ii) is performed to ensure that the desired proliferation level is achieved. For example, if step (ii) is performed for 8 days, the number of CD34+ hematopoietic stem cells in the sample of step (i) can be in the range of 30,000 to 40,000, preferably 35,000 to 36,000. In another embodiment, if step (ii) is performed for 14 to 21 days, the number of CD34+ hematopoietic stem cells in the sample of step (i) can be in the range of 5,000 to 15,000, preferably 9,000 to 10,000. In yet another embodiment, if step (ii) is performed for 14 to 21 days, the number of CD34+ hematopoietic stem cells in the sample of step (i) is 1000 to 1500.

[0022] Stem cell expansion (or expanding of stem cells as referred to in the present invention) is the process by which a stem cell divides to form two daughter cells that maintain the self-renewal stem cell properties of the parent cell, or generates two daughter cells, one of which maintains the self-renewal properties of the parent stem cell and the other differentiates into a more specialized type of cell that may not have self-renewal or may have limited self-renewal capabilities. Cell division in the former case is referred to as symmetric stem cell division, and cell division in the latter case is referred to as asymmetric cell division. Both types of cell division that lead to the expansion of stem cells with self-renewal capabilities are included in the definition of "expansion" as used herein. Stem cell expansion also includes culturing CD34+ hematopoietic stem cells under specific conditions that promote cell proliferation (e.g., mitosis), as described herein, resulting in the cells renewing and dividing to produce an expanded hematopoietic stem / progenitor cell population.

[0023] Stem cell proliferation is distinct from the process of stem cell differentiation, which involves a stem cell dividing to form two daughter cells, one of which maintains the stem cell properties of the parent stem cell for fewer cell divisions than in proliferation, and the other of which differentiates into a more specialized cell type, ultimately leading to terminal differentiation of both progeny. Cell division in stem cell differentiation, sometimes called asymmetric stem cell division, does not lead to proliferation of stem cells with self-renewal capabilities.

[0024] Self-renewal is a cell division characteristic of stem cells. When self-renewal occurs, the stem cell divides into two cells, one of which is still a stem cell (which exhibits the ability to differentiate into one or more types of differentiated cells and divides almost infinitely) and the other is a differentiated cell. The differentiated cell may or may not divide. However, the division (if any) is not self-renewal because (1) stem cells (i.e., cells with developmental potential) are not generated and (2) these divisions are finite (hence, self-renewal is infinite but controlled proliferation, and therefore not self-renewal). In other words, self-renewal is the "unlimited" division of stem cells while retaining developmental potential; in other words, "stem cell proliferation" refers to the process that leads to the self-renewal of stem cells, i.e., the propagation of unlimited divisions while retaining developmental potential.

[0025] Taking HSCs as an example, proliferation refers to the division of a parent HSC to form two identical HSCs or at least one progeny HSC that retains the same stem cell properties (the other progeny forms a progenitor cell), whereas differentiation refers to the division of a parent HSC to form two progenitor cells (such as myeloid progenitors or lymphoid progenitors).

[0026] Thus, "expanding the CD34+ hematopoietic stem cells" (step ii of the invention) includes, as a result of the method of the invention, one or more CD34+ hematopoietic stem cells dividing to form one or two daughter CD34+ hematopoietic stem cells, with at least one daughter cell maintaining the stem cell properties of the parent cell, it being understood that any daughter CD34+ hematopoietic stem cell may itself expand to produce further daughter CD34+ hematopoietic stem cells, etc.

[0027] When expanding HSCs, it is most preferred to avoid differentiation of HSCs, which results in the presence of multiple cell types in the resulting cell population. A heterogeneous cell population containing a mixture of HSCs and differentiated cells will have a lower quality of therapeutic effect when compared to a cell population containing a very high proportion of HSCs or only HSCs. The advantage of the method of the present invention is that the differentiation of HSCs can be significantly limited and / or avoided compared to other expansion methods known in the art, and the resulting cell population will exhibit a high quality of therapeutic effect. Thus, by expanding CD34+HSCs by the method of the present invention, the HSCs are not substantially differentiated or are not differentiated.

[0028] Estrogen receptors are a group of proteins that are bound and activated by estrogen. As further described below, estrogen receptors are divided into two main classes: nuclear estrogen receptors and membrane estrogen receptors. An "estrogen receptor agonist" refers to a molecule that can bind to and preferably activate an estrogen receptor. As described herein, an estrogen receptor agonist is a natural molecule or an artificially synthesized molecule. In one embodiment, an estrogen receptor agonist is a molecule that functionally mimics estrogen, and in certain embodiments, an estrogen receptor agonist is a molecule that functionally mimics estrogen fully or partially. In certain embodiments, an estrogen receptor agonist is an estrogen, as described below.

[0029] In one embodiment, the estrogen receptor agonist is a natural estrogen receptor agonist or a synthetic estrogen receptor agonist.

[0030] Generally, cell culture includes growing and maintaining cells in an in vitro environment. Thus, "culturing HSCs in the presence of one or more estrogen receptor agonists" (step ii of the present invention) includes growing and / or maintaining HSCs in the presence of one or more estrogen receptor agonists, preferably in an in vitro environment. Culturing HSCs in the presence of an estrogen receptor agonist includes allowing the estrogen receptor agonist to contact the HSCs so that the estrogen receptor agonist can activate the estrogen receptor of the HSCs. Furthermore, "culturing HSCs in the presence of one or more estrogen receptor agonists" includes adding one or more estrogen receptor agonists to the sample.

[0031] In one embodiment of the invention, the CD34+ hematopoietic stem cells further comprise one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166. In a preferred embodiment, the CD34+ hematopoietic stem cells further comprise the marker CD133 (e.g., CD34+CD133+ hematopoietic stem cells). In another preferred embodiment, the CD34+ hematopoietic stem cells further comprise the marker CD45 (e.g., CD34+CD45+ hematopoietic stem cells). In a further preferred embodiment, the CD34+ hematopoietic stem cells further comprise the markers CD133 and CD45 (e.g., CD34+CD45+CD133+ hematopoietic stem cells).

[0032] CD133 (also called prominin 1) is a pentaned membrane glycoprotein that is expressed on subpopulations of CD34+ cells obtained from various sources, including fetal liver and bone marrow, adult bone marrow, umbilical cord blood, and mobilized peripheral blood. CD133+ stem cells can reconstitute mouse myeloid and lymphopoiesis (see, for example, Non-Patent Document 5). The nucleic acid sequence of CD133 is described in Non-Patent Document 6. The amino acid sequence of CD133 is described in Non-Patent Document 7. [Non-Patent Document 5] Handgretinger R, Kujiochi S. CD133-Positive Hematopoietic Stem Cells: From Biology to Medicine. Adv Exp Med Biol. 2013;777:99-111 [Non-Patent Document 6] Katoh Y and Katoh Y: Comparative genomics on PROM1 gene encoding stem cell marker CD133. Int J Mol Med 19: 967-970, 2007 [Non-Patent Document 7] Sheri Miraglia et al., A Novel Five-Transmembrane Hematopoietic Stem Cell Antigen: Isolation, Characterization, and Molecular Cloning. Blood 1997; 90 (12): 5013_5021

[0033] CD45 is a receptor-associated protein tyrosine phosphatase present on all hematopoietic cells except erythrocytes and plasma cells. It was originally known as common leukocyte antigen or protein tyrosine phosphatase receptor type C. CD45 regulates various cellular processes such as cell growth, differentiation, the mitotic cycle, and tumorigenesis. The CD45 protein family consists of multiple members that are all products of one complex gene. CD45RA contains only the A protein domain, whereas the shortest CD45 isoform, CD45RO, lacks all three domains A, B, and C. The nucleic acid and amino acid sequences of CD45 are described in [8]. [Non-Patent Document 8] Holmes N. CD45: all is not yet crystal clear. Immunology. 2006 Feb;117(2):145-55.

[0034] CD43, also known as leukosialin or sialoglycoprotein, has been shown to be expressed on early hematopoietic cells derived from human ES cells. Like CD34, CD43 is a highly glycosylated transmembrane sialomucin that is expressed on all hematopoietic cells except mature erythrocytes. Details of CD43 are described in [Non-Patent Document 9]. The nucleic acid and amino acid sequences of CD43 are described in [Non-Patent Document 10]. [Non-Patent Document 9] Kessel KU et al., Emergence of CD43-Expressing Hematopoietic Progenitors from Human Induced Pluripotent Stem Cells. Transfus Med Hemother. 2017 Jun;44(3):143-150 [Non-Patent Document 10] Cyster J et al., Protein sequence and gene structure for mouse leukosialin (CD43), a T lymphocyte mucin without introns in the coding sequence. Eur J Immunol. 1990 Apr;20(4):875-81

[0035] CD59 is a phosphoinositol glycan-binding membrane protein that is uniformly expressed on most nucleated cells of the hematopoietic system, including all CD34+ cells derived from BM. However, CD59 is differentially expressed among CD34+ progenitor cells, with the highest levels present on subsets highly enriched for pluripotent stem cells. Details of CD59 are described in [Non-Patent Document 11]. [Non-Patent Document 11] Simmons PJ et al., A novel epitope of CD59 expressed by primitive human hematopoietic progenitors. Exp Hematol. 2001 Dec;29(12):1474-83

[0036] The nucleic acid and amino acid sequences of CD59 are described in [Non-Patent Document 12]. [Non-Patent Document 12] JG Petranka et al., Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein. Proceedings of the National Academy of Sciences Sep 1992, 89 (17) 7876-7879

[0037] CD90 or thymocyte differentiation antigen (Thy-1) is a small GPI-anchored glycoprotein of 25-35 kDa that is an important regulator of cell-cell and cell-matrix interactions. It is involved in tumor suppression, cell adhesion, migration, and apoptosis signaling. CD90 is highly expressed in highly proliferative cord blood progenitor cells and is involved in HSC development. Details of CD90 are described in [Non-Patent Document 13]. The nucleic acid sequence of CD90 is described in [Non-Patent Document 14]. The amino acid sequence of CD90 is described in [Non-Patent Document 15]. [Non-Patent Document 13] Mayani H, Lansdorp PM. Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. Blood.1994;83:2410_2417 [Non-Patent Document 14] Craig W et al., Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med. 1993 May 1;177(5):1331-42 [Non-Patent Document 15] Wollscheid B et al., Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol. 2009 Apr;27(4):378-86.

[0038] CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein that is localized on the surface of platelets, activated T cells, endothelial cells, and a subset of fetal and adult CD34+ bone marrow mononuclear cells. CD109 negatively regulates transforming growth factor beta (TGF-beta) signaling associated with HSC differentiation. For details on CD109, see [Non-Patent Document 16]. The nucleic acid and amino acid sequences of CD109 are described in [Non-Patent Document 17]. [Non-Patent Document 16] Martin Lin et al.; Cell surface antigen CD109 is a novel member of the α\och2 macroglobulin / C3, C4, C5 family of thioester-containing proteins. Blood 2002; 99 (5): 1683_1691 [Non-Patent Document 17] Lin M et al., Cell surface antigen CD109 is a novel member of the alpha(2) macroglobulin / C3, C4, C5 family of thioester-containing proteins. Blood. 2002 Mar 1;99(5):1683-91

[0039] CD117 or c-KIT is a receptor tyrosine kinase type III that binds to stem cell factor, also known as "steal factor" or "c-kit ligand," and plays an important role in cell survival, proliferation, and differentiation. CD117 is highly expressed on hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), and common myeloid progenitor cells (CMPs), and promotes cell proliferation. CD117 is described in detail in [Non-Patent Document 18]. The nucleic acid and amino acid sequences of CD117 are described in [Non-Patent Document 19]. [Non-Patent Document 18] Edling CE, Hallberg B. c-Kit--a hematopoietic cell essential receptor tyrosine kinase. Int J Biochem Cell Biol. 2007;39(11):1995-8. doi: 10.1016 / j.biocel.2006.12.005 [Non-Patent Document 19] Yarden Y et al., Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987 Nov;6(11):3341-51

[0040] CD166, also known as activated leukocyte cell adhesion molecule (Alcam), is a member of the immunoglobulin superfamily and mediates homophilic (CD166-CD166) and heterophilic (CD166-CD6) interactions. CD166 is expressed in stem and progenitor cells of all three embryonic lineages, and CD166 interaction activates key molecular pathways involved in enhancing HSC / HPC function involved in maintaining stem cell pluripotency. Details of CD166 are described in [Non-Patent Document 20]. The nucleic acid and amino acid sequences of CD166 are described in [Non-Patent Document 21]. [Non-Patent Document 20] Zhang J et al., CD166 Engagement Augments Mouse and Human Hematopoietic Progenitor Function via Activation of Stemness and Cell Cycle Pathways. Stem Cells. 2019 Oct;37(10):1319-1330 [Non-Patent Document 21] Bowen MA et al., Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand. J Exp Med. 1995 Jun 1;181(6):2213-20.

[0041] The standard procedures described above for identifying HSCs as CD34+ can also be used to identify HSCs with additional markers (e.g., CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 or CD166), including immunohistochemistry, flow cytometry and gene expression.

[0042] "The hematopoietic stem cells further comprise one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166" includes that the one or more hematopoietic stem cells express the CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166 genes.

[0043] As one skilled in the art of molecular biology and / or genetics will appreciate, expression of a particular gene (such as CD133) includes the presence of a gene product, such as mRNA and / or protein.

[0044] In one embodiment, the CD34+ hematopoietic stem cells lack one or more markers selected from the list consisting of CD38, HLA DR and Lineage markers (Lin).

[0045] CD38, also known as cyclic ADP-ribose hydrolase, is a glycoprotein on the surface of leukocytes (white blood cells) that functions in cell adhesion, signal transduction, and calcium signaling. The CD38 protein is rearranged at high levels by activated lymphocytes and is a marker of cell activation. Expression of the cell surface antigen CD38 on hematopoietic cells is activated throughout differentiation, and thus exclusion of CD38 expression on CD34+ cells is associated with long-term HSC activity. CD38 is described in more detail in

[22] . The nucleic acid and amino acid sequences of CD38 are described in

[23] . [Non-Patent Document 22] Cicuttini FM et al., Characterization of CD34+HLA-DR-CD38+ and CD34+HLA-DR-CD38- progenitor cells from human umbilical cord blood. Growth Factors. 1994;10(2):127-34 [Non-Patent Document 23] Nata K et al., Human gene encoding CD38 (ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase): organization, nucleotide sequence and alternative splicing. Gene. 1997 Feb 28;186(2):285-92

[0046] HLA-DR is a major histocompatibility complex class II (MHC class II) cell surface receptor encoded by multiple loci and genes of different functions in the human leukocyte antigen complex located in chromosome 6 region 6p21.31. The receptor / peptide complex is a ligand for the T cell receptor (TCR) and mediates the acceptance / rejection of organ / blood transplants in recipients. HLA-DR is also involved in several autoimmune diseases, disease susceptibility and disease resistance. HLA-DR is expressed in umbilical cord and peripheral blood cells. Most primitive HSCs co-express c-kit, FLT-3 and Thy-1 and are negative for HLA-DR, CD38 and lineage markers. Details of HLA-DR are described in [Non-Patent Document 24]. The nucleic acid and amino acid sequences of HLA-DR are described in [Non-Patent Document 25]. [Non-Patent Document 24] Stern J. Lawrence and Calvo-Calle Mauricio J., HLA-DR: Molecular Insights and Vaccine Design, Current Pharmaceutical Design 2009; 15(28) [Non-Patent Document 25] Das HK et al., Structure and nucleotide sequence of the heavy chain gene of HLA-DR. Proceedings of the National Academy of Sciences. 1983, 80(12):3543-3547

[0047] Lineage markers (Lin) refer to the expression of various lineage markers associated with a particular cell type. Hematopoietic stem cells give rise to various types of blood cells called myeloid and lymphoid lineages. Both myeloid and lymphoid lineages are responsible for the formation of myeloid cells (from monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes to platelets) and lymphoid cells (T cells, B cells, natural killer cells and innate lymphoid cells). Hematopoietic stem cells can be identified or separated from the surrounding blood cells using flow cytometry. In flow cytometry, a combination of one or more different cell surface markers (mentioned above) in combination with CD34 is used. Thus, HSCs that lack expression of mature blood cell markers, including but not limited to CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 and CD235a are called lineage negative (Lin-). The lack of expression of lineage markers is used in combination with the detection of several positive cell surface markers to isolate hematopoietic stem cells. Thus, in the context of the present invention, hematopoietic stem cells lacking lineage markers (Lin) lack expression of one or more markers associated with mature blood cells. Thus, in some embodiments, hematopoietic cells lack expression of one or more markers selected from CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 and CD235a. Isolation and / or enrichment of hematopoietic stem cells containing and / or lacking specific markers are further described herein.

[0048] The standard procedures described above for identifying HSCs as CD34+ can also be used to identify HSCs that lack differentiation markers such as CD38, HLA DR and / or Lin, including immunohistochemistry, flow cytometry, and gene expression.

[0049] "The hematopoietic stem cells lack one or more markers selected from the list consisting of CD38, HLA DR and / or Lin" includes that one or more hematopoietic stem cells do not contain the CD38, HLA DR and / or Lin markers and / or do not express the CD38, HLA DR and / or Lin genes described above.

[0050] In one embodiment, the CD34+ hematopoietic stem cells further comprise one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, and / or CD166, and the CD34+ hematopoietic stem cells lack one or more markers selected from the list consisting of CD38, HLA DR, and / or Lin. In a preferred embodiment, the CD34+ hematopoietic stem cells further comprise the CD133 and CD45 markers and lack the CD38 marker (e.g., CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells). In a further preferred embodiment, the CD34+ hematopoietic stem cells further comprise the CD133 marker and lack the CD38 marker (e.g., CD34+CD133+CD38- hematopoietic stem cells).

[0051] "Low / intermediate" CD45 refers to CD45 expression, and their relative levels are understandable to those skilled in molecular biology. Low / intermediate CD45 expression includes low CD45 expression and / or intermediate CD45 expression. A method to distinguish such expression is to use flow cytometry. Relative CD45 expression is relevant to the present invention, since high CD45 expression is associated with differentiated lymphocytes. Generally, in flow cytometry or other fluorescence-based separation methods, cells are gated based on the relative intensity of a fluorescent marker (such as CD45). In the selection of HSCs, high intensity CD45 expressing cells are excluded and only HSCs are selected. As will be understood by those skilled in the art, the intensity scale depends on the sensitivity of the machine (flow cytometer) and may differ between different instruments, so cells are gated based on the relative intensity within that analyzer / instrument.

[0052] In the embodiments described herein, when CD34+ hematopoietic stem cells contain additional specific markers (such as CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166) and / or do not contain differentiation markers (such as CD38, HLA DR and / or Lin), these hematopoietic stem cells are understood to be a subpopulation of the general population of CD34+ hematopoietic stem cells.

[0053] In one embodiment of the invention, the CD34+ hematopoietic stem cells are long-term repopulating hematopoietic stem cells (LT-HSC).

[0054] Long-term repopulating hematopoietic stem cells (LT-HSCs) are generally understood to be CD34+CD133+CD38- hematopoietic stem cells. Surprisingly, the present inventors have confirmed that the method of the present invention is particularly effective in expanding not only CD34+CD133+CD45+(low / intermediate)CD38- hematopoietic stem cells, but also subpopulations of this hematopoietic stem cell, as described in the Examples herein.

[0055] In certain embodiments, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells, the one or more CD34+ hematopoietic stem cells further comprising one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, and / or CD166, and / or lacking one or more markers selected from the list consisting of CD38, HLA DR, and / or Lin. In a preferred embodiment, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD133+ hematopoietic stem cells. In another preferred embodiment, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD45+(low / intermediate) hematopoietic stem cells. In a further preferred embodiment, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD45+(low / intermediate) hematopoietic stem cells and / or one or more CD34+CD133+ hematopoietic stem cells and / or one or more CD34+CD45+(low / intermediate)CD133+ hematopoietic stem cells. In another preferred embodiment, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD133+CD38- hematopoietic stem cells. In a further preferred embodiment, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD133+CD45+(low / intermediate)CD38- hematopoietic stem cells.

[0056] To describe these embodiments in another way, the sample may comprise a mixture of CD34+ hematopoietic stem cells with or without one or more additional markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166. In a preferred embodiment, the sample comprises a mixture of CD34+ hematopoietic stem cells and CD34+CD45+(low / intermediate) hematopoietic stem cells and / or CD34+CD133+ hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells. In a further preferred embodiment, the sample comprises a mixture of CD34+ hematopoietic stem cells and CD34+CD133+CD38- hematopoietic stem cells. In a further preferred embodiment, the sample comprises a mixture of CD34+ hematopoietic stem cells and CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells.

[0057] In some embodiments of the invention, when there is a mixture of CD34+ hematopoietic stem cells and CD34+ hematopoietic stem cells containing and / or lacking other specific markers, the daughter CD34+ hematopoietic stem cells of step ii) contain and / or lack the same specific markers as the parent CD34+ hematopoietic stem cells. For example, daughter hematopoietic stem cells derived from parent CD34+CD133+ hematopoietic stem cells also contain the markers CD34 and CD133, and / or daughter hematopoietic stem cells derived from parent CD34+CD133+CD38- hematopoietic stem cells also contain the markers CD34 and CD133 and lack the CD38 marker. In some other embodiments, the daughter CD34+ hematopoietic stem cells of step ii) contain or lack specific markers different from the parent CD34+ hematopoietic stem cells due to variations in conditions. For example, daughter hematopoietic stem cells derived from parent CD34+CD133+CD38- may lack the CD133 marker and / or further contain the CD38 marker.

[0058] In one embodiment, the CD34+ hematopoietic stem cells comprise a sustained stem cell phenotype. In particular, the CD34+CD133+ hematopoietic stem cells comprise a sustained stem cell phenotype.

[0059] In general, HSC stemness refers to the ability of HSCs to self-renew and generate two identical progeny, but unless sustained, the daughter cells cannot generate two further identical progeny, one of which may retain the same phenotype and the other committed to differentiation into a specific blood cell progenitor. Thus, the sustained stemness phenotype of HSCs involves differentiation-restricted self-renewal and retains the ability to differentiate into all types of blood cells when the relevant inductive signals are provided. Sustained stemness is also called maintained stemness.

[0060] In one embodiment, the sustained stemness phenotype is characterized by expanded CD34+ hematopoietic stem cells that contain the same specific markers as the CD34+ hematopoietic stem cells from which they were derived (e.g., CD34+ and / or one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, and / or CD166, and / or the absence of one or more markers selected from the list consisting of CD38, HLA DR and / or Lin).

[0061] In yet another embodiment, a sustained stem cell phenotype is characterized by the ability of expanded CD34+ hematopoietic stem cells to persist in long-term culture (eg, long-term culture on bone marrow feeder cells).

[0062] In yet another embodiment, the sustained stem cell phenotype is characterized by the ability of expanded CD34+ hematopoietic stem cells to differentiate into progenitor cells (preferably, progenitor cells capable of differentiating into any type of mature blood cell).

[0063] Methods for identifying whether HSCs can differentiate into progenitor cells (and ultimately mature blood cells) are known to those skilled in the art of cell biology, for example, this can be assessed using the methods described in the Examples, e.g., whether expanded HSCs can generate colonies of distinct progenitor cells.

[0064] In one embodiment of the invention, step ii) comprises culturing the hematopoietic stem cells in a medium containing an estrogen receptor agonist.

[0065] As is well known to those skilled in the art of cell biology, culture media (commonly referred to as cell media) is generally a liquid that contains components for growing and maintaining cells.

[0066] In one embodiment of the invention, the medium is a defined media.

[0067] As known to those skilled in the art of cell biology, a defined medium is one in which all components and their relative concentrations and / or amounts are known. This is in contrast to a medium that contains components with unknown or undefined content, concentration and / or amount. Generally, defined media do not contain components of animal origin, but instead contain chemically synthesized components and / or components of non-animal origin, allowing for greater control over the contents of the medium.

[0068] In one embodiment, the medium is serum-free media, and in a particular embodiment, the serum-free media is animal serum-free media.

[0069] As cell biologists are well aware, serum is an example of a medium component whose content, concentration and / or amount are not clearly defined, and therefore the presence of serum in a medium makes it no longer a defined medium. Furthermore, it may be desirable to avoid using media containing serum in order to avoid contamination with infectious agents (e.g., viruses) or immunization of the recipient patient with foreign proteins.

[0070] In one embodiment, the media is a xeno-free media.

[0071] As known to cell biologists, a xeno-free medium is a medium that does not contain components that are not derived from the same species as the cells being cultured; for example, when culturing human cells, the corresponding xeno-free medium would contain no non-human biological components.

[0072] In one embodiment of the invention, step ii) comprises culturing the cells of step i) without feeder cells.

[0073] As known to cell biologists, feeder cells can be co-cultured with HSCs, providing the HSCs with a mixture of undefined components.

[0074] In one embodiment of the invention, step ii) further comprises culturing the cells of step i) in the presence of one or more growth factors and / or cytokines.

[0075] Growth factors are biological substances that can regulate cellular processes, such as stimulating cell proliferation. Growth factors are often cell signaling substances and / or hormones produced by cells, although in some cases growth factors can be artificially synthesized. Cytokines are also biological substances that can mediate cellular processes, such as hematopoiesis. Cytokines are signaling molecules produced by cells and can be artificially synthesized. Methods for identifying growth factors and cytokines that can be utilized as part of the present invention are known to those skilled in the art of cell biology. Thus, growth factors and cytokines include biological substances that can stimulate cell proliferation.

[0076] In one embodiment of the invention, the one or more growth factors and / or cytokines are selected from the list consisting of: granulocyte-macrophage colony-stimulating factor (GM-CSF) (preferably recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF)); granulocyte-colony-stimulating factor (G-CSF) (preferably recombinant human granulocyte-colony-stimulating factor (rhG-CSF)); Flt-3 Ligand (preferably recombinant human Flt-3 Ligand (rhFlt-3L)); Stem Cell Factor (preferably recombinant human Stem Cell Factor (rhSCF) - also known as KIT Ligand or Steel Factor); known); IGF binding protein 2 (IGFBP2) (preferably, recombinant human insulin-like growth factor (IGF) binding protein 2 (rhIGFBP2)); Notch ligand; Notch ligand sDLL1 (preferably, recombinant human soluble DLL-1 (rhsDLL-1)), interleukin-3 (IL-3) (preferably, recombinant human interleukin-3 (rhIL-3)), interleukin-6 (IL-6) (preferably, recombinant human interleukin-6 (rhIL-6)), and / or thrombopoietin (TPO) (preferably, recombinant human thrombopoietin (rhTPO)). In a preferred embodiment, the one or more growth factors are selected from the list consisting of granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Flt-3 ligand, stem cell actor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), IGF binding protein 2 (IGFBP2) and / or notch ligand sDLL1. In a more preferred embodiment, the one or more growth factors are granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF). In another preferred embodiment, the one or more growth factors are interleukin-3 (IL-3) and interleukin-6 (IL-6).In certain embodiments, the one or more growth factors are Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3) and interleukin-6 (IL-6), which are growth factors added to the basal medium referred to herein as "FKT36 medium."

[0077] In another embodiment, Notch ligand (such as Notch ligand sDLL1) and / or IGFBP2 are not used in the methods of the invention.

[0078] In certain embodiments, the one or more growth factors are granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF), and a Notch ligand (such as the Notch ligand sDLL1) is not used in the methods of the invention.

[0079] In certain embodiments, the growth factors and / or cytokines may be in the following amounts: interleukin-3 (IL-3) from about 1 ng / mL to about 20 ng / mL (preferably about 10 ng / mL); interleukin-6 (IL-6) from about 50 ng / mL to about 150 ng / mL (preferably about 100 ng / mL); thrombopoietin (TPO) from about 50 ng / mL to about 150 ng / mL (preferably about 100 ng / mL); Flt-3 Ligand (Flt-3L) from about 50 ng / mL to about 150 ng / mL (preferably about 100 ng / mL); L (preferably, about 100 ng / mL) stem cell factor, about 1 ng / mL to about 20 ng / mL (preferably, about 10 ng / mL) granulocyte-macrophage colony-stimulating factor (GM-CSF), about 1 ng / mL to about 20 ng / mL (preferably, about 10 ng / mL) granulocyte-colony stimulating factor (G-CSF), about 50 ng / mL to about 150 ng / mL (preferably, about 100 ng / mL) insulin-like growth factor (IGF) binding protein 2 (IGFBP2), and / or about 10 ng / mL to about 100 ng / mL (preferably, about 50 ng / mL) DLL-1 (sDLL-1).

[0080] The cytokines and growth factors described herein are commercially available and are generally produced as chemical synthesis or recombinant products. Preferably, the growth factors / cytokines are derived from the same species as the starting sample cell population (preferably human) and are recombinantly expressed, and their nucleic acid and peptide sequences are available from NCBI Genbank. For example, recombinant human interleukin-3 (rhIL-3), recombinant human interleukin-6 (rhIL-6), recombinant human thrombopoietin (rhTPO), recombinant human Flt-3 ligand (rhFlt-3L), recombinant human stem cell factor (rhSCF), recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF or GMCSF), recombinant human granulocyte colony stimulating factor (rhG-CSF or GCSF), recombinant human insulin-like growth factor (IGF) binding protein 2 (rhIGFBP2), and recombinant human soluble DLL-1 (rhsDLL-1) can be purchased from Peprotech Nordic (Sweden).

[0081] The term "basal medium" refers to a medium that, when supplemented with either the serum or serum-free components of the present invention, is capable of supporting the growth of hematopoietic cells, such as CD34+ hematopoietic cells or other cells of the present invention. The basal medium should be capable of providing inorganic salts, such as sodium, iron, magnesium, calcium, potassium, trace elements, vitamins, an energy source, such as glucose, a pH buffering system, and the essential amino acids required for standard cell growth. Basal media that can be used as part of the present invention include, but are not limited to, Iscove's Modified Dulbecco's Medium, RPMI 1640, Minimum Essential Medium a (MEM-a), and other related media.

[0082] In one embodiment of the invention, the basal medium used in the invention (e.g. step i) and / or step ii) of the invention is Iscove's Modified Dulbecco's Medium (IMDM) (ThermoFisher Scientific, Merck, KGaA / Sigmaaldrich), RPMI 1640 or Minimum Essential Medium-a (MEM-a).

[0083] In one embodiment of the invention, the basal medium (such as Iscove's Modified Dulbecco's Medium (IMDM)) is further supplemented with one or more components selected from the list of components consisting of human serum albumin, ethanolamine, sodium selenite, hydrocortisone, D,L-tocopherol, human transferrin (holo), human insulin (zinc), N-acetyl-L-cysteine, 2-mercaptoethanol and / or cholesterol lipid concentrate (available from Thermo Fisher Scientific or others). In certain embodiments, each component may be in the following amounts: about 1% to about 10% (preferably about 5%) human serum albumin, about 1 mg / L to about 20 mg / L (preferably about 10 mg / L) ethanolamine, about 1 μg / L to about 10 μg / L (preferably about 5 μg / L) sodium selenite, about 20 μg / L to about 70 μg / L (preferably 50 μg / L) hydrocortisone, about 10 μg / L to about 30 μg / L (preferably about 20 μg / L) D,L-tocopherol, and about 80 mg / L to about 120 mg / L (preferably about 100 mg / L) human transferrin (holo). about 1 mg / L to about 20 mg / L (preferably about 10 mg / L) human insulin (zinc); about 140 mg / L to about 180 mg / L (preferably about 160 mg / L) N-acetyl-L-cysteine; about 1 mg / L to about 10 mg / L (preferably about 4 mg / L) 2-mercaptoethanol; and / or cholesterol lipid concentrate (about 200x to about 300x, preferably about 250x, Thermo Fisher Scientific). In a particularly preferred embodiment, the basal medium (e.g., Iscove's Modified Dulbecco's Medium (IMDM)) contains 5% human serum albumin, 10 mg / L ethanolamine, 5 μg / L sodium selenite, 50 μg / L hydrocortisone, 20 μg / L D,L-tocopherol, 100 mg / L human transferrin (holo), 10 mg / L human insulin (zinc) (a common mixture of insulin and zinc), 160 mg / L N-acetyl-L cysteine, 4 mg / L 2-mercaptoethanol, and cholesterol lipid concentrate (250X, ThermoFisher Scientific).

[0084] In one embodiment of the invention, one or more CD34+ hematopoietic stem cells are cultured with one or more growth factors before and / or simultaneously with and / or after culturing the cells with one or more estrogen receptor agonists.

[0085] In one embodiment of the present invention, the sample is one or more selected from the group consisting of a sample comprising isolated CD34+ hematopoietic stem cells, a sample comprising isolated hematopoietic stem cells comprising CD34+ hematopoietic stem cells, a medium comprising CD34+ hematopoietic stem cells, a blood sample and / or a bone marrow sample. In a preferred embodiment, the blood sample comprises CD34+ hematopoietic stem cells. In another preferred embodiment, the bone marrow sample comprises CD34+ hematopoietic stem cells.

[0086] In embodiments where the sample is a culture medium containing CD34+ hematopoietic stem cells, step ii) comprises adding an estrogen receptor agonist to the culture medium.

[0087] In one embodiment of the invention, the blood sample is one or more selected from the list consisting of peripheral blood, mobilised peripheral blood, placental blood and / or umbilical cord blood. Preferably, the sample is a blood sample which is umbilical cord blood and / or placental blood. Preferably, the sample is a human sample and / or a sample derived from a human.

[0088] The blood samples described herein are well known to those skilled in medicine and / or hematology, and methods for identifying and obtaining such samples are well known. Blood samples can be obtained from the subject or from commercial sources. For example, umbilical cord blood can be obtained from Zen Bio, USA. Peripheral blood is circulating within the subject's body and is not sequestered, for example, in the lymphatic system, spleen, liver, or bone marrow. Thus, "peripheral blood" includes blood obtained or obtainable from blood circulating within the subject's body. Exemplary methods for obtaining peripheral blood include venipuncture and / or lymphopheresis. Mobilized peripheral blood is peripheral blood that has been treated with so-called "mobilizing agents," which are agents that increase the number of stem cells (particularly HSCs) in peripheral blood. Examples of mobilizing agents include granulocyte colony-stimulating factor and plerixafor. The subject is provided with the mobilizing agent prior to collection of peripheral blood. Thus, "mobilized peripheral blood" includes blood obtained or derived from blood circulating in a subject to which a mobilizing agent was initially provided and / or administered. Umbilical cord blood is blood obtained from the umbilical cord, and placental blood is blood obtained from the placenta, which are often rich sources of stem cells, particularly HSCs. These blood samples are typically taken shortly after the birth of the baby. Thus, "umbilical cord blood" includes blood samples obtained or derived from the umbilical cord. Thus, "placental blood" includes blood samples obtained or derived from the placenta.

[0089] Bone marrow samples are well known to those skilled in the art of medicine and / or hematology, as are methods for identifying and obtaining such samples. Bone marrow is tissue found within the bones of a subject and is often a rich source of stem cells, particularly HSCs. One method for obtaining bone marrow is a bone marrow aspiration, in which a hollow needle is inserted through the bone, into the bone marrow, and withdrawn into an attached syringe. Bone marrow may also be obtained from commercial sources. "Bone marrow sample" includes a sample obtained or obtainable from bone marrow.

[0090] As described herein, the preferred blood sample is umbilical cord blood and / or placental blood. These blood samples can be obtained by any procedure known in the art. Using umbilical cord blood / placental blood as a stem cell source has several advantages, such as easy blood sample acquisition without trauma to the donor. For an overview of umbilical cord blood and placental blood collection at birth, see, for example, U.S. Patent No. 5,004,681. [Patent Document 1] U.S. Pat. No. 5,004,681

[0091] Collection of blood samples (such as umbilical cord blood and / or placental blood) is performed under sterile conditions and the blood is immediately mixed with an anticoagulant.

[0092] Thus, in one embodiment of the invention, the sample in step i) of the method of the invention comprises an anticoagulant and / or step i) comprises contacting the sample with an anticoagulant, e.g. adding an anticoagulant to the sample.

[0093] The anticoagulant used in the present invention can be any known in the art, such as those described herein or those described in

[26] , which is incorporated herein by reference. [Non-Patent Document 26] 1968, Storage of Blood, Academic Press, New York, pp. 26-160

[0094] In one embodiment of the invention, the anticoagulant is one or more anticoagulants selected from the list including ACD (acid citrate dextrose), CPD (citrate-phosphate-dextrose), De Gowin solution (Non-Patent Document 27), Alsever solution (Non-Patent Document 28), Rous-Turner solution (Non-Patent Document 29), Edglugate-Mg (Non-Patent Document 30), other glucose mixtures, heparin and / or ethyl biscoumatate. [Non-Patent Document 27] De Gowin, et al., 1940, J. Am. Med. Ass. 114:850); Alsever's solution (Alsever et al., 1941, NY St. J. Med. 41:126 [Non-Patent Document 28] Alsever et al., 1941, NY St. J. Med. 41:126); Rous-Turner Solution (Rous and Turner, 1916, J. Exp. Med. 23:219) [Non-Patent Document 29] Rous and Turner, 1916, J. Exp. Med. 23:219 [Non-Patent Document 30] Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573

[0095] In one embodiment, the sample (such as a blood sample) is free of contamination, such as contamination with an infectious agent.

[0096] In one embodiment of the invention, the infectious agent is one or more infectious agents selected from the list consisting of human immunodeficiency virus-1, human immunodeficiency virus-2, hepatitis B, hepatitis C, T-cell lymphotropic virus I (HTLV-I), T-cell lymphotropic virus II (HTLV-II), West Nile virus, cytomegalovirus and / or syphilis.

[0097] In one embodiment of the invention, the method includes a step (preferably prior to step i)) of testing the sample for contamination, for example contamination with an infectious agent.

[0098] Testing for contamination is known to those skilled in the art, particularly those skilled in the art of microbiology.

[0099] In certain embodiments, testing for contamination includes bacterial culture and / or pathogenic microorganism screening.

[0100] "Bacterial culture" includes the cultivation of bacteria in aerobic and anaerobic environments.

[0101] "Pathogen screening" includes various diagnostic tests that can be performed to confirm the absence of certain pathogenic microorganisms that are transmitted through blood. For example, a sample (such as a blood sample) can be screened for the presence of human immunodeficiency virus-1 or 2 (HIV-1 or HIV-2). In another example, a collected sample can also be screened for human hepatitis B, hepatitis C, T-cell lymphotropic virus I and II (HTLV-I and HTLV-II), West Nile virus, cytomegalovirus, syphilis, and other communicable diseases.

[0102] Prior to collection of the blood, particularly where the blood sample is umbilical cord blood and / or placental blood, the maternal health history may be evaluated to identify risk of transmission of infectious or genetic diseases such as leukemia, immune disorders, cancer, hepatitis, neurological disorders, viral diseases, AIDS, etc. The collected blood sample may be screened for one or more of total nucleated cell count, cell viability, CD34+ cell count, HLA type, and ABO / Rh type.

[0103] As will be appreciated, if the sample contains contaminants it may not be usable in the methods of the invention and / or the methods of the invention include a step (preferably prior to step i)) of removing the contaminants.

[0104] After collection of a sample (such as a blood sample, particularly umbilical cord blood and / or placental blood), the sample can be treated to enrich for hematopoietic stem / progenitor cells (particularly CD34+ hematopoietic stem cells) as described herein.

[0105] In one embodiment of the invention, the estrogen receptor agonist is one or more molecules selected from the list consisting of endoestrogens, phytoestrogens, steroid ligands, tamoxifen analogues, synthetic estrogens, xenoestrogens and / or non-steroidal ligands.

[0106] An endoestrogen is a molecule that is capable of binding to and preferably activating an estrogen receptor and that is endogenously produced by a subject, for example a human subject.

[0107] In one embodiment of the invention, the endoestrogen is one or more molecules selected from the list consisting of estrogen E2 (also known as E2, estradiol and / or estradiol) and its derivatives, estrogen E3 (also known as E3, estriol and / or estriol) and its derivatives, estrogen E1 (also known as E1, estrone and / or estrone) and its derivatives, estrogen E4 (also known as E4, estetrol and / or estetrol) and its derivatives, Δ(5)-androstenediol and its derivatives, 5α-androstanediol and its derivatives, 3β-androstanediol (3β-adiol) and its derivatives and / or 27-hydroxycholesterol and its derivatives. Most preferably, the estrogen receptor agonist is an endoestrogen, i.e. estrogen 2.

[0108] Estrogens 1 to 4 described herein are all steroid hormones metabolically produced from cholesterol through a series of catalytic reactions. The chemical structures of estrogens are known in the art and are described in [Non-Patent Document 31]. [Non-Patent Document 31] Nagai MA, Brentani MM. Gene expression profiles in breast cancer to identify estrogen receptor target genes. Mini Rev Med Chem. 2008 May;8(5):448-54.

[0109] Estrogens are hormones that are metabolically produced from cholesterol through a series of catalytic reactions involving several enzymes, including cytochrome P450 aromatase (CYP19A1), and their nucleic acid and amino acid sequences are known. Detailed information on the chemical structures of estrogens is provided in

[32] . [Non-Patent Document 32] Nagai MA, Brentani MM. Gene expression profiles in breast cancer to identify estrogen receptor target genes. Mini Rev Med Chem. 2008 May;8(5):448-54. doi: 10.2174 / 138955708784223503. PMID: 18473934

[0110] Phytoestrogens are molecules capable of binding to and preferably activating estrogen receptors and are nonsteroidal polyphenolic compounds produced naturally by plants.

[0111] In one embodiment, the phytoestrogen is one or more molecules selected from the list consisting of isoflavones, coumestans, lignans, genistein, S-equol and / or liquiritigenin.

[0112] Phytoestrogens, also called "dietary estrogens," are found in certain edible plants, including soybean, flaxseed, sesame, licorice, hops, and ginseng, with the highest abundance in soybean (isoflavones). Phytoestrogens are similar to endoestrogens and exhibit both genomic and non-genomic effects of phytoestrogens, including selective but weak binding to estrogen receptors. Phytoestrogens may have beneficial effects on menopausal symptoms, bone health, and cardiovascular health (33). [Non-Patent Document 33] North American Menopause Society (2011), NAMS 2011 Flavones Report, Menopause, 18:732_753; Cederroth & Nef (2009), Molecular and Cellular Endocrinology, 304: 30_42.

[0113] A steroidal ligand is a molecule that is capable of binding to and preferably activating the estrogen receptor, and is a steroidal molecule.

[0114] In one embodiment, the steroidal ligand is one or more molecules selected from the list consisting of 11β-ether-17α-ethynyl-3,17β-estradiol, combretastatin A4 analogs (preferably combretastatin A4 analogs on a steroid framework) and / or C6-piperazine substituted purine steroidal nucleosides.

[0115] A tamoxifen analog is a molecule capable of binding to and preferably activating the estrogen receptor which is tamoxifen or which is structurally and / or functionally similar to tamoxifen.

[0116] In one embodiment of the invention, the tamoxifen analogue is one or more molecules selected from the list consisting of 4-hydroxytamoxifen (4-HT), tamoxifen and / or raloxifene and / or toremifene. Tamoxifen, raloxifene and toremifene are used in the treatment of estrogen-sensitive breast cancer and in the prevention of osteoporosis in postmenopausal women (34). [Non-Patent Document 34] Jordan (2003), Nature Reviews Drug Discovery, 2:205_213; Barrett-Connor et al. (2006), New England Journal of Medicine, 355:125_137

[0117] Synthetic estrogens are molecules capable of binding to and preferably activating estrogen receptors, and are chemically modified forms of the natural hormone estradiol. Synthetic estrogens are used in various medical applications, such as hormone replacement therapy (HRT) and oral contraceptives, and have been demonstrated to alleviate menopausal symptoms, prevent osteoporosis, and treat certain reproductive disorders (Non-Patent Document 35). In one embodiment of the present invention, the synthetic estrogen is one or more molecules selected from the list consisting of ethinyl estradiol, mestranol, and conjugated equine estrogens. [Non-Patent Document 35] Lobo (2017) Nature Reviews Endocrinology, 13:220_231

[0118] In one embodiment of the present invention, the estrogen receptor agonist is a xenoestrogens. Xenoestrogens are synthetic or natural compounds that mimic the effects of endogenous estrogens and are found in some industrial chemicals, pesticides, and plastics, such as bisphenol A (BPA). Xenoestrogens may affect human health due to their estrogenic activity (Non-Patent Document 36). [Non-Patent Document 36] Gore et al. (2015), Endocrine Reviews, 36: E1·E150

[0119] A non-steroidal ligand is a molecule that is capable of binding to and preferably activating the estrogen receptor, and is a non-steroidal molecule.

[0120] In one embodiment of the invention, the steroid ligand is one or more molecules selected from the list consisting of benzopyran derivatives (e.g., LY-500307 (also known as elteberel), LY-3201 (also known as (3aS,4R,9bR)-2,2-difluoro-4-(4-hydroxyphenyl)-3,3a,4,9b-tetrahydro-1H-cyclopenta(c)chromen-8-ol), diarylpropionitrile (DPN) and / or propylpyrazoletriol (PPT)), 2,3-diarylisoquinolinone derivatives, diphenylmethane backbone derivatives, deoxybenzoin analogues, hydrazide derivatives, fluoren-3-one derivatives, triphenylethylene-coumarin hybrid derivatives, fulvestrant, 1,1,2-triarylolefin derivatives, 17α-(carboranylalkyl)estradiol and / or fluorinated carboarylphenols.

[0121] LY500307 is a synthetic ligand that selectively binds to and activates the estrogen receptor beta (ERβ) subtype. LY500307 is neuroprotective and reduces the accumulation of amyloid beta, a protein associated with Alzheimer's disease (37). It also reduces inflammation and improves cognitive function in a rat model of traumatic brain injury (38). [Non-Patent Document 37] Zhao et al. (2011), Neurobiology of Aging, 32:1949-1963 [Non-Patent Document 38] Barreto et al. (2009), European Journal of Neuroscience, 29:1997-2006

[0122] LY3201 is a synthetic ligand that selectively binds to and activates the estrogen receptor beta (ERβ) subtype. Activation of ERβ is known to have potential beneficial effects on several pathologies, including neurodegenerative diseases, inflammation, and certain types of cancer (39). [Non-Patent Document 39] Paterni et al. (2014), Expert Opinion on Drug Discovery, 9:467-481

[0123] PPT is a synthetic ligand that selectively binds to and activates the estrogen receptor alpha (ERα) subtype. It has been shown to have agonistic effects on bone and uterine tissues that are primarily mediated by ERα (Non-Patent Document 40). [Non-Patent Document 40] Stauffer et al. (2000), Journal of Medicinal Chemistry, 43:4934-4947

[0124] DPN is a synthetic ligand that selectively binds to and activates the estrogen receptor beta (ERβ) subtype. DPN has been shown to have anti-inflammatory, neuroprotective, and antitumor effects in various experimental models (Non-Patent Document 41). [Non-Patent Document 41] Meyers et al. (2001), Journal of Medicinal Chemistry, 44:4230-4251

[0125] In one embodiment, the estrogen receptor agonist is a nuclear estrogen receptor agonist and / or a membrane-bound estrogen receptor agonist.

[0126] The terms nuclear estrogen receptor and membrane-bound estrogen receptor are known to cell biology specialists, and not only can such receptors be identified, but also molecules capable of acting as their agonists can be identified. Generally speaking, nuclear estrogen receptors are intracellular receptors to which estrogen binds and which are preferably activated by estrogen. When activated by an estrogen receptor agonist, the nuclear estrogen receptor translocates to the nucleus of the cell, where it regulates the activity of genes. Thus, "nuclear estrogen receptor agonists" include molecules capable of binding to and preferably activating the intracellular estrogen receptor, the nuclear estrogen receptor. Generally, membrane-bound estrogen receptors are estrogen receptors located or embedded in the membrane of the cell, which are bound by estrogen and which are preferably activated by estrogen. Thus, "membrane-bound estrogen receptor agonists" include molecules capable of binding to and preferably activating the membrane-bound estrogen receptor. Membrane-bound estrogen receptors are estrogen receptors located or embedded in the membrane of the cell. A common type of membrane-bound estrogen receptor is the estrogen G protein-coupled receptor. Specific examples of membrane-bound estrogen receptors include GPRC6A (also known as G protein-coupled receptor family C group 6 member A, encoded by the GPRC6A gene in human subjects), ER-X and / or Gq-mER (also known as Gq-coupled membrane estrogen receptor).

[0127] In one embodiment, the nuclear estrogen receptor agonist is an estrogen receptor alpha (ERα) agonist and / or an estrogen receptor beta (ERβ) agonist.

[0128] Estrogen receptor alpha (ERα), also known as nuclear receptor subfamily 3, group A, member 1 (NR3A1), is encoded by the gene ESR1 (estrogen receptor 1) in human subjects. Estrogen receptor beta (ERβ), also known as nuclear receptor subfamily 3, group A, member 2 (NR3A2), is encoded by the gene ESR2 (estrogen receptor 2) in human subjects.

[0129] In one embodiment, the ERα agonist is one or more molecules selected from the group consisting of estrogen E2, estrogen E1, estrogen E3, estrogen E4 and / or propylpyrazoletriol (PPT).

[0130] In one embodiment, the ERβ agonist is one or more molecules selected from the group consisting of estrogen E2, estrogen E1, estrogen E3, estrogen E4, diarylpropionitrile (DPN), LY3201, and / or LY-500307.

[0131] In some embodiments, the agonist described above as an ERα agonist or an ERβ agonist may bind to both ERα and ERβ. A particular agonist is described as an ERα agonist if it has a stronger binding affinity to ERα than ERβ, i.e., if it binds to ERα with a higher potency. Similarly, a particular agonist is described as an ERβ agonist if it has a stronger binding affinity to ERβ than ERα. Those skilled in the art are aware of standard techniques in the art for measuring the binding affinity of a particular compound to a receptor.

[0132] In one embodiment of the invention, the membrane-bound estrogen receptor agonist is one or more molecules selected from the group consisting of GPRC6A agonists, ER-X agonists and / or Gq-mER agonists.

[0133] In one embodiment of the invention, the estrogen receptor agonist is present at low doses / concentrations as described below. Surprisingly, the inventors have discovered that low doses / concentrations of the estrogen receptor agonist result in surprisingly high levels of CD34+ hematopoietic stem cell expansion, particularly CD34+CD133+ hematopoietic stem cell expansion with sustained stemness.

[0134] In one embodiment of the invention, the estrogen receptor agonist is present at a concentration of about 0.5 nM to about 25 nM, preferably about 1 nM to about 10 nM. As described in the Examples, the inventors have determined that these concentrations are particularly advantageous in the methods of the invention.

[0135] In yet another embodiment, the estrogen receptor agonist is about 0.5 nM or more, e.g., about 1 nM or more, about 1.5 nM or more, about 2 nM or more, about 2.5 nM or more, about 3 nM or more, about 3.5 nM or more, about 4 nM or more, about 4.5 nM or more, about 5 nM or more, about 6 nM or more, about 7 nM or more, about 8 nM or more, about 9 nM or more, about 10 nM or more, about 11 nM or more, about 12 nM or more, e.g., about 1 nM or more, about 14 nM or more, about 16 nM or more, about 18 nM or more, about 19 nM or more, about 20 nM or more, about 21 nM or more, about 22 nM or more, about 23 nM or more, about 24 nM or more, about 25 nM or more, about 26 nM or more, about 27 nM or more, about 28 nM or more, about 29 nM or more, about 30 nM or more, about 31 nM or more, about 32 nM or more, about 33 nM or more, about 34 nM or more, about 35 nM or more, about 36 nM or more, about 37 nM or more, about 38 nM or more, about 39 nM or more, about 40 ...1 nM or more, about 42 nM or more, about 43 nM or more, about 44 nM or more, about 45 nM or more, about 46 nM or more, about 47 nM The nucleic acid is present at a concentration of about 10 nM or more, about 13 nM or more, about 14 nM or more, about 15 nM or more, about 16 nM or more, about 17 nM or more, about 18 nM or more, about 19 nM or more, about 20 nM or more, about 21 nM or more, about 22 nM or more, about 23 nM or more, about 24 nM or more, about 25 nM or more, about 26 nM or more, about 27 nM or more, about 28 nM or more, about 29 nM or more, or about 30 nM or more.

[0136] In some other embodiments, the estrogen receptor agonist is present at a concentration of about 100 nM or more, e.g., about 1000 nM or more, about 2,000 nM or more, about 3,000 nM or more, about 4,000 nM or more, about 5,000 nM or more, about 6,000 nM or more, about 7,000 nM or more, about 8,000 nM or more, about 9,000 nM or more, or about 10,000 nM (10 oM).

[0137] In one embodiment of the invention, the method further comprises the step of isolating and / or enriching CD34+ hematopoietic stem cells.

[0138] In one embodiment of the invention, the step of isolating CD34+ hematopoietic stem cells preferably comprises separating CD34+ hematopoietic stem cells from the sample after step i) and before step ii). In a particular embodiment, the step of separating CD34+ hematopoietic stem cells from the sample comprises separating CD34+ hematopoietic stem cell subpopulations (e.g., CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) as described herein.

[0139] Hematopoietic stem cells (especially CD34+ hematopoietic stem cells) can be enriched for higher expression of certain markers on hematopoietic stem cells or hematopoietic stem and progenitor cells compared to other hematopoietic cell types. Hematopoietic stem cells are identified as CD34+, which can also be used to identify HSCs as having these additional markers (e.g., lacking one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD166, and / or CD38, HLADR, and / or Lin). Preferably, the enriched hematopoietic cells are CD34+CD45+(low / intermediate)CD38- or CD34+CD45+(low / intermediate)CD133+, as described herein.

[0140] In one embodiment of the invention, the method includes (preferably prior to step i)) a step of enriching for CD34+ hematopoietic stem cells in the sample and / or isolating CD34+ hematopoietic stem cells (e.g., isolating CD34+ hematopoietic stem cells from a sample, such as a blood sample). In embodiments in which CD34+ hematopoietic stem cells are isolated, step i) can include providing one or more isolated CD34+ hematopoietic stem cells.

[0141] Isolation and / or enrichment of CD34+ hematopoietic stem cells can be performed using flow cytometry with fluorochrome-conjugated monoclonal antibodies and / or immunomagnetic cell separation with magnetic bead-conjugated monoclonal antibodies and / or immunopanning and / or affinity chromatography with antibodies attached to a solid matrix in a culture plate or bag. The isolation method should not cause excessive damage to the cells and should maximize the viability of the isolated cells. Isolation techniques depend on the availability of equipment, the efficiency of separation, the ease of using the method, the time it takes to separate the cells and the damaging effects (if any) of the method on the separated cell population.

[0142] Preferably, the separation and / or enrichment of CD34+ hematopoietic stem cells is accomplished using magnetic bead-bound CD34 monoclonal antibodies and a magnetic cell separation device. In another embodiment, as described above, one or more magnetic bead-bound monoclonal antibodies of other surface markers can be used alone or in combination with them, for example, CD45, CD133, to separate subpopulations of CD34+ hematopoietic stem cells by retaining the positive population. To separate a subpopulation of CD34+ cells lacking CD38, the separated cells can be further exposed to magnetic bead-bound CD38 monoclonal antibodies, retaining the negative population.

[0143] Thus, in one embodiment of the invention, the enrichment and / or isolation step comprises one or more methods selected from the list consisting of flow cytometry as described herein, immunomagnetic cell separation as described herein, immune panning as described herein and / or affinity chromatography as described herein.

[0144] In another embodiment, the sample is a previously cryopreserved sample (such as a blood sample, particularly cord blood and / or placental blood), a frozen sample (such as a blood sample, particularly cord blood and / or placental blood) or a freshly collected sample (such as a blood sample, particularly cord blood and / or placental blood). Preferably, the sample is a freshly collected sample.

[0145] In a preferred embodiment, cryopreserved (e.g., cryopreserved in nitrogen vapor) cord blood samples are processed to separate / enrich CD34+ hematopoietic stem cells using magnetic beads conjugated to anti-CD34 antibodies and a magnetic cell separator, e.g., the MiniMACS or MidiMACS or OctoMACS or CliniMACS® cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). This method utilizes nano-sized superparamagnetic particles consisting of iron oxide and dextran conjugated to a specific monoclonal antibody.

[0146] In another embodiment, the method of the present invention can include a frozen cord blood sample, the method further comprising a step of thawing the cord blood sample (preferably preceding step i). Thus, the frozen cord blood sample can be provided in a sample bag, an example of a thawing method in this case is to remove the sample bag from the nitrogen vapor storage and inspect the bag for damage. The sample bag is quickly placed in a sterile plastic zipper bag and quickly thawed in a 37°C water bath for 5 minutes or less. Alternatively, any thawing method known in the art can be utilized, including but not limited to a blood thawing system, a plasma thawing system, a microcontroller-based thawing system, a dry rapid plasma thawing device, a ThawStar cryobag thawing system, etc. After the sample is thawed, the bag is gently massaged and removed from the zipper bag, and the injection port is disinfected with a 70% alcohol wipe or any disinfectant known in the art, such as 70% isopropanol, Decidos, 2% chlorhexidine and / or betadine. Blood is collected in a 50 ml sterile tube using an appropriate sized spike connector (e.g., Braun blood spike adapter) depending on the blood bag. The thawed blood sample is diluted with a thawing solution made of any volume expanding solution of crystalloid or colloid known in the art. The thawing solution includes, but is not limited to, 5-10% dextran-40, 3-6% hydroxyethyl starch (HES), Hespan, Volven, Vollyte and / or Plasmalyte. Optionally, the thawing solution is supplemented with a non-animal derived natural or synthetic albumin solution known in the art. The solution includes, but is not limited to, human serum albumin, recombinant human albumin, synthetic serum substitutes and / or knockout serum substitutes. Preferably, human serum albumin is added to the thawing solution. Alternatively, a xeno-free knockout serum substitute can be added to the thawing solution. Based on prior information known in the art, it is preferred to use an isotonic thawing solution of 10% dextran-40 and 5% human serum albumin.

[0147] A portion of the thawed blood sample can be diluted with any one or more of the above thawing solutions, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. Preferably, the thawed blood is first diluted 1:1 with isotonic 10% dextran 40 and 5% human serum albumin, then diluted 1:3 by adding two more volumes of the same solution, and then diluted 1:5 by adding two more volumes of the same solution. After adding the thawing solution at each dilution step, the sample is stabilized for 15 minutes each. After dilution with the thawing solution at each dilution step, or once after the final dilution, the sample can be stabilized for any time known in the art, for example, about 1 minute or more, for example, about 2 minutes or more, about 3 minutes or more, about 4 minutes or more, about 5 minutes or more, about 10 minutes or more, about 15 minutes or more, about 30 minutes or more, about 40 minutes or more, or about 45 minutes or more. In certain embodiments, at each dilution step or immediately after the final dilution, the sample can be stabilized for about 1 minute to about 1 hour after dilution with thawing solution. In certain embodiments, at each dilution step or immediately after the final dilution, the sample can be stabilized for a total of 1 hour or less after dilution with thawing solution. In a preferred embodiment, the sample is stabilized for 15 minutes after each dilution step. The sample obtained by this exemplary thawing method can be used for isolating and / or enriching hematopoietic stem cells.

[0148] In certain embodiments, the method of the invention includes enrichment and / or separation of CD34+ hematopoietic stem cells by retaining the CD34 positive hematopoietic stem cell population (e.g., using an anti-CD34 antibody, preferably an anti-CD34 antibody bound to magnetic beads). Optionally, the method of the invention further includes a step of separating CD38- hematopoietic stem cells from CD34+ hematopoietic stem cells (e.g., using an anti-CD38 antibody, preferably an anti-CD38 antibody bound to magnetic beads, where cells that do not bind to the anti-CD38 antibody form a CD38- subpopulation of CD34+ hematopoietic stem cells). Similarly, a CD133+ subpopulation of CD34+ hematopoietic stem cells can be separated, e.g., using an anti-CD133 antibody bound to magnetic beads, to retain CD133+ cells. Antigen-specific antibodies can also be used to select cells expressing CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD166, HLA DR, or a combination of one or more markers. Preferably, CD34+CD38- hematopoietic stem cells are enriched and / or isolated.

[0149] In one embodiment of the invention, one or more suitable samples (such as blood samples, particularly cord blood and / or placental blood) can be combined prior to enrichment and / or separation of hematopoietic stem cells (preferably prior to step i). In another embodiment, individually enriched cord blood hematopoietic stem cell samples from donors of the same race, e.g., African American, Caucasian, Asian, Hispanic, Native American, Australian Aboriginal, Inuit, Pacific Islander, etc., and / or donors of the same ethnicity, e.g., Irish, Italian, Indian, Japanese, Chinese, Russian, etc., can be combined. Selection of suitable cord blood samples for pooling can be based on partial or complete HLA matching, as known in the art.

[0150] In one embodiment, where the sample is a blood sample, red blood cells (RBCs) and / or plasma can be removed, for example, before hematopoietic stem cell isolation / enrichment and / or cryopreservation and / or after thawing of the cryopreserved blood sample. In yet another embodiment, white blood cell fraction (WBCs) can be isolated, for example, after RBC removal for hematopoietic stem cell isolation and / or enrichment. In another embodiment, the thawed and diluted blood sample can be centrifuged at any g-force or relative centrifugal force (RCF), for example, 400xg, 450xg, 500xg, 550xg, 600xg, 650xg, 700xg, 750xg, 800xg or higher. Preferably, the g-force or relative centrifugal force (RCF) used is 650xg. The supernatant (thawing solution), including plasma, is removed and the resulting cell pellet is reconstituted to the original sample volume before thawing with any isotonic solution known in the art. Examples of isotonic solutions include, but are not limited to, phosphate buffered saline, plasmaLyte, Dulbecco's modified phosphate buffered saline (DPBS), Hanks' balanced salt solution (HBSS), Ringer's solution, etc. Preferably, Dulbecco's modified phosphate buffered saline (DPBS) is used for reconstitution of the cell pellet.

[0151] Removal of RBCs can be accomplished by centrifugation techniques or the use of density gradient media (e.g., FICOLL®, FICOLL-PAQUE®, PERCOLL® (GE Healthcare, Piscataway, NJ), LYMPHOPREP® (Alere Technologies), in combination with centrifugation, or the use of hemagglutinating agents (e.g., 6% hydroxyethyl starch (HES), Hespan (B. Braun Medical, Inc.), Hextend (Hospira, Lake Forest, IL), Hetasep® (Stemcell Technologies Inc)), or the use of RBC-specific antibodies coupled to magnetic beads and a choice of magnetic devices (e.g., EasySep (Stemcell Technologies The separation of white and red blood cell fractions can be performed by any method known in the art, such as separation of white and red blood cell fractions ... Inc. can be used with LYMPHOPREP®. SEPMATE® tubes are preferably used for density gradient centrifugation. After centrifugation, several layers form in the tube, identifiable from top to bottom as plasma, electrolytes and proteins, the buffy coat, a dense opaque band of mononuclear cells including white blood cells, and finally red blood cells (RBCs) and granulocytes as a thick pellet in the LYMPHOPREP®. The top layer is reduced to a volume of 4-5 ml by carefully aspirating the plasma to remove platelets.Finally, pour the upper layer and buffy coat contents of the SEPMATE tube into another conical tube and centrifuge to obtain a cell pellet, wash further with Dulbecco's modified phosphate-buffered saline (DPBS) supplemented with 1% human serum albumin, and resuspend the cell pellet in the same solution.

[0152] If desired, a small representative aliquot of fresh or thawed blood can be tested for, eg, total nucleated cell count, cell viability and CD34+ frequency / content prior to isolation / enrichment of CD34+ cells.

[0153] In one embodiment, all or half of the fresh blood can be processed to separate CD34+ or CD34+CD38- or CD34+CD133+ cells and recover the CD34+ positive enriched hematopoietic stem cell fraction and the CD34- cell fraction. If half of the fresh blood is used, the CD34- cell fraction can be mixed with half of the fresh cord blood and used for immediate transplantation or cryopreserved.

[0154] In another embodiment, CD34+ or CD34+CD38- or CD34+CD133+ hematopoietic stem cell enriched fractions from fresh or thawed whole blood samples can be processed sequentially prior to expansion, e.g., the hematopoietic stem cells can be resuspended in an appropriate cell expansion or culture medium for transport or storage.

[0155] In one embodiment of the invention, the cell culture medium can be any suitable medium for in vitro growth of cells known in the art, including but not limited to basal Iscove's Modified Dulbecco's Medium (IMDM) or basal Roswell Park Memorial Institute Medium (RPMI1640) or basal Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12) or STEMSPAN® Serum-Free Growth Medium (StemCell Technologies, Vancouver, British Columbia) or StemMACS HSC Growth Medium (Miltenyi Biotec GmbH, Germany) or Gibco® StemPro® 34SFM (ThermoFisher Scientific). If one of these basal media is used, it should be supplemented with either fetal bovine serum (FBS) or knockout serum supplement (ThermoFisher Scientific) or human serum albumin. Additionally, these basal media can be supplemented with several other nutrients, lipids, proteins, and antioxidants known in the prior art, such as ethanolamine, sodium selenite, hydrocortisone, D,L-tocopherol, human transferrin (holo), human insulin (zinc), N-acetyl-L-cysteine, 2-mercaptoethanol, monothioglycerol, magnesium L-ascorbyl-2-phosphate, etc., to make them serum-free media, but are not limited to these. When using one of the basal or serum-free media, it is necessary to supplement it with one or more hematopoietic stem cell growth factors and cytokines known in the prior art.This includes, but is not limited to, IL-3 (such as recombinant human interleukin-3 (rhIL-3)), IL-6 (such as recombinant human interleukin-6 (rhIL-6)), TPO (such as recombinant human thrombopoietin (rhTPO)), Flt-3 Ligand (such as recombinant human Flt-3 Ligand (rhFlt-3L)), stem cell factor (such as recombinant human stem cell factor (rhSCF), also known as KIT ligand or steel factor), GMCSF (such as recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF or GMCSF)), and / or GCSF (such as recombinant human granulocyte-colony stimulating factor (rhG-CSF or GCSF), optionally IGFBP2 (such as recombinant human insulin-like growth factor (IGF) binding protein 2 (rhIGFBP2)), and / or DLL (such as human soluble DLL-1 (rhsDLL-1))). Preferably, the base medium used for the growth / cell culture of CD34+ hematopoietic stem cells is Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 5% human serum albumin, 10 mg / L ethanolamine, 5 μg / L sodium selenite, 50 μg / L hydrocortisone, 20 μg / L D,L-tocopherol, 100 mg / L human transferrin (holo), 10 mg / L human insulin (zinc), 160 mg / L N-acetyl-L-cysteine, 4 mg / L 2-mercaptoethanol and cholesterol lipid concentrate (250X, ThermoFisher Scientific).The culture medium is further supplemented with one or more of 10 ng / mL recombinant human interleukin-3 (rhIL-3), 100 ng / mL recombinant human interleukin-6 (rhIL-6), 100 ng / mL recombinant human thrombopoietin (rhTPO), 100 ng / mL recombinant human Flt-3 ligand (rhFlt-3L), 100 ng / mL recombinant human stem cell factor or KIT ligand or steel factor (rhSCF), 10 ng / mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF or GMCSF) and / or 10 ng / mL recombinant human granulocyte colony-stimulating factor (rhG-CSF or GCSF), and optionally, 100 ng / mL recombinant human insulin-like growth factor (IGF) binding protein 2 (rhIGFBP2) and / or 50 ng / mL recombinant human soluble DLL-1 (rhsDLL-1).

[0156] In yet another embodiment, the step of isolating and / or enriching CD34+ hematopoietic stem cells comprises, preferably after and / or during step ii), isolating CD34+ hematopoietic stem cells from the stem cells expanded in step ii). In a particular embodiment, the step of isolating and / or enriching CD34+ hematopoietic stem cells from the stem cells expanded in step ii) comprises isolating a CD34+ hematopoietic stem cell subpopulation (e.g., CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells). In a further specific embodiment, the step of separating and / or enriching CD34+ hematopoietic stem cells from the expanded stem cells in step ii) comprises separating a CD34+ hematopoietic stem cell subpopulation (e.g., CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) from the CD34+ hematopoietic stem cells.

[0157] In certain embodiments, the step of separating and / or enriching CD34+ hematopoietic stem cells comprises separating CD34+ hematopoietic stem cells from the sample and separating CD34+ hematopoietic stem cells from the expanded stem cells of step ii).

[0158] "Isolating CD34+ hematopoietic stem cells" includes separating CD34+ hematopoietic stem cells from other cells and / or medium and / or components, e.g., other cells and / or medium and / or components in a sample. The resulting isolated CD34+ hematopoietic stem cells can be in a solution that consists essentially of CD34+ hematopoietic stem cells or in which the only cells present are CD34+ hematopoietic stem cells.

[0159] In one embodiment of the invention, isolation and / or enrichment of CD34+ hematopoietic stem cells comprises the use of flow cytometry and / or immunomagnetic cell separation and / or immune panning, as further described below.

[0160] In one embodiment of the invention, step ii) is carried out over a period of about 1 hour or more, such as about 2 hours or more, or about 3 hours or more, or about 4 hours or more, or about 5 hours or more, or about 6 hours or more, or about 7 hours or more, or about 8 hours or more, or about 9 hours or more, or about 10 hours or more, or about 12 hours or more, or about 14 hours or more, or about 16 hours or more, or about 18 hours or more, or about 20 hours or more, or about 22 hours or more, or about 1 day or more, or about 2 days or more, or about 3 days or more, or about 4 days or more, or about 5 days or about 6 days or more, or about 7 days or more, or about 8 days or more, or about 9 days or more, or about 10 days or more, or about 11 days or more, or about 12 days or more, or about 13 days or more, or about 14 days or more, or about 15 days or more, or about 16 days or more, or about 17 days or more, or about 18 days or more, or about 19 days or more, or about 20 days or more, or about 21 days or more, or 22 days or more, or 23 days or more, or 24 days or more, or 25 days or more. In certain embodiments, step ii) is carried out for a period of about 8 days or more, for example, about 14 days or more or about 21 days or more. In a preferred embodiment, step ii) is carried out for a period of about 7 days or more. In another preferred embodiment, step ii) is carried out for a period of about 8 days or more. In a preferred embodiment, step ii) is carried out for a period of about 14 days or more. In a preferred embodiment, step ii) is carried out for about 21 days or more, most preferably for about 21 days.

[0161] In one embodiment of the invention, step ii) is carried out for a period of about 21 days or less, for example, about 20 days or less, about 19 days or less, about 18 days or less, about 17 days or less, about 16 days or less, about 15 days or less, about 14 days or less, about 13 days or less, about 12 days or less, about 11 days or less, about 10 days or less, about 9 days or less, about 8 days or less, about 7 days or less. In a preferred embodiment, step ii) is carried out for a period of about 7 days or less. In another preferred embodiment, step ii) is carried out for a period of about 8 days or less. In a preferred embodiment, step ii) is carried out for a period of about 14 days or less. In a preferred embodiment, step ii) is carried out for a period of about 21 days or less.

[0162] In one embodiment of the invention, step ii) is carried out over a period of about 7 days to about 21 days, such as about 8 days to about 21 days, about 8 days to about 14 days, or about 14 days to about 21 days. In a preferred embodiment, step ii) is carried out over a period of about 7 days to about 21 days.

[0163] In one embodiment of the present invention, during step ii) of the method of the present invention, the medium containing one or more estrogen receptor agonists (and one or more growth factors and / or cytokines as options) can be replaced or added at least once with an equivalent (or identical) medium containing one or more estrogen receptor agonists (and one or more growth factors and / or cytokines as options). For example, the medium is replaced or added at least twice, the medium is replaced or added at least three times, the medium is replaced or added at least four times, the medium is replaced or added at least five times, and / or the medium is replaced or added at least six times. Preferably, about half of the volume of the medium is replaced or added with an equal volume of medium. In a particular embodiment, the volume of the medium replaced or added is about 50 ml to about 100 ml, preferably about 70 ml.

[0164] In certain embodiments in which step ii) is carried out for about 21 days, medium containing one or more estrogen receptor agonists (and optionally one or more growth factors and / or cytokines) is replaced or added on days 4, 8, 11, 14, 17 and 19, preferably on days 7 and / or 14.

[0165] In one embodiment, the or each step (preferably step ii)) of the present invention is carried out in a sealed container, such as a sealed cell culture bag.

[0166] In one embodiment of the invention, in the expanded CD34+ hematopoietic stem cells of step ii), the proportion of one or more CD34+ hematopoietic stem cells of the CD34+ hematopoietic stem cell subpopulation (preferably CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+low / intermediate)CD133+CD38- hematopoietic stem cells) is about 2 to about 40 CD34+ hematopoietic stem cells. For example, the proportion of one of the CD34+ hematopoietic stem cells of the subpopulation (preferably CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is about 2 or about 3 or about 4 or about 5 or about 6 or about 7 or about 8 or about 9 or about 10 or about 15 or about 20 or about 25 or about 30 or about 35 or about 40 CD34+ hematopoietic stem cells. In a preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), the proportion of one or more CD34+ hematopoietic stem cells of the CD34+ hematopoietic stem cell subpopulation (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+low / intermediate)CD133+CD38- hematopoietic stem cells) is about 2 to about 20 CD34+ hematopoietic stem cells. In a further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), the proportion of one or more CD34+ hematopoietic stem cells of the CD34+ blood stem cell subpopulation (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is about 4 CD34+ hematopoietic stem cells. In another further preferred embodiment, the expanded CD34+ hematopoietic stem cells of step ii) comprise a proportion of CD34+ hematopoietic stem cells of one or more of the CD34+ hematopoietic stem cell subpopulations (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) of about 15CD34+ hematopoietic stem cells.In yet another preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), the proportion of CD34+ hematopoietic stem cells of one or more of the CD34+ hematopoietic stem cell subpopulations (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is about 20CD34+ hematopoietic stem cells.

[0167] One skilled in cell biology will appreciate that the ratio of CD34+ hematopoietic stem cells to a particular CD34+ hematopoietic stem cell subpopulation can be further defined by reference to the period of time that step ii) of the present invention was performed. Thus, the ratios described herein can be combined with the period of time that step ii) was performed as described herein. One skilled in cell biology will also appreciate that with longer periods of time, the ratios can increase over time, as HSC populations with higher starting ratios will expand at a faster rate than HSC populations with lower starting ratios.

[0168] In a further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), the proportion of CD34+ hematopoietic stem cells of one of the CD34+ hematopoietic stem cell subpopulations (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is 4CD34+ hematopoietic stem cells when step ii) is performed for about 8 days. In yet another preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), the ratio of CD34+ hematopoietic stem cells in one of the CD34+ hematopoietic stem cell subpopulations (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is about 15CD34+ hematopoietic stem cells when step ii) is performed for about 14 days. In yet another preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), the ratio of CD34+ hematopoietic stem cells of one of the CD34+ hematopoietic stem cell subpopulations (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is about 20CD34+ hematopoietic stem cells when step ii) is performed for about 21 days.

[0169] In one embodiment of the invention, the fold change of the expanded CD34+ hematopoietic stem cells in step ii) is about 88 fold or more, for example, about a 90 fold or more change, about a 95 fold or more change, about a 100 fold or more change, about a 110 fold or more change, about a 120 fold or more change, about a 130 fold or more change, about a 140 fold or more change, about a 150 fold or more change, about a 160 fold or more change, about a 170 fold or more change, about a 180 fold or more change, about a 190 fold or more change, about a 200 fold or more change, about a 300 fold or more change, about a 400 fold or more change, about a 500 fold or more change, about a 600 fold or more change, about a 700 fold or more change, about a 800 fold or more change, about a 900 fold or more change, about a 100 fold or more change, about a 120 fold or more change, about a 130 fold or more change, about a 140 fold or more change, about a 150 fold or more change, about a 160 fold or more change, about a 170 fold or more change, about a 180 fold or more change, about a 190 fold or more change, about a 200 fold or more change, about a 300 fold or more change, about a 400 fold or more change, about a 500 fold or more change, about a 600 fold or more change, about a 700 fold or more change, about a 800 fold or more change, about a 100 fold or more change, about a 100 fold or more change, about a 100 fold or more change, about a a fold change of about 1,000 or more, a fold change of about 1,100 or more, a fold change of about 1,200 or more, a fold change of about 1,300 or more, a fold change of about 1,400 or more, a fold change of about 1,500 or more, a fold change of about 1,600 or more, a fold change of about 1,700 or more, a fold change of about 1,800 or more, a fold change of about 1,900 or more, a fold change of about 2,000 or more, a fold change of about 2,100 or more, a fold change of about 2,200 or more, a fold change of about 2,300 or more, a fold change of about 2,400 or more, or a fold change of about 2,500.

[0170] It will be appreciated by those skilled in the art of cell biology that the fold change in CD34+ hematopoietic stem cells can be further defined by reference to the period over which step ii) of the present invention was performed, and thus the fold change as described herein can be combined with the period over which step ii) as described herein was performed.

[0171] In one embodiment of the invention, the number of expanded CD34+ hematopoietic stem cells (particularly long-term repopulating hematopoietic stem cells) in step ii) is increased by a fold change of about 90 or more when step ii) is performed for about 8 days. In yet another embodiment, the number of expanded CD34+ hematopoietic stem cells in step ii) is increased by a fold change of about 1,500 or more when step ii) is performed for about 21 days.

[0172] In one embodiment of the invention, the number of expanded CD34+ hematopoietic stem cells in step ii) is increased by about 8,800% or more, for example by about 9,000% or more, about 9,100% or more, about 9,200% or more, about 9,300% or more, about 9,400% or more, about 9,500% or more, about 9,600% or more, about 9,700% or more, about 9,800% or more, about 9,900% or more, about 10,000% or more, about 11,000% or more, about 12,000% or more, about 13,000% or more, about 14,000% or more, or about 15,000% or more.

[0173] Those skilled in cell biology will appreciate that the percentage change in CD34+ hematopoietic stem cells can be further defined by reference to the period during which step ii) of the present invention was performed, and therefore the percentage change as described herein can be combined with the period during which step ii) as described herein was performed.

[0174] The quality, purity and / or success of the expansion of CD34+ hematopoietic stem cells can be assessed by a cell biology expert. In one embodiment of the invention, the total number of cells, viable cells, viable CD34+ hematopoietic stem cells, viable CD34+CD38- hematopoietic stem cells and / or CD34+CD133+ hematopoietic stem cells are analyzed after hematopoietic stem cell expansion to determine the efficacy of the expanded cell population, e.g., efficacy in providing a hematopoietic effect. In yet another embodiment, viable cells can be additionally and / or alternatively assessed during step ii) of the invention. In one exemplary embodiment, during step ii), representative samples are taken from the expansion culture at days 7, 14 and 21 to quantify the total number of viable nucleated cells and the total number of CD34+ cells, which can be counted (optionally using flow cytometry) to obtain the percentage of CD34+ hematopoietic stem / progenitor cells. Similarly, a representative aliquot of the expanded hematopoietic stem cells can be taken to count the total number of nucleated cells and the percentage of CD34+ cells before cryopreservation or after thawing, as further described herein.

[0175] In certain embodiments, the total viable CD34+ hematopoietic stem cells or CD34+CD38- hematopoietic stem cells can be assessed by any method known in the art, such as trypan blue or 7-AAD exclusion. The percentage of viable CD34+ hematopoietic stem cells can be determined by the use of dyes that are excluded from viable cells in a flow cytometry assay. The relative increase in CD34+ cells can be assessed as the total viable CD34+ cells after expansion / total viable CD34+ cells seeded at the start of expansion. This ratio is commonly referred to as the fold expansion. The CD34 antigen can be used alone or in combination with other antigens in this assay, as known in the art.

[0176] As already mentioned, the inventors have confirmed that the inventive method of expanding CD34+ hematopoietic stem cells improves the quality of the resulting CD34+ hematopoietic stem cell population and also increases the quantity of CD34+ hematopoietic stem cells compared to conventional methods. This is of therapeutic importance: several studies have shown that the total amount of CD34+ cells in the nucleated cells used in a treatment (e.g., in a graft) is positively correlated with a better outcome (e.g., better platelet and neutrophil engraftment, reduced incidence of graft failure and graft-versus-host disease after cord blood transplantation). Examples of methods that can assess quality include the outcome of transplantation and / or engraftment in a subject, which can be a human or, more preferably, an animal model. Animal models for assessing the purity of hematopoietic stem cells are known and include immunodeficient humanized mice (lacking species-specific immune cells). In this animal model, serial transplants of human HSCs expanded at different doses are performed and immune reconstitution is assessed by flow cytometry of blood and / or bone marrow. Another way to assess the quality, purity and / or success of CD34+ hematopoietic stem cell expansion is to examine the percentage of CD34+ hematopoietic stem cells (or CD34+ hematopoietic stem cell subpopulation) relative to (compared to) the total number of cells obtained from step ii) of the method of the present invention.

[0177] The potency of expanded hematopoietic stem cells can also be assessed by their ability to differentiate into myeloid and lymphoid lineages. Colony forming unit (CFU) assay is one of the methods used to study the proliferation and differentiation potential of hematopoietic stem / progenitor cells by their ability to form colonies in semi-solid medium. The number and morphology of colonies generated by a given number of hematopoietic stem / progenitor cells provide information on the potency of the expanded cell population. A relatively high number of CFUs from a given number of input hematopoietic stem cells is considered to predict better engraftment potential.

[0178] In one embodiment, the ability of the expanded CD34+ hematopoietic stem cells of step ii) to differentiate is substantially the same as the ability of one or more CD34+ hematopoietic stem cells in the sample. In another embodiment, the ability of the expanded CD34+ hematopoietic stem cells of step ii) to successfully transplant and / or engraft in a subject is substantially the same as the ability of the CD34+ hematopoietic stem cells in the sample to differentiate.

[0179] In one embodiment of the invention, the percentage of CD34+ hematopoietic stem cells compared to the total number of cells obtained from step ii) of the invention is about 20% or more, for example, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more.

[0180] In some embodiments, other cell types included in the total number of cells obtained from step ii) of the present invention include, but are not limited to, lymphoid and / or myeloid progenitor cells, their progenitor derivatives, and mature hematopoietic cells, which can be identified by the expression of one or more mature cell markers, including, but not limited to, CD4, CD7, CD8, CD14, CD15, CD16, CD56.

[0181] The functionality of the CD34+ hematopoietic stem cells obtained from step ii) of the present invention can be tested using an animal model, for example a mouse model, an example of such a mouse model is described in

[42] . [Non-Patent Document 42] Ishikawa et al., Development of functional human blood and immune systems in NOD / SCID / IL2 receptor {gamma} chain(null) mice. Blood. 2005 Sep 1;106(5):1565-73

[0182] In one embodiment of the invention, the method is an ex vivo method and / or an in vitro method.

[0183] In one embodiment of the invention, the method further comprises the step of administering the expanded CD34+ hematopoietic stem cells of step ii) to a subject in need thereof, preferably the subject is a human subject.

[0184] In one embodiment of the present invention, the method further comprises a step of differentiating the expanded CD34+ hematopoietic stem cells of step ii) into one or more cells derived from the CD34+ hematopoietic stem cells, in particular, the method further comprises a step of administering one or more cells derived from the CD34+ hematopoietic stem cells of step iii) to a subject in need thereof, preferably the subject is a human subject.

[0185] In one embodiment of the invention, the one or more cells derived from a CD34+ hematopoietic stem cell are cells selected from the list consisting of myeloid progenitor cells, lymphoid progenitor cells, erythroid cells, erythroid cells, mast cells, megakaryocyte cells, thrombocytes, mast cells, myeloblast cells, basophil cells, neutrophil cells, eosinophil cells, monocyte cells, macrophage cells, dendritic cells, natural killer cells, T lymphocytes, and / or B lymphocytes.

[0186] In a further version of the first aspect of the invention, step ii) of the method of the first aspect of the invention may comprise culturing the cells of step i) in the presence of an agent capable of increasing expression of the estrogen receptor and / or an agent capable of increasing a downstream effector of the estrogen receptor signalling pathway in addition to, or instead of, one or more estrogen receptor agonists.

[0187] The expanded CD34+ hematopoietic stem cells obtained by the methods described herein and / or one or more cells derived from the CD34+ hematopoietic stem cells obtained by the methods described herein can be cryopreserved or frozen, particularly for use in transplantation / infusion into a human patient at a later date.

[0188] Thus, the methods described herein may include a step (preferably after step ii)) of cryopreserving or freezing the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells.

[0189] In one embodiment of the invention, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells can be distributed into one or more bags or cryogenic tubes or units and cryopreserved at cryogenic temperatures in liquid nitrogen. In another embodiment, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells can be divided into several units and cryopreserved.

[0190] Cryopreservation can be any method known in the art that allows cells to be frozen in a live / viable state at ultra-low temperatures. Generally, freezing cells causes intracellular water to form ice crystals upon cooling, which damage the cells by causing damage due to osmotic effects on the cell membrane with dehydration and increased solute concentration. For a detailed description, see "Cell Cryopreservation: A Novel Method for Freezing Cells in a Viable State," 2009, pp. 1171-1175, 2009, which is incorporated herein by reference. [Non-Patent Document 43] Mazur, P., 1977, Cryobiology 14:251-272

[0191] Generally, these deleterious effects can be avoided by using cryoprotectants, controlling the rate of freezing, and storing frozen cells at ultra-low temperatures to minimize damage. Thus, in one embodiment, cryopreservation involves contacting the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells with one or more cryoprotectants.

[0192] Cryoprotectants that can be used include polyvinylpyrrolidine (Rinfret, 1960, Ann. NY Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548), glycerol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157), methanol, acetamide, glycerol monoacetate (Lovelock, 1954, Biochem. J. 56:265), D-sorbitol, inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), and dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 196:520). 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), amino acids (Phan The Tranz and Bender, 1960, Exp. Cell Res. 20:651), inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388), etc. are included, but are not limited to these. Preferably, DMSO, which is non-toxic at low concentrations, is used as a cryoprotectant that easily penetrates cell membranes to modify the freezing pattern of water molecules and protect intracellular organelles by preventing damage caused by ice crystal formation. In addition, the cryoprotective effect of DMSO can be further enhanced by supplementing the cryoprotective medium with serum albumin or plasma, e.g., serum albumin, to a concentration of 20-40%.

[0193] In one embodiment, after addition of cryoprotective medium, the cells can be placed at 0° C. until frozen, since DMSO concentrations above 1% become toxic above 4° C. Slow and controlled cooling of the cells is important, and this process can be carried out in an insulated box or using a programmed freezing machine that ensures freezing at a controlled rate of −1° C. / min.

[0194] A controlled-rate freezing box is an alcohol-free cell freezing container with a highly insulated outer material to control the heat removal rate and provide reproducible cell cryopreservation. Controlled-rate freezing boxes such as CoolCell (Corning Inc, Arizona, USA) provide reproducible cell cryopreservation. For example, for most stem cells in a cryoprotective medium consisting of 10% DMSO and 25% serum albumin, the optimal cooling rate is -1° to -3°C per minute from 0°C to -80°C.

[0195] In a preferred embodiment, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells can be cooled at this rate in sealed cryogenic vials / tubes (e.g., Nunc or Corning) for small quantities, or frozen between metal plates in ethyl vinyl acetate (e.g., Pall Corporation CA, USA, Thermogenesis holdings Inc. CA, USA) bags for larger quantities.

[0196] After freezing the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells at -80°C, the cells can be transferred to a cryogenic tank for long-term storage. Preferably, the cells can be cryopreserved in the liquid (-190°C) or vapor (-170°C) phase of nitrogen.

[0197] After cryopreservation as described herein, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells, the frozen samples can be stored in a cryopreservation facility, which can be a bank that can be located in one or more physical locations with one or more temperature-controlled freezers that use liquid nitrogen to maintain ultra-low temperatures. Thus, in one embodiment, the method of the invention includes storing, after cryopreservation or freezing, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells in a cryopreservation bank or cryopreservation repository.

[0198] The cryopreserved collection can include any number of samples of expanded hematopoietic stem cells, each sample can be obtained from a single blood sample, or multiple samples can belong to a single blood sample from a human. In another embodiment, the cryopreserved bank can consist of each frozen expanded cell sample from two or more independent cord blood or blood samples, such as the pooled samples described above. The expanded hematopoietic stem cell samples are stored at ultra-low temperatures below -20°C, -80°C, or preferably obtained with liquid nitrogen (-190°C) or nitrogen vapor (-170°C).

[0199] Cryopreserved and frozen expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells can be rapidly thawed, as needed, for example in a water bath or vial thawing system (e.g., ThawStar, BIOLIFE Solutions, WA, USA) or anhydrous cryobag thawing device (e.g., Thaw star CB, BIOLIFE Solutions, WA, USA) maintained at 35°C to 37°C. In a particular embodiment, the cryovial of frozen cells is immersed 3 / 4 in a bath of warm water with the neck of the tube out of the water, and the vial is constantly gently rotated to ensure proper heat transfer from the surface to the ice mass inside and mixing of the cell suspension. Thawing is rapid, with small ice pellets remaining in the vial to prevent the temperature of the cell suspension from exceeding 4°C, as DMSO in the cryoprotective medium can damage cells above this temperature. Once thawing is complete, the vial can be immediately placed on ice for further use, left thawed, or infused in part into a human patient to perform hematopoietic functions. Several methods are available for further processing of the thawed hematopoietic stem cells as deemed appropriate by the skilled artisan.

[0200] It may be necessary to remove cryoprotectants such as DMSO from the thawed hematopoietic stem cells due to their toxicity. It may also be desirable to prevent the cells from clumping together using any procedure known in the art. Such means include, but are not limited to, hydroxyethyl starch (

[44] ), low molecular weight dextran and HSA (

[45] ), and DNase (

[46] ). [Non-Patent Document 44] HES, Zhu et al., 2015, Cytotherapy 17(12): 1813-1819, doi: 10.1016 / j.jcyt.2015.08.007 [Non-Patent Document 45] Berz et al., 2007, Am J Hematol 82(6): 463-472, doi:10.1002 / ajh.20707 [Non-Patent Document 46] Garc≡ocha-Pineres et. al., 2006, J Immunol methods 30;313(1-2):209-213, doi: 10.1016 / j.jim.2006.04.004

[0201] Also, prior to infusion into the required human patient, it may be desirable to wash the cells by adding an appropriate isotonic medium known in the art (including but not limited to 5-10% dextran 40, 3-6% hydroxyethyl starch (HES), Hespan, Volven, Vollyte or Plasmalyte), followed by one or more centrifugation steps to concentrate the cells at the bottom of the tube, remove the cryoprotectant and diluent as the supernatant, and resuspend the cells in the above isotonic medium to obtain the final cell product for infusion into the human patient to provide hematopoietic function. This procedure should be performed gradually to minimize abrupt changes in the osmotic gradient and prevent cell damage due to removal of DMSO.

[0202] If necessary, a small representative aliquot of the thawed hematopoietic stem cells can be taken before use in therapy to check the total number of nucleated cells, cell viability and percentage of CD34+ hematopoietic stem cells. The procedures used to evaluate these parameters can be any method known in the art, depending on suitability. Preferably, the same procedures as those described for the samples in step i) of the method of the present invention can be used.

[0203] In a third aspect, the present invention provides a population of CD34+ haematopoietic stem cells obtainable and / or obtained by the methods and / or uses of the present invention.

[0204] It can be understood that the expanded CD34+ hematopoietic stem cells obtained or obtainable by the present invention can be distinguished from hematopoietic stem cells obtained by different methods based on the CD34+ hematopoietic stem cells of the present invention expressing genes known to be associated with estrogen receptor activity. As can be understood by those skilled in cell biology, such gene expression can be identified by measuring messenger RNA (mRNA) and / or the resulting proteins and / or microRNAs (miRNA). Genes associated with estrogen receptor activity can be identified using public databases (for example, as described in [Non-Patent Document 47]). It is also possible to identify the expanded CD34+ hematopoietic stem cells obtained or potentially obtainable by the present invention because their intracellular DNA is physically associated with the nuclear estrogen receptor. [Non-Patent Document 47] Jin et al., ERTargetDB: an integral information resource of transcription regulation of estrogen receptor target genes. J Mol Endocrinol. 2005 Oct;35(2):225-30

[0205] In one embodiment of the invention, the expanded CD34+ hematopoietic stem cells of step ii) contain gene expression associated with estrogen receptor activity. In yet another embodiment, the expanded CD34+ hematopoietic stem cells of step ii) contain nuclear estrogen receptor physically associated with their DNA. In some embodiments, one or more of the following genes associated with estrogen receptor activity may be expressed or suppressed in CD34+ hematopoietic cells obtained or potentially obtainable from the present invention: HSPC111, GREB1, CCNG2, IGFBP4, SLC38A1, H11, CXCl12, FZD8, NF3L1, IGSF4, PGR, NRIP1, PTGES, CTSD, BRI3BP, LOR, MPP3, JAK1, NOL5A, PADI3, ADCY9, NMA, NDRG1, NFIA, SERPINE1, EPHA4, NMES1, RFPL2, F10, CD7, REA, LMCD1, MKNK2, CRABP2, CTBS, SNK, PAFH1B1, HIG2, CENTG1, BMP7, AHCYL1.

[0206] In some additional or alternative embodiments, the expanded CD34+ hematopoietic stem cells obtained or obtainable from the present invention can be distinguished from hematopoietic stem cells obtained by a different method based on a higher percentage of CD34+CD133+ cells in the population of CD34+ hematopoietic stem cells obtained by the method of the present invention compared to populations of CD34+ hematopoietic stem cells obtained by other methods. In some embodiments, the cells obtained by the method of the present invention are CD34+CD133+CD38-. In some embodiments, a higher percentage of CD34+CD133+ and / or CD34+CD133+CD38- cells in the population obtained or potentially obtainable by the method of the present invention is associated with a higher quality stem cell population and / or greater clinical potential, and such cells are of higher quality as would be understood by a medical professional (Non-Patent Document 48, Non-Patent Document 49, and Non-Patent Document 50). [Non-Patent Document 48] Wynter EA, et al., CD34+AC133+ Cells Isolated from Cord Blood are Highly Enriched in Long-Term Culture-Initiating Cells, NOD / SCID-Repopulating Cells and Dendritic Cell Progenitors, Stem Cells, Volume 16, Issue 6, November 1998, Pages 387_396 [Non-Patent Document 49] Ishikawa F, et al., Human cord blood long-term engrafting cells are CD34+ CD38-. Leukemia. 2003 May;17(5):960-4 [Non-Patent Document 50] Andre G≡ochrgens, Stefan Radtke, Peter Horn & Bernd Giebel (2013) New relationships of human hematopoietic lineages facilitate detection of multipotent hematopoietic stem and progenitor cells, Cell Cycle, 12:22, 3478-3482, DOI: 10.4161 / cc.26900

[0207] In one embodiment, the expanded CD34+ hematopoietic stem cells of the invention have no significant difference in their surface phenotype compared to the CD34+ hematopoietic stem cells provided in step i) of the method described herein. In a particular embodiment, the expanded CD34+ hematopoietic stem cells of the invention have no significant difference in their surface phenotype compared to the CD34+ hematopoietic stem cells provided in step i) of the method described herein. The inventors have confirmed that this feature of the invention demonstrates the advantages of the method, particularly in providing expanded CD34+ hematopoietic stem cells with sustained stemness.

[0208] In a fourth aspect, the present invention provides one or more cells derived from CD34+ haematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein.

[0209] In one embodiment of the fourth aspect of the invention, the one or more cells derived from a CD34+ hematopoietic stem cell are cells selected from the list consisting of myeloid progenitor cells, lymphoid progenitor cells, erythroid cells, erythroid cells, mast cells, megakaryocyte cells, thrombocytes, mast cells, myeloblast cells, basophil cells, neutrophil cells, eosinophil cells, monocyte cells, macrophage cells, dendritic cells, natural killer cells, T lymphocytes and / or B lymphocytes.

[0210] In one embodiment, the T lymphocytes are chimeric antigen receptor (CAR)-T cells.

[0211] In a fifth aspect, the present invention provides a composition comprising a population of CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein and / or as described herein.

[0212] The HSCs for transplantation can be mixed with any isotonic, buffered intravenous crystalloid solution having a physiochemical composition that closely reflects human plasma, some examples of which include, but are not limited to, Plasmalyte, Normosol, and lactated Ringer's solution (LR). Thus, in some embodiments, a composition comprising CD34+ hematopoietic cells obtained by the methods described herein comprises an isotonic, buffered intravenous crystalloid solution as described above. The preparation and components of such solutions for inclusion in a composition are well known in the art.

[0213] In a sixth aspect, the present invention provides a kit of parts comprising a population of CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein and / or as described herein.

[0214] In a sixth alternative aspect, the present invention provides a kit of parts for expanding CD34+ hematopoietic stem cells comprising one or more estrogen receptor agonists and / or a sample comprising one or more CD34+ hematopoietic stem cells and / or means for obtaining a sample comprising one or more CD34+ hematopoietic stem cells. The kit of parts of the sixth alternative aspect of the present invention may comprise instructions describing the steps described herein in connection with the method of the first aspect of the present invention and / or the use of the second aspect of the present invention. The instructions may detail how to expand the cells in a Good Manufacturing Practice (GMP) compliant cord blood bank environment using tissue culture incubators (5% CO2, 98% humidity), cell culture bags and a centrifuge.

[0215] In some embodiments, the kit further comprises a growth factor and / or cytokine as described herein (provided in admixture with one or more estrogen receptor agonists or as separate components). The kit further comprises a serum-free medium suitable for the growth of hematopoietic stem cells. The composition of this medium is well known in the art.

[0216] In a seventh aspect, the present invention provides a CD34+ hematopoietic stem cell obtainable and / or obtained by the methods and / or uses described herein and / or a population of CD34+ hematopoietic stem cells as described herein for use in medicine.

[0217] In another seventh aspect, the present invention provides CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein and / or one or more cells obtained from the population of CD34+ hematopoietic stem cells described herein for use in medicine.

[0218] In an eighth aspect, the present invention provides CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein and / or one or more cells obtained from the population of CD34+ hematopoietic stem cells described herein for use in the treatment and / or prevention of a condition in a subject in need thereof.

[0219] In another eighth aspect, the present invention provides a population of CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein and / or as described herein, for use in the treatment and / or prevention of a condition in a subject in need thereof.

[0220] In a ninth aspect, the present invention provides the use of a population of CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein in a medicament for treating and / or preventing a condition in a subject in need thereof.

[0221] In another ninth aspect, the present invention provides one or more cells derived from one or more cells derived from a CD34+ hematopoietic stem cell obtained by and / or obtainable by the methods and / or use described herein in the manufacture of a medicament for treating and / or preventing a condition in a subject in need thereof and / or derived from a population of CD34+ hematopoietic stem cells as described herein.

[0222] In a tenth aspect, the present invention provides a method for treating and / or preventing a condition in a subject in need thereof comprising administering to a subject in need thereof CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein and / or a population of CD34+ hematopoietic stem cells as described herein.

[0223] In another tenth aspect, the present invention provides a method for treating and / or preventing a condition in a subject in need thereof comprising administering to the subject in need thereof one or more cells derived from a CD34+ hematopoietic stem cell and / or a population of CD34+ hematopoietic stem cells obtainable and / or obtained by the methods and / or uses described herein.

[0224] In yet another tenth aspect, the present invention provides a method of treating and / or preventing a condition in a subject in need thereof, comprising the steps of: a) providing a sample comprising one or more CD34+ hematopoietic stem cells; b) culturing the cells of step a) in the presence of a nuclear estrogen receptor agonist to expand the CD34+ hematopoietic stem cells and, optionally, to differentiate the expanded CD34+ hematopoietic stem cells into one or more cells derived from the CD34+ hematopoietic stem cells; c) administering the cells of step b) to a subject in need thereof.

[0225] The method of the tenth further aspect of the invention may comprise the steps as described herein in relation to the method of the first aspect of the invention and / or the use of the second aspect of the invention. It will be understood that step a) of the tenth further aspect of the invention corresponds to step i) of the first aspect and step b) of the tenth further aspect of the invention corresponds to step ii) of the first aspect.

[0226] In one embodiment of the invention, the above conditions are conditions requiring transplantation, hematological conditions and / or hematological malignancies, conditions resulting from failure or dysfunction of normal blood cell production and maturation, immunosuppression in subjects with malignant and / or solid tumors, autoimmune diseases and / or immune conditions, genetic diseases, cancers, tumors, osteopetrosis, myelosclerosis, acquired hemolytic anemia, infections causing primary or secondary immune deficiencies, acquired immune deficiencies, neutrophil actin deficiencies, neutrophil membrane GP-180 deficiencies, bacterial infections (e.g., brucellosis infections, listeriosis infections, endocannabinoid system infections, inflammatory bowel disease, osteoporosis ... The condition is one or more conditions selected from the list consisting of: leprosy, leprosy, leukemia, leprosy, leprosy and / or leprosy), parasitic infections (e.g., malaria and / or leishmaniasis), fungal infections, diseases involving lymphocyte set imbalance and / or immune dysfunction due to aging, phagocyte disorders, Kostmann agranulocytosis, chronic granulomatous diseases, Chediak-Higachi syndrome, Wiskott-Aldrich syndrome, metabolic storage diseases, mucopolysaccharidoses, mucolipidoses, alpha1-antitrypsin deficiency, neurological diseases, and / or other diseases involving the immune mechanism.

[0227] In one embodiment of the invention, the transplant is an allogeneic or autologous transplant.

[0228] In one embodiment of the invention, the condition resulting from failure or dysfunction of normal blood cell production and maturation is one or more conditions selected from the list consisting of pancytopenia (such as radiation and / or chemotherapy induced pancytopenia), aplastic anemia, thrombocytopenia, agranulocytosis, red blood cell aplasia, hyperproliferative stem cell disorder, Blackfan-Diamond syndrome, Blackfan-Diamond syndrome due to drug use, Blackfan-Diamond syndrome due to radiation, Blackfan-Diamond syndrome due to infection, and / or idiopathic Blackfan-Diamond syndrome.

[0229] In one embodiment of the invention, the hematological disease and / or hematopoietic malignancy is one or more diseases selected from the list consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic (lymphocytic) leukemia, acute myeloid leukemia, acute malignant myelosclerosis, chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, myelodysplasia of unknown etiology, Waldenstrom's macroglobulinemia, and / or polycythemia vera.

[0230] In one embodiment of the invention, the immunosuppression in a subject with a malignant and / or solid tumor is one or more conditions selected from the list consisting of ovarian cancer, breast cancer, malignant melanoma, testicular cancer, gastric cancer, small cell lung cancer, rhabdomyosarcoma, Ewing's sarcoma, retinoblastoma, glioblastoma, neuroblastoma and / or lymphoma.

[0231] In one embodiment of the invention, the autoimmune disease and / or immune condition is one or more conditions selected from the list consisting of type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, chronic hepatitis and / or multiple sclerosis.

[0232] In one embodiment of the invention, the genetic disease is anemia, familial aplastic anemia, Bloom's syndrome, Fanconi syndrome, pure red cell aplasia (PRCA), dyskeratosis congenita, Blackfan-Diamond syndrome, congenital dyserythroid syndromes I-IV, Hwachman-Diamond syndrome, dihydrofolate reductase deficiency, formaminotransferase deficiency, pyruvate kinase deficiency, sickle cell disease, thalassemia alpha, thalassemia beta, thalassemia gamma, methemoglobinemia, Lesch-Nyhan syndrome, congenital spherocytosis, congenital elliptocytosis, congenital stomatocytosis, congenital Rh deficiency, congenital erythropoietin sensitivity deficiency, paroxysmal nocturnal hemoglobinuria, G6PD (glucose-6-phosphate dehydration) In one embodiment, the condition is one or more selected from the list consisting of: erythropoietin deficiency, erythropoietin deficiency syndrome ...

[0233] In one embodiment, the genetic disease is one or more conditions selected from the list consisting of cerebral palsy, autism spectrum disorder, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, stroke, motor neuron disease, traumatic brain injury, spinal cord injury, amyotrophic lateral sclerosis, hypoxic-ischemic brain injury, eye injury and / or epilepsy.

[0234] The term "treatment" includes the administration of one or more CD34+ hematopoietic stem cells and / or cells derived from CD34+ hematopoietic stem cells as described herein in an amount, manner and / or mode effective to ameliorate a condition, symptom, or parameter associated with a condition, or to prevent the progression or worsening of a condition (including secondary damage caused by a condition) to a statistically significant extent or to a degree detectable by a medical professional.

[0235] The term "prevention" includes the administration of one or more CD34+ hematopoietic stem cells and / or cells derived from CD34+ hematopoietic stem cells described herein in an amount, manner and / or mode effective to prevent the onset of a condition, symptom, or parameter associated with a condition to a statistically significant extent or to a degree detectable by a medical professional.

[0236] The expanded CD34+ stem cell population generated by the method described herein can be administered by any convenient route known in the art, such as systemically or locally, by infusion or bolus injection, and can be administered together with other biologically active agents, such as any isotonic buffered intravenous crystalloid solution with a physiochemical composition that closely reflects human plasma. Some examples include, but are not limited to, Plasmalyte, Normosol, and lactated Ringer's solution (LR). Preferably, the expanded CD34+ stem cell population is administered by infusion.

[0237] The subject (patient) can be administered with the agent alone (monotherapy) or in combination with other agents (combination therapy), mixed, or administered separately, simultaneously or sequentially. In the case of combination therapy, the dosage and administration time can be, for example, an amount and time that produces an additive or synergistic therapeutic effect. Furthermore, administration can be used as a first-line treatment (e.g., first-line treatment) or a second-line treatment (e.g., treatment for subjects who have not responded adequately to previously administered treatments).

[0238] In one embodiment of the invention, the one or more estrogen receptor agonists (and / or other growth factors or cytokines, if used) are removed or diluted (such as by washing or dilution) from the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells prior to administration to the subject. EXAMPLES

[0239] The following examples are presented to illustrate specific embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors and found to work well in the practice of the invention, and are therefore believed to constitute preferred modes for the practice of the invention. However, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed and changes can be made without departing from the spirit and scope of the invention while still achieving the same or similar results.

[0240] summary Hematopoietic stem cell (HSC) transplantation for the treatment of various hematological, oncological and immunological disorders holds great promise but is often limited in terms of availability of matched donors. Umbilical cord blood (UCB) contains HSCs with low maturity and long telomeres, making it a better alternative, but its clinical use in adults is often limited by low stem cell numbers and slow engraftment. Ex vivo expansion of UCB-HSCs to clinically relevant doses can overcome this hurdle and benefit patients. Several approaches and methods have been adopted by research groups worldwide, including the use of hematopoietic growth factors, cytokines and differentiation inhibitors such as Notch ligands, copper chelators, nicotinamide and aryl hydrocarbon receptor antagonists. Estrogen (E2) and estrogen receptor (ER) have been shown to be involved in the proliferation of pluripotent and lineage-committed progenitor cells, but have not yet been tested in UCB-HSC expansion. Interestingly, E2, which is readily available in placenta and normal umbilical cord blood, has been shown to be involved in lineage-committed progenitor growth and differentiation in rodents. To date, little is known about the role of E2 in HSC expansion and self-renewal with long-term repopulating potential. E2 has not yet been tested in the expansion of human UCB-derived hematopoietic stem cells in a completely xeno-free environment using serum-free defined growth media. Reliable and clinically useful UCB-HSC expansion is not only beneficial for allogeneic transplantation, but also provides a suitable platform to induce effector immune cells for in vitro expansion and / or genetic modification to improve cellular immunotherapies such as UCB-derived chimeric antigen receptor (CAR)-T cells and NK cells. These enhanced antitumor cells are provided as an off-the-shelf product, minimizing the time to treatment by not requiring modification of the patient's own cells.

[0241] We have developed a novel xeno-free method to efficiently expand UCB-HSPCs in vitro using a proliferation supplement containing estrogen ligands. It allows for a unique and adequate therapeutic dose to be achieved, thereby facilitating the clinical use of UCB-HSCs for both autologous and allogeneic transplantation, and opening up the possibility of using UCB CAR-T cells as an effective adoptive cellular immunotherapy. Our method is robust and unique in that it reliably expands UCB-HSCs in a limited time and provides a higher number of therapeutic cells with a sustained stem cell phenotype compared to other reported examples. Interestingly, our method is efficient in expanding CD34+HSPCs and long-term repopulating HSCs (LT-HSCs) up to 1500-fold and 90-fold, respectively, in 21 days, without affecting their multilineage differentiation potential. This has not been observed before with other methods, and these cells demonstrate the advantages of UCB-HSCs expanded in a clinical setting.

[0242] background Hematologic and non-hematological diseases can be treated with autologous or allogeneic hematopoietic stem cell (HSC) transplantation, but its application is limited by identifying suitable donors. Umbilical cord blood (UCB) contains less mature HSCs with longer telomere lengths, making it a better alternative source for HSC transplantation, but adult UCB transplantation can be clinically limited by lower total nucleated cell numbers (~1 billion, TNC) and CD34+ progenitor cell numbers (~1.2 million) compared with bone marrow or mobilized peripheral blood stem cell transplantation, resulting in slower / delayed engraftment. Most healthy full-term births provide more than 1 million hematopoietic stem cells (HSCs) in the umbilical cord blood, and as a result, most public cord blood banks do not consider a significant number of UCB units to be optimal when donations fall below the threshold. A higher nucleated (≥25–50 million cells / kg) and CD34+ (1–1.5x105 / kg) cell dose per kg body weight is associated with better cord blood transplant outcomes (1) and can overcome delayed engraftment. Thus, ex vivo expansion of umbilical cord blood hematopoietic stem cells (UCB-HSCs) may help to increase the cell dose available for transplantation. Ex vivo expansion of stem cells requires symmetric division, so that both daughter cells maintain stem cell properties. The precise dosage of cytokines, media, serum, and other small molecules used for expansion is also crucial. The ultimate goal of cord blood expansion should aim to generate clinically safe and transplantable HSCs that can reliably repopulate the recipient. Another acceptable alternative is to ex vivo generate more mature progenitor cells that can rapidly engraft. The first efforts on HSC expansion were initiated in the 1970s, and since then, many efforts have been made to expand cells using cytokines or stromal cells or a combination of both to maintain stemness and limit differentiation. However, there have been mixed successes and failures, and the quest to stably expand reliable populations of HSCs from umbilical cord blood continues worldwide.

[0243] 1, Hematopoietic stem cell (HSC) phenotype and self-renewal pathways HSCs are a rare subpopulation of the hematopoietic system that can generate all mature blood cells throughout life. (2) Thus, a unique feature of HSCs is the dynamic balance between quiescence, self-renewal, and differentiation that is regulated by various mechanisms, including genetic and epigenetic regulation and microenvironmental (niche) factors.

[0244] Routine flow cytometric CD34 analysis counts hematopoietic stem and progenitor cells regardless of subpopulation, but this does not necessarily predict true engraftment kinetics and immune reconstitution. Over the past two decades, the CD34 antigen has been widely used to define human hematopoietic stem cells. Experiments to identify human HSCs using umbilical cord blood have yielded mixed results using CD34 as the sole marker for long-term repopulating HSCs (LT-HSCs). Ishikawa et. al. (3) strongly suggested that human CB stem cells are phenotypically CD34+CD38-, whereas exclusively CD34+ positive cells represent not only LT-HSCs but also short-term hematopoietic stem cells (ST-HSCs) and progenitor cell subpopulations (HSPCs) in the hematopoietic system hierarchy. That is, primitive progenitor cells (LT-HSCs) lack or only weakly express the cell surface antigen CD38, in contrast to which CD38 is abundantly expressed on more mature progenitor cells. Furthermore, Gorgens et al. have meticulously demonstrated that all HSCs with long-term potential, including those that can be engrafted into NOD / SCID or NSG mice, segregate into the CD133+CD34+CD38- fraction (4), i.e., long-term potential UCB-HSCs are phenotypically characterized as CD133+CD34+CD38-.

[0245] Monoclonal antibody recognition has also been used to identify the presence or absence of cell surface protein markers to recognize and isolate hematopoietic stem cells. Such markers include, but are not limited to, Lin, CD34, CD38, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLADR, and combinations thereof. Further details on this subject are provided in Chapter 2 of the US Department of Health and Human Services

[51] . [Non-Patent Document 51] Regenerative Medicine, Department of Health and Human Services, August 2006 (http: / / stemcells.nih.gov / info / scireport / 2006 report.htm

[0246] Functional readouts of HSCs and HSPCs are associated with cell survival, self-renewal, proliferation, differentiation, and migration (homing, migration, and chemotaxis) (10). These processes involve several intracellular signaling molecules, including but not limited to p21cip1 / waf1, p27kip1, Stat3, Stat5, Notch, Wnt, β-catenin, GSK-3, sonic hedgehog, bone morphogenetic proteins, members of the Hox family (e.g., Hoxb4), FoxO, Pu.1, GATA-1, Sirt1 (and other members of the sirtuin family of deacetylases), HIF-1α, Rheb2, and the rapamycin-sensitive m-TOR pathway (reviewed in (17)).

[0247] 2. Hematopoietic growth factors in self-renewal and proliferation Hematopoietic growth factors are released under inflammatory or cytopenic conditions and induce the mobilization and proliferation of HSCs in vivo. These growth factors are also known as colony-stimulating factors (CSFs) and include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (EPO), and TPO (5). In the late 1990s, many of these cytokines were introduced into cell culture media and UCB-HSCs were expanded in vitro to elucidate their capacity for self-renewal and reconstitution in mice. The bulk of experimental evidence indicates that a combination of multiple cytokines, such as stem cell factor (SCF), FLT-3 / FLK-2 ligand (Flt3 ligand), and thrombopoietin (TPO), appears to be essential for the expansion of progenitor cells. Of these growth factors, SCF has been recognized as essential for the proliferation of stem and progenitor cells and has even been shown to be a critical factor for the survival of hematopoietic progenitor cells. Using this cytokine cocktail, CD34+ cells can be expanded ex vivo to several fold over pre-expansion values. The crux of ex vivo HSC expansion is not the proliferation of both differentiated and undifferentiated cells, but the increase in undifferentiated stem cells. Mohamed et.al. (6) expanded CB stem cells ex vivo for 7 and 11 days using SCF, GM-CSF, IL-3 and TPO under specific conditions. They reported better proliferation of CD34+ cells at 7 days and the addition of more cytokines (IL-6 and FLT-3) did not affect the CD34+ cell count but resulted in the appearance of apoptotic cells. Yao et. al. used a statistically formulated cytokine-restricted serum-free medium and reported a 20-fold expansion of CD34+CD133+ cells, a 723-fold expansion of CD34+CD38+ cells and an 8-fold expansion of long-term culture-initiating cells after 7 days of culture. Madkaikar et. al. (7) combined various cytokines (SCF, IL-3, GM-CSF) with stromal cell support for the expansion of HSCs and achieved up to 21-fold expansion of CD34+ and nucleated cells.Another group (Wei et. al.) defined seven culture condition groups for the expansion of CD34+ cells, including a basic combination of SCF, IL-3, and IL-6 for 3, 7, and 14 days. The proliferation rate of CD34+ cells was 10-50 times higher compared to fresh UCB. Although there are abundant recombinant cytokines used for the expansion of primitive hematopoietic progenitor cells, the optimal combination for use in clinical settings has not yet been approved. The most commonly used factors are stem cell factor (SCF), IL-3 and IL-6, G-CSF, thrombopoietin (TPO), and Flt-3 ligand (8, 9). It should be noted that in addition to the factor set, their concentration and the order of use are also important.

[0248] Fetal calf serum (FCS), which contains a natural cocktail of growth factors, adhesion mediators, minerals, lipids and hormones, is a standard component for the culture of most human cell types, including HSCs. However, there is no consensus in clinical practice on the feasibility of using cells after FCS-supported expansion. The disadvantages of serum include the difficulty of standardizing its composition, the possibility of viral contamination and the high risk of immunization of the recipient with foreign proteins (10, 11). Therefore, excluding FCS for the in vitro expansion of HSPCs and using specific serum-free media would be of great help in optimizing the cytokine combination and accepting the expanded cells for transplantation (12, 13).

[0249] 3. Ex vivo expansion strategies for cord blood HSCs Many ex vivo expansion methods have been developed to increase UCB cell mass, but the results of traditional UCB expansion methods using cytokines alone have been disappointing due to variable concentrations of each cytokine.

[0250] Another approach is to use inhibitors of HSC differentiation such as nicotinamide analogues, copper chelators, induction of constitutive Notch signaling or the use of aryl hydrocarbon receptor antagonists (StemReginin1). Many of these methods result in significant expansion of total nucleated cells and CD34+ cells and significantly improved time to neutrophil or platelet engraftment in patients transplanted with the expansion product compared to unmanipulated CBT recipients. These studies differ not only in terms of the expansion method but also in terms of the cytokines used, patient population, conditioning regimen, transplantation method, etc. Some of these methods start with unselected cell populations from umbilical cord blood, while others use selected subsets such as CD34+ and CD133+. Another UCB cell expansion technique expanded TNCs 2.4-fold and CD34+ cells 0.5-fold using computer-automated "continuous perfusion" culture for 12 days.

[0251] 3.1 Copper Chelating Agents Tetraethylenepentamine (TEPA), a polyamine copper chelator, administered together with the cytokines FLt-3, IL-6, TPO, and SCF for 3 weeks has been reported to expand CD133+ selected HSCs by inhibiting cytokine-induced differentiation without affecting the subsequent differentiation and proliferation of mature committed cells (14), resulting in an 89-fold expansion of CD34+ cells, a 30-fold expansion of CD34+CD38-, and a 172-fold expansion of colony forming units (CFUs).

[0252] 3.2 Notch Signaling Another way to inhibit EPC differentiation is the induction of constitutive Notch signaling, which allows the establishment of immortalized cell lines that can be cultured in liquid medium containing cytokines. Interestingly, in both invertebrates and vertebrates, cell fate choice is controlled by Notch signaling (15, 16), which inhibits certain differentiation pathways and allows cells to differentiate along alternative pathways or to self-renew. Hematopoietic progenitor cells express the Notch receptor, and Notch signaling enhances the in vitro generation of human and mouse hematopoietic progenitors (17-19) and also controls the specification of T-cell or B-cell lineages from common lymphoid progenitors (20). Delaney et al. (21) used CD34+ selected CB cells, introduced a modified Notch ligand (Delta1ext-IgG) and cultured them in the presence of IL-3, IL-6, TPO, SCF, and flt-3 ligand for 16 days, resulting in an average expansion of CD34+ cells 222-fold.

[0253] 3.3 Nicotinamide Another technique to inhibit HSC differentiation is the use of NAM. NAM is a member of the sirtin family of protein deacetylases (SIRT1) that plays an important role in determining the life span of many lower organisms (22). It also plays a key role in the self-renewal of mouse embryonic stem cells. NAM is known to inhibit HPC differentiation and plays an important role in stem cell adhesion, migration, and proliferation (23). CD34+Lin-selected CB cells were expanded in a medium containing SCF, FLT-3, IL-6, and TPO, containing NAM. After 3 weeks of culture, TNCs expanded an average of 486-fold, and CD34+ cells expanded an average of 72-fold (24).

[0254] 3.4 Aryl hydrocarbon receptor antagonism A group from the University of Minnesota published the results of a phase I / II study of CB expansion using StemReginin1 (SR1), an aryl hydrocarbon receptor antagonist that helps expand CD34+ cells without differentiation in the presence of SCF, flt-3 ligand, TPO, and IL-6. The average expansion of CD34+ cells was 328-fold over 21 days of culture (25).

[0255] 3.5 estrogen The female hormone estrogen (E2) plays a key role in female and male sexual differentiation and reproduction. A key question is whether stem cells are targets of sex hormone regulation in tissues that lack sex-specific morphological differences. The terminal differentiation and function of some committed hematopoietic cells are regulated by sex hormones (26, 27). However, the function of hematopoietic stem cells (HSCs) is largely similar in both sexes. Apart from its importance in reproduction, E2 has been shown to control the proliferation and differentiation of pluripotent and multipotent stem cells (28, 29). In mice, E2 has been shown to be involved in promoting the circulation of bone marrow hematopoietic multipotent progenitor cells (MPPs) and their differentiation into megakaryocyte-erythroid progenitors (MEPs) (30). E2 has also been reported to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stromal cells (BM-MSCs) by activating Notch signaling and upregulating the estrogen receptor (ER) (31). Interestingly, E2, which is readily available in the placenta and normal umbilical cord blood, has been shown to be involved in the growth and differentiation of lineage-committed progenitor cells in rodents.

[0256] E2 acts on target cells by binding to nuclear receptors. Two nuclear receptors have been identified: estrogen receptor alpha (ERα) and beta (ERβ) (32). The E2-ER complex binds to specific promoter regions on DNA and regulates the transcription of hundreds of target genes. This is known as the genomic action of E2. E2 can also mediate rapid nongenomic actions by interacting with signaling cascades in the cytoplasm. ERα has been detected in nonhematopoietic cells, such as mouse bone marrow and B lymphoid progenitors (33) and human hematopoietic multipotent progenitor cells (MPPs) (34). ERβ has also been found in nonhematopoietic cells in mouse bone marrow and human spleen (33), but there have been no reports on the status of ERβ in UCB-HSCs. We recently showed that loss of ERβ disrupts neural stem cell proliferation. This effect was likely propagated via Notch-Hes signaling, which was deregulated by loss of ERβ (35). Interestingly, ERβ can mediate the expression of p21, p27, and Notch1 in both ligand-dependent and -independent manners (36, 37) and has been shown to regulate NOTCH1 transcription in squamous cell carcinoma cells (38). Ligand-mediated actions of ERα are often associated with cell proliferation, whereas ERβ actions are associated with multiple cell cycle regulating antiproliferative events, often countering excessive proliferation (antitumor effects).

[0257] Illing et. al. (39) reported that estradiol treatment of mice increases the number of progenitor cells in the bone marrow vascular compartment but not long-term (LT) repopulating HSCs, independent of estrogen-induced bone growth. Interestingly, this study showed that the action of estradiol on stromal cells strongly promotes HSPC cell frequency. Furthermore, the proliferative effect of E2 on BM HSCs was due to enhanced cycling, resulting in an increase in short-term (ST) HSCs and a depletion of LT-HSCs, as evidenced by a significant reduction in LT-HSCs obtained from E2-treated donors when administered to tertiary recipient mice. However, it appears that the effect of E2 on these cell proliferations is not mediated through classical ERs.

[0258] Placental trophoblast cells increase maternal E2 levels up to 1000-fold during pregnancy (40). This may be due to maternal adaptation to the increased blood demand associated with fetal nutrition and physiological blood loss during birth. Based on these findings, Nakada et. al. (41) observed that the replicative activity of HSCs was elevated in pregnant female mice. They reported that E2 promoted cell cycle entry of HSCs and multipotent progenitors (MPPs) and increased differentiation into megakaryocytic erythroid progenitors (MEPs). The number of HSCs in pregnant mice was not different from that in control or male mice, despite increased HSC proliferation and production of red blood cells and platelets. These effects of estrogen were mediated through ERα, suggesting that estrogen primarily promotes asymmetric division of HSCs rather than symmetric self-renewal in the bone marrow. Notably, however, the authors found no difference in HSC numbers after ovariectomy in both males and females, yet loosely attributed this to HSC self-renewal. BrdU incorporation in HSCs in their experiments may not reflect self-renewal (generation of identical HSCs) but rather enhanced cell division, leading to more committed progenitor cells.

[0259] Nakada et. al. (42) determined the effect of E2 on specific HSPC subpopulations in mice and found that ERα and ERβ were differentially expressed in LT-HSCs, MPPs, and committed progenitor cells, which may suggest distinct roles of E2 signaling in different cell types. This study suggests that ERα activation by selective ER modulators induces apoptosis in part through Myc induction in ST-HSCs and MPPs. In contrast, E2-mediated proliferation of quiescent LT-HSCs is associated with a loss of self-renewal activity, which leads to a loss of the ability to reconstitute hematopoiesis and promotes differentiation.

[0260] Kim et. al.(43) recently showed that supplementation of the growth medium with E2 improved hematopoietic differentiation of human pluripotent stem cells (hPSCs) in vitro via an ERα-mediated pathway. In this study, ERα was reported to be persistently expressed during the differentiation process and to be present only in the hematopoietic phenotype. However, E2 signaling did not significantly affect the functional capacity of differentiated hematopoietic cells. Furthermore, the authors suggested that E2 signaling is involved in the fate decision process during early hematopoiesis, as a large number of hemangioblasts were generated by E2 treatment. Furthermore, E2 alone had no effect on the generation of hematopoietic cells from hPSCs, but the generation of cells was improved two-fold in the presence of hematopoietic growth factors. Contradictory, the authors reported that erythroid differentiation from hPSCs was increased after E2 supplementation. Interestingly, they also reported that the number of CD34+ blood cells from human umbilical cord could be increased two-fold when cultured for 10 days in the presence of E2. The authors' conclusion is that E2 enhances the proliferation of hematopoietic progenitor cells by maintaining ER positivity and functional capacity. However, the study reported by Kim et al. mainly focuses on the generation of hematopoietic cells from human pluripotent stem cells, and there are many pitfalls that do not clearly support the claim that E2 exerts a proliferative effect on HSCs but not on late progenitor cells. Some of the questionable findings are listed below.

[0261] 1. The HSC markers used in the study, such as CD34+CD45+, do not accurately reflect LT-HSCs with long-term repopulation capacity, and therefore may not be able to reconstitute hematopoiesis over the long term. (See 1. Hematopoietic stem cell (HSC) phenotype and self-renewal pathway.) 2. Even if the use of identification markers is allowed in the study, simple E2 supplementation has only achieved a maximum of 8% cells expressing hematopoietic progenitor markers in the study, resulting in low cell yields that are not suitable for therapeutic use. 3. Although the authors use the terms hematopoietic stem and progenitor cells interchangeably for CD34+CD45+ cells in their experiments, the actual hematopoietic stem cell fraction is only CD133+CD34+CD38-, with the rest being multipotent or committed progenitor cells. CD133+CD34+CD38- was not investigated in this study. 4. Studies have focused primarily on the specification of hematopoietic fates from pluripotent stem cells, and no experimental evidence has been provided for the further proliferation of differentiated HSCs in the presence of E2. 5. The study is consistent with other studies performed in mouse models, which reported that E2 promotes the proliferation of progenitor cells but not HSC proliferation and subsequent differentiation into erythroid cells. The bias in progenitor cells observed under the above experimental conditions is not beneficial for the expansion of HSCs and their use in transplantation, since these expanded fractions cannot adequately reconstitute the recipient. 6. The authors claim that UCB hematopoietic progenitors expanded up to 2-fold in 10 days, but this is of little clinical utility. Furthermore, no information is provided about other growth factors used in combination with E2, and there is no mention of the number of progenitors used in the assay, so the assessment of the multilineage potential of E2-expanded progenitors is not accurate, and the CFU numbers shown are technically not reproducible. 7. Studies have widely used fetal bovine serum as a medium supplement for culturing umbilical cord blood stem and progenitor cells, and these cells may contain some unknown growth factors, hormones, matrix proteins and other factors that may have contributed to the significant increase in differentiation of hematopoietic progenitor cells despite the two-fold expansion. Furthermore, the use of animal components for in vitro expansion is not suitable for therapeutic purposes due to the risk of exposure to non-human molecules and pathogens that may induce a strong immune response in humans.

[0262] Thus, the above studies have mainly focused on the effect of E2 on the proliferation of hematopoietic progenitor cells and promoting their differentiation, but have never demonstrated that HSCs can be significantly expanded while maintaining self-renewal. Furthermore, the above studies have taken the opportunity to define hematopoietic development under the influence of E2, rather than the proliferation of hematopoietic stem cells themselves.

[0263] Considering the in vivo rodent studies above, which show that estrogen increases, decreases, or has no effect on HSC numbers depending on the methodology / markers used to define the cell population, animal-based studies do not translate well to the human environment due to differences in cell surface marker phenotype and growth factor requirements and telomere activity that may lead to more frequent cell cycles and cell divisions in mice.

[0264] Although the proliferative potential of E2 has been well described in several mature tissues, little is known about its role in the proliferation and self-renewal of human HSCs with long-term repopulating potential. Moreover, E2 has not yet been tested in the expansion of human UCB-derived hematopoietic stem cells in a completely xeno-free environment using a therapeutically relevant defined serum-free growth medium.

[0265] US Patent No. 5,999,943 describes improvements for effecting and promoting recovery after hematopoietic stem cell transplantation. [Patent Document 1] U.S. Patent Application No. US20210386756A1

[0266] In [Patent Document 2], E2 and E4 were used to evaluate the engraftment and fold increase of CD34+CD38- cells. [Patent Document 2] U.S. Patent Application No. US20210386756A1

[0267] Fananas-Baquero et al. (see above) have shown that E2 and E4 proliferate human hematopoietic stem and progenitor cells.

[0268] Tomellini et al. (see above) utilize UM171 to expand cord blood cells.

[0269] Yao et al. (see above) have proposed a simple, well-defined, stoma-free, serum-free culture system consisting of Iscove's modified Dulbecco's medium, a cytokine cocktail, and serum replacement without the use of stromal receptor modulators. result

[0270] Since large amounts of HSCs are required for therapeutic effects, a few of the above studies have suggested that estrogen may mediate a proliferative effect on cord blood-derived HSCs with high clinical relevance. However, these studies do not fully address this issue as they do not distinguish between HSCs and more differentiated progenitors, leaving a large gap in interpretation. Furthermore, the above studies mainly used rodent models, which may not be ideal conditions for human HSCs, including differences in cell surface markers. To fully determine the capacity of E2 for therapeutic HSC expansion, the inventors investigated the effect of E2 on primitive (long-term) HSC populations, i.e., CD34+CD133+ cells. Furthermore, they used a more therapeutically relevant serum-free medium containing carefully selected chemically defined additives and a unique combination of growth factors, cytokines and ER-selective ligands. Beyond expectations, a significant increase in the number of CD34+ cells (CD34+CD133+ or CD34+CD38-) that maintained stemness with low doses of E2 was observed. The inventors have investigated the dose response of E2 and / or ER subtype-specific ligands in combination with known cytokines and growth factors at specific concentrations in defined serum-free medium.

[0271] Experiment 1 UCB HSCs were outsourced to a commercial supplier (Zen-Bio Inc., USA), and the cells were 80–95% CD34+CD133+HSCs. HSCs were cultured without feeder cells in defined serum-free medium containing an optimized cocktail of growth factors and cytokines. 17β-estradiol (E2) and ER-specific estrogen ligand were added at various concentrations ranging from 0.5 nM to 25 nM. Cells were cultured for 8 days and then counted with trypan blue to exclude dead cells. Cell numbers were quantified and fold proliferation compared to the control group was calculated. The fold proliferation of HSCs cultured for 8 days in the presence of E2 at doses of 1 and 10 nM was significantly higher at 129 ± 0.79 and 121.1 ± 3.98, respectively, compared to 94.23 ± 6.97 in untreated cells (Figure 1). Interestingly, the proliferation of UCB-HSCs was quite good even in the absence of E2 compared to ex vivo expansion methods reported by other researchers, suggesting that our specific growth factor formulation may strongly promote HSC proliferation. In any case, the addition of E2 to this medium leads to even higher proliferation of the cells. We next evaluated the HSC stemness of the ex vivo expanded cells using flow cytometry analysis. Following the ISHAGE recommendation, we gated the cells for CD45 expression and confirmed that all treatment and control conditions contained 70-80% CD34+ cells and 24-26% CD34+CD133+ cells (Figure 2). The number of CD34+CD133+ was surprisingly high, which further increases the reliability of our expansion method. Quantification of CD34+ cells during expansion cultures revealed that E2 treatment at 1 nM and 10 nM doses resulted in significantly higher proliferation of 91.94 ± 3.11 and 95.07 ± 1.47 fold, respectively, compared to 74.66 ± 6.68 fold in the control group (Figure 3). This represents the highest CD34+ cell proliferation in the shortest period (8 days) to date compared to proliferation reported in other studies (186 fold in 14 days, 222 fold in 16 days, 118 fold in 18 days, and 425 fold in 21 days).

[0272] Importantly, no significant differences were observed in the surface phenotype of untreated and E2-treated populations, indicating that the expanded cells retained their stemness and that the treatment conditions did not affect the phenotype. This supports our hypothesis that estrogen ligands can be effectively used to promote robust in vitro expansion of HSC populations from umbilical cord blood without affecting the HSC phenotype (Figure 4) (see Hematopoietic Stem Cell (HSC) Phenotype and Self-Renewal Pathway in 1). However, their in vitro differentiation capacity towards myeloid and lymphoid lineages and their ability to repopulate recipients require further investigation.

[0273] Experiment 2 In another experiment, to fully evaluate the estrogen effect on the proliferation of UCB-HSC in the absence of Notch ligands, magnetically selected CD34+CD38- cells (80-90%) from umbilical cord blood (Zen Bio, USA) were expanded with various growth factors, cytokines, estrogen and ER subtype-specific ligands for 14 and 21 days, respectively. The cells were analyzed by flow cytometry as described in experiment 1. The results of this experiment showed that the addition of GM-CSF and G-CSF to the culture medium significantly increased the number of expanded cells (in millions) compared to the control group (7.26±0.11 vs. 3.7±0.17) (Figure 5a). The addition of E2 to this cytokine cocktail significantly expanded the cells compared to the control group (5.86±0.11). Interestingly, the addition of different concentrations of Notch ligand (sDLL1) and IGF binding protein 2 (IGFBP2) to the standard medium recipe (FKT36, i.e. Flt-3, SCF, TPO, IL-3 and Il-6) had no significant effect on cell proliferation (Fig. 5a). However, the addition of both ligands to medium containing GM-CSF and G-CSF increased the number of cells compared to the control at day 14 (5.6 ± 0.72), but not at day 21 (Fig. 5a). Surprisingly, the addition of a 0.5 nm dose of E2 with the ERβ-selective LY3201 (0.5 nm) and ERα-selective PPT (1 nm) ligands significantly increased the number of cells proliferated at both culture periods (days 14 and 21). A similar effect was seen with regard to the fold proliferation of the cells, where the addition of estrogen and its ligand in the absence of Notch ligands significantly increased the fold proliferation at both culture periods (days 14 and 21). Furthermore, when E2 and its ligand were added in the presence of a Notch ligand, the cell proliferation fold increased compared to the control group (Fig. 5b).

[0274] The expanded cells were evaluated by flow cytometry for phenotypic characterization. As expected from the above observations, the fold expansion of CD34+ HSPCs on day 14 was significantly higher in the GG-supplemented group compared to the control group (Fig. 6a). However, there was no significant difference in the fold expansion of CD34+ cells among all treatment groups on day 21. This may be because at this point the self-renewal capacity of HSCs was exhausted and reached a plateau. Interestingly, the percentage of CD34+ cells among the total expanded viable cells did not change during both culture periods (Fig. 6b). These findings indicate that the expansion of UCB-HSCs in these different culture conditions does not affect their phenotype when expanded ex vivo. We further analyzed the fold expansion of CD34+CD133+ cells, also defined as CD38-, which are often referred to as LT-HSCs, which are not very different from our starting cell population (CD34+CD38-HSCs). Surprisingly, the fold expansion of CD34+CD133+ cells was higher in GM-CSF and G-CSF-supplemented media, while the addition of Notch ligand had no significant effect on both days 14 and 21 of culture (Figure 7). As expected from the above observations, the addition of estrogen with or without Notch ligand significantly increased the fold expansion of CD34+CD133+ cells at both culture periods. Addition of an ER-selective ligand to media containing Notch ligand also resulted in significant proliferation at day 21, but not at day 14. This may be due in part to the highly selective nature of this ligand, which may take some time to reach optimal effect in vitro.

[0275] Functional assessment and characterization of ex vivo expanded HSPCs for repopulating capacity and long-term engraftment Ex vivo expanded HSCs need to be characterized and quantified for stem cell activity. Single HSCs require self-renewal and the ability to efficiently differentiate into all types of hematopoietic cells to regenerate the entire hematopoietic system (37). However, direct assessment of HSC function in humans is quite limited, so alternative in vitro assays for functional assessment of immature HSCs have been developed (38). Flow cytometric analysis is a widely accepted method to prospectively identify and isolate HSCs. It can be used to assess regenerative potential, although it does not provide functional data (39). The colony-forming cell assay (CFC) is a superior in vitro method to identify hematopoietic progenitors, taking advantage of their ability to form multilineage colonies, which requires both differentiation potential and some degree of self-renewal capacity. Functional assessment of the most primitive HSC populations can be performed using in vitro cobblestone area-forming cells (CAFC) and long-term culture-initiating cells (LTC-IC) assays (40), both of which require more extensive self-renewal capacity than the CFC assay. However, the best evaluation of HSCs requires in vivo transplantation into immunodeficient mice, such as NOD-SCID mice, and assays for long-term multilineage and serial reconstitution ( 41 ). We expanded cord blood HSCs and assessed cell surface phenotype in culture and stem cell activity of the resulting cells by in vitro CFC assays.

[0276] The CFC assay in the above experiment (#2) showed that cells from all experimental groups (growth factor / ligand combinations) had the functional capacity of hematopoietic progenitors in addition to proliferation, and therefore retained differentiation potential. Furthermore, all groups treated with estrogen ligand showed the ability of HSPCs to differentiate into myeloid (CFU-GEMM, CFU-GM) and erythroid (CFU-E and BFU-E) lineages. Interestingly, there was no tendency for HSCs grown in estrogen ligand-supplemented medium to be biased towards megakaryocytic-erythroid lineages.

[0277] Experiment 3 In another experiment, to fully evaluate the estrogen effect on the proliferation of UCB-HSCs from cryopreserved cord blood units, D34+CD38- cells from cryopreserved cord blood units (Nationella navelström ochngsblodbanken, Sahlgrenska Universitetssjukhuset Sweden) were magnetically selected for 14 and 21 days, respectively, in various commercial and laboratory-made culture media containing combinations of growth factors, cytokines, estrogen and ER subtype-specific ligands. Cells were analyzed by flow cytometry as described in experiment 1 and experiment 2. The results of this experiment showed that both serum-free medium (SFM / SPro34) and serum-containing medium (FBS medium) without standard growth factors known for HSC proliferation (FKT) did not promote cell proliferation at all. Surprisingly, in the absence of FKT factors, all viable cells died. However, the addition of these factors, with or without the addition of E2, significantly promoted cell proliferation in both serum-free and serum-containing medium. Further addition of cytokines IL3 and IL6 (marked as 36) almost doubled cell proliferation compared to FKT alone medium. Further addition of GM-CSF and G-CSF to various serum-free media (SFM / SPro34, Invitrogen Corp.; StemSpanII, StemCell, Technologies Inc.; StemMACS HSC medium, Miltenyi Biotec; KISCO-H1, in-house; KISCO-H2, in-house) with or without estrogen ligands significantly increased the number of proliferated cells (in millions) compared to the control group (SFM / SPro34+FKT36, 3.7±0.1106) (Fig. 9a). Surprisingly, addition of E2 (10 nm dose) to different serum-free media containing FKT36GG cocktail significantly proliferated cells compared to the control group (SFM / SPro34+FKT36, 3.7±0.1106). Furthermore, addition of the ERβ-selective ligand LY3201 (1 nm) and the ERα-selective ligand PPT (1 nm) also increased the number of proliferated cells during the 14- and 21-day culture periods (FIG. 9a).A similar effect was observed with respect to cell fold expansion, where addition of estrogen and its ligand together with the optimized growth factor / cytokine cocktail FKT36GG significantly increased fold expansion over both 14- and 21-day culture periods (Fig. 9b).

[0278] The expanded cells were evaluated by flow cytometry for phenotypic characterization. As expected from the above observations, the expansion fold of CD34+HSPCs was significantly higher in all serum-free media supplemented with FKT36GG compared to FBS medium or standard FKT-supplemented SFM / SPro34 medium on days 14 and 21 (Fig. 10a). Various serum-free media performed relatively better with the optimized growth factor cocktail (FFKT36GG) supplemented with estrogen ligand. We further analyzed the expansion fold of CD34+CD38 cells, which are often referred to as long-term culture-initiating cells (LTC-ICs) and are also considered primitive hematopoietic stem cells with long-term reconstitution potential. Considering this fact, the starting population for this expansion experiment was also CD34+CD38-HSCs. Surprisingly, serum-free medium supplemented with FKT36GG+E2 (except StemMACS medium) increased the proliferation fold of CD34+CD38- cells more than two-fold (41.31-110.37-fold) on day 14 compared to SFM / SPro34 medium with FKT36GG alone (21.3-fold). Meanwhile, this effect was not evident on day 21. However, our proprietary serum-free medium increased the proliferation of CD34+CD38-LTC-ICHSCs 223.79-fold (KISCO-H2) or 173.50-fold (KISCO-H1) compared to the control medium (65.02-fold, SFM / SPro34+FKT36GG) (Fig. 10b).

[0279] Functional assessment and characterization of ex vivo expanded HSPCs for repopulating capacity and long-term engraftment As described in experiment 2 above, the cell surface phenotype and stem cell activity of the resulting cells during culture were assessed by in vitro CFC assay.

[0280] CFC assays from experiment (#3) demonstrated that cells from all experimental groups (growth factor / ligand combinations) had the functional capacity for hematopoietic progenitors in addition to proliferation, and therefore retained differentiation potential. Furthermore, all estrogen ligand-treated groups had higher total colony forming units (CFUs) of myeloid (CFU-GEMM, CFU-GM) and erythroid (CFU-E and BFU-E) lineages compared to the original cord blood units after thawing (Figure 11, dashed lines).

[0281] conclusion Ex vivo expansion of UCB-derived HSCs to therapeutically relevant numbers is promising for the treatment of malignant and non-malignant hematological diseases. Many approaches to expand cells have been reported, but reliable expansion in the short and long term has not been fully demonstrated. We have shown that with a unique and optimal composition of growth factors and ligands, UCB-HSCs can be sufficiently expanded in the short (8 days) and long term (14 and 21 days) without affecting their pluripotency. Furthermore, the presented medium supplement is remarkably effective in expanding pluripotent LT-HSCs, with the addition of estrogenic compounds that help to enhance cycling and maintain self-renewal. Using the method of the present invention, CD34+HSPCs and CD34+CD133+LT-HSCs expand more than 500-fold and 30-fold, respectively, in 14 days. Interestingly, this expansion is significantly increased when the cells are cultured for 21 days, with CD34+HSPCs and CD34+CD133+LT-HSCs expanding more than 1500-fold and 90-fold, respectively. Thus, this study provides a reliable method for both short-term and long-term (1-3 weeks) expansion of umbilical cord blood HSCs ex vivo without affecting their differentiation potential. The number of expanded cells far exceeds that of any other method, demonstrating the advantages of expanded UCB-HSCs in a clinical setting.

[0282] [Table 1]

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[0284] 20. Radtke F, et al. (1999) / / Immunity 10(5):547-558. 21. Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger RL, Bernstein ID. Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat Med (2010) 16:232_6. doi:10.1038 / nm.2080 22. Bordone L, Guarente L. Calorie restriction, SIRT1 and metabolism: under- standing longevity. Nat Rev Mol Cell Biol (2005) 6:298_305. doi:10.1038 / nrm1616 23.Peled T, Shoham H, Aschengrau D, Yackoubov D, Frei G, Rosenheimer GN, et al. Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Exp Hematol (2012) 40(342_355):e341. doi:10.1016 / j.exphem.2011.12.005 24. Horwitz ME, Chao NJ, Rizzieri DA, Long GD, Sullivan KM, Gasparetto C, et al. Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment. J Clin Invest (2014) 124:3121_8. doi:10.1172 / JCI74556 25.Wagner JE, Brunstein C, McKenna D, Sumstad D, Maahs S, Laughlin M, et al. StemRegenin-1 (SR1) expansion culture abrogates the engraftment barrier associated with umbilical cord blood transplantation (UCBT). Blood (2014) 124:728 26. Thurmond TS, et al. (2000) / / Endocrinology 141(7):2309-231. 27. Fish EN (2008) / / Nat Rev Immunol 8(9):737-744. 28. Heo HR, et al. (2015) / / Int J Stem Cells 8(1):18-23. 29. Ray R, et al. (2008) / / Mol Med 14(7-8):493-501.

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[0287] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Preferences and options with respect to a particular aspect, feature or parameter of the invention should be considered as disclosed in combination with all preferences and options with respect to all other aspects, features and parameters of the invention, unless the context dictates otherwise. For example, any defined estrogen receptor agonist can be combined with any defined sample.

[0288] A summary of the invention is provided below in numbered paragraphs. 1. A method for expanding CD34+ hematopoietic stem cells, comprising the steps of: i) providing a sample comprising one or more CD34+ hematopoietic stem cells; ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonists to expand the CD34+ hematopoietic stem cells. 2. The method of paragraph 1, wherein the CD34+ hematopoietic stem cells further comprise one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166. 3. The method of paragraph 2, wherein the CD34+ hematopoietic stem cells further comprise the marker CD133. 4. The method of paragraph 2, wherein the CD34+ hematopoietic stem cells further comprise the marker CD45(low / intermediate).

[0289] 5. The method of any one of paragraphs 1 to 4, wherein the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD133+ hematopoietic stem cells. 6. The method of any one of paragraphs 1 to 5, wherein the CD34+ hematopoietic stem cells lack one or more markers selected from the list consisting of CD38 and / or Lin. 7. The method of any one of paragraphs 1 to 6, wherein the CD34+ hematopoietic stem cells further comprise one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166, and the CD34+ hematopoietic stem cells lack one or more markers selected from the list consisting of CD38 and / or Lin. 8. The method of any one of paragraphs 1-7, wherein the CD34+ hematopoietic stem cells comprise long-term repopulating hematopoietic stem cells (LT-HSCs). 9. The method of any one of paragraphs 1 to 8, wherein the CD34+ hematopoietic stem cells comprise a sustained stemness phenotype.

[0290] 10. The method according to any one of paragraphs 1 to 9, wherein the medium is a serum-free medium. 11. The method according to any one of paragraphs 1 to 10, wherein step ii) comprises culturing the cells of step i) in the absence of feeder cells. 12. The method according to any one of paragraphs 1 to 11, wherein step ii) further comprises culturing the cells of step i) in the presence of one or more growth factors and / or cytokines. 13. The method of paragraph 12, wherein the one or more growth factors and / or cytokines are selected from the list consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), IGF-binding protein 2 (IGFBP2), Notch ligand, Notch ligand sDLL1, interleukin-3 (IL-3), interleukin-6 (IL-6), Flt-3 ligand, stem cell factor or KIT ligand or steel factor (SCF) and / or thrombopoietin (TPO). 14. The method of any one of paragraphs 1 to 13, wherein the sample is one or more selected from the group consisting of a sample comprising isolated CD34+ hematopoietic stem cells, a sample comprising isolated hematopoietic stem cells comprising CD34+ hematopoietic stem cells and / or a blood sample and / or a bone marrow sample.

[0291] 15. The method of paragraph 14, wherein the blood sample is one or more selected from the list consisting of peripheral blood, mobilized peripheral blood, or umbilical cord blood. 16. The method according to any one of paragraphs 1 to 15, wherein the estrogen receptor agonist is a nuclear estrogen receptor agonist and / or a membrane-bound estrogen receptor agonist. 17. The method of paragraph 16, wherein the nuclear estrogen receptor agonist is an estrogen receptor alpha (ERα) agonist and / or an estrogen receptor beta (ERβ) agonist. 18. The method of paragraph 17, wherein the ERα agonist is one or more molecules selected from the group consisting of estrogen E2, estrogen E1, estrogen E3, estrogen E4 and / or propylpyrazoletriol (PPT). 19. The method of paragraph 17, wherein the ERβ agonist is one or more molecules selected from the group consisting of estrogen E2, estrogen E1, estrogen E3, estrogen E4, diarylpropionitrile (DPN), LY3201 and / or LY-500307.

[0292] 20. The method of paragraph 16, wherein the membrane-bound estrogen receptor agonist is one or more molecules selected from the group consisting of a GPRC6A agonist, an ER-X agonist and / or a Gq-mER agonist. 21. The method of any one of paragraphs 1 to 20, wherein the estrogen receptor agonist is present at a concentration of from about 0.5 nM to about 25 nM, preferably from about 1 nM to about 10 nM. 22. The method of any one of paragraphs 1-21, further comprising the step of isolating CD34+ hematopoietic stem cells. 23. The method of paragraph 22, wherein isolating CD34+ hematopoietic stem cells comprises the use of flow cytometry and / or immunomagnetic cell separation and / or immune panning. 24. The method of any one of paragraphs 1 to 23, wherein step ii) is carried out for about 8 days or more, such as about 14 days or more or about 21 days or more.

[0293] 25. The method according to any one of paragraphs 1 to 24, wherein in the CD34+ hematopoietic stem cells expanded in step ii), the change in the proportion of one or more CD34+ hematopoietic stem cells of the CD34+ hematopoietic stem cell subpopulations (preferably CD34+D133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) is about 2 to about 20 CD34+ hematopoietic stem cells. 26. The method according to any one of paragraphs 1 to 25, wherein the number of CD34+ hematopoietic stem cells expanded in step ii) is increased by about 88-fold or more. 27. The method according to any one of paragraphs 1 to 26, wherein the number of CD34+ hematopoietic stem cells expanded in step ii) is increased by about 8,800% or more. 28. The method according to any one of paragraphs 1 to 27, further comprising administering the expanded CD34+ hematopoietic stem cells in step ii) to a subject in need thereof. 29. The method according to any one of paragraphs 1 to 27, further comprising differentiating the CD34+ hematopoietic stem cells expanded in step ii) into one or more cells derived from the CD34+ hematopoietic stem cells.

[0294] 30. The method of paragraph 29, further comprising the step of administering one or more cells derived from the CD34+ hematopoietic stem cells of step iii) to a subject in need thereof. 31. The method according to any one of paragraphs 1 to 30, wherein the method is an ex vivo method and / or an in vitro method. 32. Use of an estrogen receptor agonist to expand one or more CD34+ hematopoietic stem cells. 33. A population of CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 and / or a use as defined in paragraph 32. 34. One or more cells derived from CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31.

[0295] 35. The cell or cells of paragraph 34 or the method of any one of paragraphs 1 to 31, wherein the cell or cells derived from a CD34+ hematopoietic stem cell are cells selected from the list consisting of myeloid progenitor cells, lymphoid progenitor cells, erythroid cells, erythroid cells, mast cells, megakaryocytic cells, thrombocytes, mast cells, myeloblast cells, basophilic cells, neutrophilic cells, eosinophilic cells, monocytic cells, macrophage cells, dendritic cells, natural killer cells, T lymphocytes and / or B lymphocytes. 36. The cell or cells of paragraph 35 or the method of paragraph 35, wherein the T lymphocytes are CAR-T cells. 37. A composition comprising a population of CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 or as defined in paragraph 33. 38. A kit of parts comprising CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells as defined in paragraph 33. 39. A kit of parts for expanding CD34+ hematopoietic stem cells comprising one or more estrogen receptor agonists and a sample comprising one or more CD34+ hematopoietic stem cells and / or a means for obtaining a sample comprising one or more CD34+ hematopoietic stem cells.

[0296] 40. CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells as defined in paragraph 33, for use in medicine. 41. One or more cells derived from a CD34+ hematopoietic stem cell obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells as defined in paragraph 33, for use in medicine. 42. CD34+ hematopoietic stem cells obtainable and / or obtained by the method defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells as defined in paragraph 33, for use in treating and / or preventing a condition in a subject in need thereof. 43. One or more cells obtained from a CD34+ hematopoietic stem cell obtainable and / or obtained by a method defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells defined in paragraph 33, for use in the treatment and / or prevention of a condition in a subject in need thereof. 44. Use of CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31, or a population of CD34+ hematopoietic stem cells as defined in paragraph 33, in the manufacture of a medicinal product for the treatment and / or prevention of a condition in a subject in need thereof.

[0297] 45. Use of CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 or one or more cells obtained from a population of CD34+ hematopoietic stem cells as defined in paragraph 33 in the manufacture of a medicament for treating and / or preventing a condition in a subject in need thereof. 46. ​​A method for treating and / or preventing a condition in a subject in need thereof comprising administering to a subject in need thereof one or more cells obtained from a population of CD34+ hematopoietic stem cells obtainable and / or obtained by a method as defined in any one of paragraphs 1 to 31 or CD34+ hematopoietic stem cells as defined in paragraph 33. 47. A method for treating and / or preventing a condition in a subject in need thereof, comprising administering to a subject in need thereof CD34+ hematopoietic stem cells obtainable and / or obtained by a method defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells as defined in paragraph 33.

[0298] 48. A method of treating and / or preventing a condition in a subject in need thereof, comprising the steps of: a) providing a sample comprising one or more CD34+ hematopoietic stem cells; b) culturing the cells of step a) in the presence of a nuclear estrogen receptor agonist to expand the CD34+ hematopoietic stem cells and, optionally, differentiate the expanded CD34+ hematopoietic stem cells into one or more cells derived from the CD34+ hematopoietic stem cells; c) administering the cells of step b) to a subject in need thereof.

[0299] 49. CD34+ hematopoietic stem cells for use in the use according to any one of paragraphs 40 to 43, the use according to any one of paragraphs 44 to 45 or the method according to any one of paragraphs 46 to 48, wherein said condition is a condition requiring transplantation, a hematological condition and / or hematopoietic malignancy, a condition resulting from failure or dysfunction of normal blood cell production and maturation, immunosuppression for subjects with malignant and / or solid tumors, an autoimmune disease and / or immune condition, a genetic disease, a cancer, a tumor, osteopetrosis, myelosclerosis, acquired hemolytic anemia, an infection causing a primary or secondary immune deficiency, an acquired immune deficiency, CD34+ hematopoietic stem cells with one or more conditions selected from the list consisting of neutrophil actin deficiency, neutrophil membrane GP-180 deficiency, bacterial infections (such as brucellosis, listeriosis, tuberculosis, leprosy), parasitic infections (such as malaria, leishmaniasis), fungal infections, diseases with lymphocyte population imbalance and / or age-related immune dysfunction, phagocyte disorders, Kostmann agranulocytosis, chronic granulomatous diseases, Chediak-Higachi syndrome, Wiskott-Aldrich syndrome, metabolic storage diseases, mucopolysaccharidoses, mucolipidoses, alpha1-antitrypsin deficiency and / or other diseases involving immune mechanisms. 50. Methods, uses, populations of CD34+ hematopoietic stem cells, one or more cells derived from CD34+ hematopoietic stem cells, compositions, kits of parts, for the treatment and / or prevention of conditions substantially as described herein and / or with reference to the drawings.

Claims

1. A method for expanding CD34+ hematopoietic stem cells, comprising the steps of: i) providing a sample containing one or more CD34+ hematopoietic stem cells; and ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonists to expand the CD34+ hematopoietic stem cells.

2. 2. The method of claim 1, wherein the CD34+ hematopoietic stem cells further comprise one or more markers selected from the list consisting of CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166.

3. The method of claim 2, wherein the CD34+ hematopoietic stem cells further comprise the marker CD133.

4. The method according to any one of claims 1 to 3, wherein the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and one or more CD34+CD133+ hematopoietic stem cells.

5. The method of any one of claims 1 to 4, wherein the CD34+ hematopoietic stem cells lack one or more markers selected from the list consisting of CD38 and / or Lin.

6. The method of any one of claims 1 to 5, wherein step ii) further comprises culturing the cells of step i) in the presence of one or more growth factors and / or cytokines.

7. 7. The method of any one of claims 6, wherein the one or more growth factors and / or cytokines are selected from the list consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), IGF-binding protein 2 (IGFBP2), Notch ligand, Notch ligand sDLL1, interleukin-3 (IL-3), interleukin-6 (IL-6), Flt-3 ligand, stem cell factor or KIT ligand or steel factor (SCF) and / or thrombopoietin (TPO).

8. The method according to any one of claims 1 to 7, wherein the sample is one or more selected from the group consisting of a sample containing isolated CD34+ hematopoietic stem cells, a sample containing isolated hematopoietic stem cells comprising CD34+ hematopoietic stem cells, and / or a blood sample and / or a bone marrow sample.

9. 9. The method of claim 8, wherein the blood sample is one or more selected from the list consisting of peripheral blood, mobilized peripheral blood, or umbilical cord blood.

10. The method according to any one of claims 1 to 9, wherein the estrogen receptor agonist is a nuclear estrogen receptor agonist and / or a membrane-bound estrogen receptor agonist.

11. 11. The method of claim 10, wherein the nuclear estrogen receptor agonist is an estrogen receptor alpha (ERα) agonist and / or an estrogen receptor beta (ERβ) agonist.

12. 12. The method of claim 11, wherein the ERα agonist is one or more molecules selected from the group consisting of estrogen E2, estrogen E1, estrogen E3, estrogen E4 and / or propylpyrazoletriol (PPT).

13. 12. The method of claim 11, wherein the ERα agonist is one or more molecules selected from the group consisting of estrogen E2, estrogen E1, estrogen E3, estrogen E4, diarylpropionitrile (DPN), LY3201 and / or LY-500307.

14. The method of any one of claims 1 to 13, wherein the estrogen receptor agonist is present at a concentration of about 0.5 nM to about 25 nM, preferably about 1 nM to about 10 nM.

15. 15. The method of any one of claims 1 to 14, wherein step ii) is carried out for about 8 days or more, such as about 14 days or more, or about 21 days or more.

16. The method according to any one of claims 1 to 15, wherein in the expanded CD34+ hematopoietic stem cells of step ii), the ratio of one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+(low / intermediate)CD133+CD38- hematopoietic stem cells) in the CD34+ hematopoietic stem cell subpopulation is from about 2 to about 20 CD34+ hematopoietic stem cells.

17. The method according to any one of claims 1 to 16, wherein the number of expanded CD34+ hematopoietic stem cells in step ii) is increased by a factor of about 88 or more.

18. The method according to any one of claims 1 to 17, wherein the number of expanded CD34+ hematopoietic stem cells in step ii) is increased by about 8,800% or more.

19. Use of an estrogen receptor agonist to expand one or more CD34+ hematopoietic stem cells.

20. A population of CD34+ hematopoietic stem cells obtainable and / or obtained by the method as defined in any one of claims 1 to 18 and / or the use as defined in claim 19.

21. One or more cells derived from CD34+ hematopoietic stem cells obtainable and / or obtained by the method defined in any one of claims 1 to 18.

22. A composition comprising a population of CD34+ hematopoietic stem cells obtained and / or obtained by a method as defined in any one of claims 1 to 18 or CD34+ hematopoietic stem cells as defined in claim 20.

23. A population of CD34+ hematopoietic stem cells as defined in claim 20, obtained and / or obtained by a method as defined in any one of claims 1 to 18, for use in medicine.

24. 20. A population of CD34+ hematopoietic stem cells obtained and / or obtained by a method as defined in any one of claims 1 to 18 or CD34+ hematopoietic stem cells as defined in claim 20 for use in the treatment and / or prevention of a condition in a subject in need thereof.

25. CD34+ hematopoietic stem cells, wherein the condition is one or more conditions selected from the list consisting of conditions requiring transplantation, hematological conditions and / or hematopoietic malignancies, conditions resulting from failure or dysfunction of normal blood cell production and maturation, immunosuppression in subjects with malignant and / or solid tumors, autoimmune diseases and / or immune conditions, genetic diseases, cancers, tumors, osteopetrosis, myelosclerosis, acquired hemolytic anemia, infections causing primary or secondary immunodeficiencies, acquired immunodeficiencies, neutrophil actin deficiency, neutrophil membrane GP-180 deficiency, bacterial infections (e.g., brucellosis infection, listeriosis infection, tuberculosis infection, and / or leprosy).

25. The CD34+ hematopoietic stem cells for use according to claim 23 or 24, which are CD34+ hematopoietic stem cells for the treatment of parasitic infections (such as malaria and leishmaniasis), fungal infections, diseases associated with an imbalance in lymphocyte sets or a decline in immune function due to aging, phagocytic disorders, Kostmann agranulocytosis, chronic granulomatous diseases, Chediak-Higachi syndrome, Wiskott-Aldrich syndrome, metabolic storage diseases, mucopolysaccharidoses, mucolipidoses, alpha-antitrypsin deficiency and / or other diseases involving the immune system.

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