Methods

Abstract

The present invention provides methods for the expansion of CD34+ hematopoietic stem cells (HSCs), and associated uses, cells produced by such methods and medical treatments.

Description

[0001]Methods The present invention provides methods for the expansion of CD34+ hematopoietic stem cells (HSCs), and associated uses, cells produced by such methods and medical treatments. The transplantation of HSCs has great potential for medical treatments for a wide variety of illnesses, including various hematologic, oncological and immune disorders. However, the practical application of such treatments is limited due to the low availability of HSCs, often due to the lack of availability of HSCs donors matched to the potential patient. The resultant low doses of HSCs available for transplantation leads to slow engraftments, which can lead to the treatment failure. In order to overcome the low availability of HSCs, ex vivo expansion of HSCs (in particular, HSCs from umbilical cord blood) shows great potential by allowing those cells to be prepared in amounts that are more clinically relevant, and more likely to allow for successful treatments. Although several approaches to the ex vivo expansion of HSCs have been developing using molecules such as hematopoietic growth factors, cytokines and blockers of differentiation (such as Notch ligands, copper chelators, nicotinamide and aryl hydrocarbon receptor antagonist) there is the need for additional and improved expansion methods. For example, expansion of hematopoietic stem / progenitor cells described in Liedtke et. al., 2019, Stem Cell Translational Medicine 8: S20-S20, doi:10.1002 / sctm.12563. In a specific example, hematopoietic stem / progenitor cells can be thawed, enriched and cultured in serum free media 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), 100 ng / mL stem cell factor or KIT ligand or steel factor (SCF) for 3-4 days. In another example, expansion of hematopoietic stem / progenitor cells is set forth in Rogers et. al., 2008, biology of Blood and Marrow transplantation, 14:927-937, doi:10.1016 / j.bbmt.2008.06.002. In a specific example, hematopoietic stem / progenitor cells can be cultured in serum free media containing IMDM, 1% bovine serum albumin, 10 mg / mL bovine pancreatic insulin, 200 mg / mL human transferrin, 10-4M b-mercaptoethanol, 2 mM L-glutaminem, 40 mg / mL low-density lipoproteins, and growth factors; 25 ng / mL FGF-4, 25 ng / mL IL-3, 25 ng / mL SCF, 25 ng / mL FLT3 ligand, plus heparin 25 ng / mL. Cells were grown for 4 days to 28 days with medium and growth factor changes made 3 times / week. All cells were grown in tissue culture treated 6, 12, or 24 well plates. However, neither of the methods of Liedtke et. al., 2019, supra or Rogers et. al., 2008 supra provide as high quality amounts of HSCs as the present invention. Estrogen has been used as a media supplement for growth of lineage committed progenitors and differentiation of stem cells. However, the use of estrogen has not been considered, or identified, as a molecule that could be used for HSCs’ expansion. Against this background, the inventors have identified, for the first time, the use of estrogen receptor agonists (including estrogen) for the expansion of medically highly- relevant HSCs which express the CD34 marker (for example, CD34+ HSCs), as well as a sub-populations of CD34+ HSCs such as those also expressing the CD133 marker (for example, CD34+ CD133+ HSCs). Surprisingly, methods of expansion of CD34+ HSCs and CD34+ CD133+ HSCs using estrogen receptor agonists leads to expanded populations of cells of high quality which makes them very valuable for medical treatments. In a first aspect, the 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; and ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonist, to expand the CD34+ hematopoietic stem cells. In a second aspect, the invention provides a use of an estrogen receptor agonist for expanding one or more CD34+ hematopoietic stem cells. The use of the second aspect of the invention can comprise the steps described herein in relation to the method of the first aspect of the invention. Hematopoietic stem cells (HSCs) are a type of multipotent stem cell that are capable of self-renewal and differentiation into progenitor cells, such as myeloid progenitor cells and / or lymphoid progenitor cells, which can ultimately differentiate into any blood cell (such as erythrocytes) and / or cell of the immune system (such as T-lymphocytes and B-lymphocytes). HSCs can be found in vivo in bone marrow, as well as umbilical cord blood. HSCs expressing the cell surface marker CD34 are known as CD34+ HSCs. CD34 is a transmembrane phosphoglycoprotein protein. The nucleic acid sequence of CD34 is known, and is described in, for example, Satterthwaite AB et al., Structure of the gene encoding CD34, a human hematopoietic stem cell antigen, Genomics, 1992 Apr;12(4):788-794. The amino acid sequence of CD34 is described in 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. CD34 can be used to identify and isolate CD34+ HSCs, using methods such as flow cytometry (for example, fluorescence-activated cell sorting (FACS)), immunomagnetic cell separation and / or immunopanning. The concepts of HSCs and CD34+ HSCs, as well as their identification and isolation, would be known to persons skilled in medicine and hematology. For example, standard procedures for identifying HSCs as being CD34+ include immunohistochemistry, flow cytometry, and gene expression. Accordingly, by “one or more CD34+ hematopoietic stem cells” we include one or more hematopoietic stem cells comprising the CD34 marker and / or expressing the CD34 gene. By “one or more” CD34+ hematopoietic stem cells, we include about two or more CD34+ hematopoietic stem cells, which might also be referred to as a population of CD34+ hematopoietic stem cells. In one embodiment, the about two or more CD34+ hematopoietic stem cells and / or population of CD34+ hematopoietic stem cells is 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; about 900 or more; about 1,000 or more; about 1,500 or more; about 2,000 or more; about 2,500 or more; about 3,000 or more; about 3,500 or more; about 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 16,000 or more; about 17,000 or more; about 18,000 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 300,000 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. In some embodiments, the sample comprises about 100 CD34+ hematopoietic stem cells. In some other embodiments, the sample comprises about 100 or more 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 about 1,000 or more 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 about 3,000,000 or more CD34+ hematopoietic stem cells. In some other embodiments, the sample comprises up to about 3,000,000 CD34+ hematopoietic stem cells. For example, the “one or more” CD34+ hematopoietic stem cells and / or population of CD34+ hematopoietic stem cells is about 1,000,000 or more CD34+ hematopoietic stem cells; for example, 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 between about 100 and about 3,000,000 CD34+ hematopoietic stem cells. The number of CD34+ hematopoietic stem cells included in the sample of step (i) may be dependent on how long the culturing step of (ii) is to be carried out for, to ensure a desired level of expansion is achieved. For example, if step (ii) is to be carried out for 8 days, the number of CD34+ hematopoietic stem cells included in the sample of step (i) may be from 30,000-40,000, preferably in the region 35,000-36,000 cells. In another example, if step (ii) is to be carried out for 14 to 21 days, the number of CD34+ hematopoietic stem cells included in the sample of step (i) may be from 5,000 to 15,000 cells, preferably in the region 9,000 to 10,000 cells. In a further example, if step (ii) is to be carried out for 14 to 21 days, the number of CD34+ hematopoietic stem cells included in the sample of step (i) may be from 1000-1500. Stem cell expansion (or the expanding of stem cells, as referred to in the present invention) is the process by which a stem cell divides to form either two daughter cells both maintaining self-renewing stem cell properties of the parent cell, or producing two daughter cells, one of which retains the self-renewing property of the parent stem cell whilst the other differentiates into a more specialised type of cell that may have none or limited potential to self-renew. Cell division in the former case is sometimes referred to as symmetric stem cell division whilst the latter case is referred to as asymmetric cell division. Both types of cell division that lead to the propagation of stem cells with the capacity for self-renewal are included in the definition of “expansion” as used herein. By stem cell expansion we further include that the CD34+ hematopoietic stem cells are cultivated under specific conditions, as described herein, that promote cell growth (for example, mitosis) such that the cells will reproduce and divide giving rise to expanded hematopoietic stem / progenitor cell population. Stem cell expansion is different to the process of stem cell differentiation. Differentiation includes when a stem cell divides to form two daughter cells, one of which maintains the stem cell properties of the parent stem cell for fewer cell divisions than in the case of expansion whilst the other differentiates into a more specialised type of cell, eventually leading to terminal differentiation of both progenies. Cell division in stem cell differentiation is may sometimes be referred to as asymmetric stem cell division; however it does not lead to the propagation of stem cells with the capacity for self-renewal. Self-renewal is cell division specific to stem cells. When self-renewal occurs, a stem cell divides into two cells, one of which is still a stem cell (which can display differentiation potential into one or more types of differentiated cells and divides almost indefinitely), and another is a differentiated cell, which may or may not divide, but its divisions (if any) are not self-renewal because: (1) no stem cell (i.e. a cell with developmental potential) is produced, and; (2) these divisions are finite (so they are not self-renewal as self-renewal is indefinite yet controlled proliferation). In sum, self- renewal is a stem cell's 'unlimited' division while retaining its developmental potential. In other words, “stem cell expansion” as referred to herein refers to stem cell self- renewal, i.e. any process that leads to propagation of unlimited division whilst retaining development potential. To use HSCs as an example, expansion would be the division of a parent HSC to form two identical HSCs or at least one progeny HSC retaining the same stem cell properties (with the other progeny forming a progenitor cell), whilst differentiation would be the division of a parent HSC to form two progenitor cells (such as a myeloid progenitor cell or a lymphoid progenitor cell). Accordingly, by “to expand the CD34+ hematopoietic stem cells” (as described in step ii) of the present invention) we include that a result of the method is that the one or more CD34+ hematopoietic stem cells divide 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. As will be appreciated, any daughter CD34+ hematopoietic stem cells can themselves become expanded to generate further daughter CD34+ hematopoietic stem cells, and so on. When expanding HSCs it is most preferable to avoid HSC differentiation, as that differentiation will lead to multiple cell types being present in the resulting cell population. A heterogeneous population of cells, including a mixture of HSCs and differentiated cells, provides a lower quality of therapeutic effect when compared to a population of cells with a very high proportion of HSCs or a population of cells only including HSCs. An advantage of the inventors’ method is that it greatly limits and / or avoids HSC differentiation, when compared to other expansion methods known in the art, leading to a resulting cell population that will provide a high quality therapeutic. Therefore, by expanding CD34+ HSCs by the method of the invention we include not substantially differentiating HSCs or not differentiating HSCs. Estrogen receptors are a group of proteins that are bound by and activated by estrogen. As described further herein, estrogen receptors can largely be divided into two classes – nuclear estrogen receptors and membrane estrogen receptors. By “estrogen receptor agonist”, we include a molecule that is capable of binding to, and preferably activating, an estrogen receptor. As described herein, estrogen receptor agonists can be naturally occurring molecules or artificially synthesised molecules. In one embodiment, the estrogen receptor agonist is a molecule that functionally mimics estrogen, in a particular embodiment an estrogen receptor agonist is a molecule that functionally mimics estrogen completely or partially. In a particular embodiment, the estrogen receptor agonist is an estrogen, as described herein. In one embodiment, the estrogen receptor agonist is a naturally occurring estrogen receptor agonist or a synthetic estrogen receptor agonist. In general terms, culturing cells includes growing and maintaining cells in an in vitro environment. Accordingly, by “culturing the HSCs in the presence of one or more estrogen receptor agonist” (as described in step ii) of the present invention) we include growing and / or maintaining the HSCs in the presence of one or more estrogen receptor agonist, preferably in an in vitro environment. The culturing of the HSCs in the presence of the estrogen receptor agonist includes that the estrogen receptor agonist is allowed to come into contact with the HSCs in a manner which will allow the estrogen receptor agonist to activate the estrogen receptors of the HSCs. We further include that “culturing the HSCs in the presence of one or more estrogen receptor agonist” can include adding one or more estrogen receptor agonist to the sample. 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. In a preferred embodiment, the CD34+ hematopoietic stem cells further comprise the marker CD133 (for example, CD34+ CD133+ hematopoietic stem cells). In an alternative preferred embodiment, the CD34+ hematopoietic stem cells further comprise the marker CD45 (for example, CD34+ CD45+ hematopoietic stem cells). In a further preferred embodiment, the CD34+ hematopoietic stem cells further comprise the markers CD133 and CD45 (for example, CD34+ CD45+ CD133+ hematopoietic stem cells). CD133 (also known as prominin-1), is a pentaspan membrane glycoprotein, and is expressed on a subpopulation of CD34+ cells obtained from different sources including fetal liver and bone marrow, adult bone marrow, cord blood, and mobilized peripheral blood. CD133+ stem cells can reconstitute myelo- and lymphopoiesis in mice (see for example Handgretinger R, Kuçi S. CD133-Positive Hematopoietic Stem Cells: From Biology to Medicine. Adv Exp Med Biol. 2013;777:99-111. The nucleic acid sequence of CD133 is described in Katoh Y and Katoh Y: Comparative genomics on PROM1 gene encoding stem cell marker CD133. Int J Mol Med 19: 967-970, 2007. The amino acid sequence of CD133 is described in Sheri Miraglia et al., A Novel Five-Transmembrane Hematopoietic Stem Cell Antigen: Isolation, Characterization, and Molecular Cloning. Blood 1997; 90 (12): 5013–5021. CD45 is a receptor linked protein tyrosine phosphatase present in all cells of the hematopoietic lineage except erythrocytes and plasma cells. It was originally known as common leukocyte antigen or protein tyrosine phosphatase receptor type C. CD45 regulates a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. The CD45 protein family consists of multiple members that are all products of a single complex gene. CD45RA includes only the A protein region whilst CD45RO, the shortest CD45 isoform lacks all three of the A, B, and C regions. The nucleic acid and amino acid sequences of CD45 are described in Holmes N. CD45: all is not yet crystal clear. Immunology. 2006 Feb;117(2):145-55. CD43, also known as leukosialin or sialoglycoprotein, has been shown to be expressed on early hematopoietic cells derived from hESCs. Comparable to CD34, CD43 is a strongly glycosylated transmembrane sialomucin, expressed on all hematopoietic cells, except mature erythrocytes. CD43 is described further in 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. The nucleic acid and amino acid sequences of CD43 are described in 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. CD59 is a phosphoinositol glycan–linked membrane protein that is uniformly expressed by most nucleated cells of the hematopoietic system including all the BM-derived CD34+ cells. However, CD59 is differentially expressed among CD34+ progenitors, with the highest level present on a subset that is highly enriched for pluripotent stem cells. CD59 is further described in Simmons PJ et al., A novel epitope of CD59 expressed by primitive human hematopoietic progenitors. Exp Hematol. 2001 Dec;29(12):1474-83. The nucleic acid and amino acid sequences of CD59 are described in J G 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. CD90, or Thymocyte differentiation antigen (Thy-1), is a small 25^35 kDa GPI anchored glycoprotein, and is an important regulator of cell-cell and cell-matrix interaction. It is involved in tumor suppression, cell adhesion, migration and apoptosis signaling. CD90 is highly expressed on cord blood primitive cells of high proliferation capacity and participates in HSC development. CD90 is further described in 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. The nucleic acid sequence of CD90 is described in Craig W et al., Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med. 1993 May 1;177(5):1331-42. The amino acid sequence of CD90 is described in Wollscheid B et al., Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol. 2009 Apr;27(4):378-86. CD109 is a glycosyl phosphatidylinositol (GPI)-linked glycoprotein that localizes to the surface of platelets, activated T-cells, and endothelial cells and a subset of fetal and adult CD34+ bone marrow mononuclear cells. CD109 negatively regulates transforming growth factor beta (TGF-beta) signalling associated with HSC differentiation. CD109 is discussed further in Martin Lin et al.; Cell surface antigen &'^^^^ LV^ D^ QRYHO^PHPEHU^ RI^ WKH^ Į^^PDFURJOREXOLQ^&^^^&^^^&^^ IDPLO\^ RI^ WKLRHVWHU- containing proteins. Blood 2002; 99 (5): 1683–1691. The nucleic acid and amino acid sequences of CD109 are described in 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. CD117 or c-KIT, a receptor tyrosine kinase type III, binds to a stem cell factor also known as "steel factor" or "c-kit ligand" and plays a key role in cell survival, proliferation, and differentiation. CD117 is highly expressed on hematopoietic stem cells (HSC), multipotent progenitors (MPP), and common myeloid progenitors (CMP) and promotes proliferation of cells. CD117 is discussed further in 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. Epub 2007 Jan 20. PMID: 17350321. The nucleic acid and amino acid sequences of CD117 are described in 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. CD166, also known as activated leukocyte cell adhesion molecule (Alcam), a member of the immunoglobulin superfamily, mediates homophilic (CD166-CD166) and heterophilic (CD166-CD6) interactions. CD166 is expressed on stem and progenitor cells of all three embryonic lineages, and CD166 interactions led to activation of key molecular pathways involved in enhancing HSC / HPC functions implicated in maintenance of stem cell pluripotency. CD166 is further described in 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. The nucleic acid and amino acid sequences of CD166 are described in 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. The standard procedures for identifying a HSC as being CD34+ discussed above can also be used to identify the HSCs as having these further markers (for example: CD133; CD45; CD43; CD45RO; CD45RA; CD59; CD90; CD109; CD117; or CD166), which includes immunohistochemistry, flow cytometry, and gene expression. By 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” we include that the one or more hematopoietic stem cells express the CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, and / or CD166 genes. As would be appreciated by one skilled in molecular biology and / or genetics, expression of a particular gene (such as CD133) includes the presence of a gene product, such as mRNA and / or protein. 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). CD38, also known as cyclic ADP ribose hydrolase, is a glycoprotein found on the surface of leucocytes (white blood cells) and functions in cell adhesion, signal transduction and calcium signalling. The CD38 protein, rearranged at high levels by activated lymphocytes, is a marker of cell activation. The expression of cell surface antigen CD38 in hematopoietic cells is activated through differentiation, and thus exclusion of CD38 expression on CD34+ cells is related with long-term HSC activity. CD38 is discussed further in 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. The nucleic acid and amino acid sequences of CD38 are described in 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. HLA-DR is major histocompatibility complex class II (MHC class II) cell surface receptor encoded by several loci and genes of different function of the human leukocyte antigen complex on chromosome 6 region 6p21.31. The receptor / peptide complex is a ligand for T-cell receptor (TCR) and mediates acceptability / rejection of organ / blood transplants in recipients. HLA-DR is also involved in several autoimmune conditions, disease susceptibility and disease resistance. HLA-DR is expressed in the umbilical cord and peripheral blood cells. The most primitive HSCs co-express c-kit, FLT-3 and Thy- 1, being negative for HLA-DR, CD38 and lineage markers. HLA-DR is discussed further in Stern J. Lawrence and Calvo-Calle Mauricio J., HLA-DR: Molecular Insights and Vaccine Design, Current Pharmaceutical Design 2009; 15(28). The nucleic acid and amino acid sequences of HLA-DR described in 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. Lineage (Lin) markers refers to the expression of various lineage markers associated with a certain cell type. Hematopoietic stem cells give rise to different types of blood cells, termed as myeloid and lymphoid. Myeloid and lymphoid lineages both are involved in the formation of myeloid cells (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets) and lymphoid cells (T cells, B cells, natural killer cells, and innate lymphoid cells). Hematopoietic stem cells can be identified or isolated from the surrounding blood cells using flow cytometry where the combination of one or more different cell surface markers (mentioned above) in conjunction with CD34 are used. HSCs lacking the expression of mature blood cell markers, including but not limited to CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a, are thus called Lineage negative (Lin-). Lack of expression of lineage markers is used in combination with detection of several positive cell-surface markers to isolate hematopoietic stem cells. Therefore, 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. In some embodiments, the hematopoietic cells therefore lack expression of one or more markers selected from: CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a. The isolation and / or enrichment of hematopoietic stem cells including and / or lacking particular markers is described further herein. The standard procedures for identifying a HSC as being CD34+ discussed above can also be used to identify the HSCs as lacking differentiation markers including CD38, HLA DR, and / or Lin, which includes immunohistochemistry, flow cytometry, and gene expression. By the “hematopoietic stem cells lack one or more markers selected from the list consisting of: CD38, HLA DR and / or Lin” we include that the one or more hematopoietic stem cells do not comprise the CD38, HLA DR and / or Lin markers and / or do not express the CD38, HLA DR and / or Lin genes discussed above. 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 (for example, 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 (for example, CD34+ CD133+ CD38- hematopoietic stem cells). The “low / intermediate” CD45 relates to CD45 expression, and those relative levels would be understood by one skilled in molecular biology. By low / intermediate CD45 expression, we include low CD45 expression and / or intermediate CD45 expression. A method of identifying such expression is using flow cytometry. The relative CD45 expression is relevant to the present invention because high CD45 expression is related to differentiated lymphocytes. Generally in flow cytometry or other fluorescence based isolation methods, cells are gated based on the relative intensity of fluorescence marker, e.g. CD45. For HSC selection, high intensity CD45 expressing cells are excluded to select only HSCs. As would be appreciated by the skilled person, the intensity scales depend on the sensitivity of the machine (flow cytometer) and could differ between different equipment and for this reason cells are gated based on their relative intensity within that analyzer / equipment. In the embodiments described herein where the CD34+ hematopoietic stem cells comprise additional specified markers (such as CD133; CD45; CD43; CD45RO; CD45RA; CD59; CD90; CD109; CD117; and / or CD166) and / or do not comprise differentiation markers (such as CD38, HLA DR and / or Lin), these hematopoietic stem cells are understood to be sub-populations of a general population of CD34+ hematopoietic stem cells. In one embodiment, the CD34+ hematopoietic stem cells are long-term repopulating hematopoietic stem cells (LT-HSCs). Long-term repopulating hematopoietic stem cells (LT-HSCs) are generally understood to be CD34+ CD133+ CD38- hematopoietic stem cells. The inventors have surprisingly identified that their methods are particularly effective at expanding this sub-population of hematopoietic stem cells as well as the CD34+ CD133+ CD45+(low / intermediate)CD38- hematopoietic stem cells, as described in the Examples herein. In a particular embodiment, the CD34+ hematopoietic stem cells comprise one or more CD34+ hematopoietic stem cells and 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 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 comprise one or more CD34+ hematopoietic stem cells and one or more CD34+ CD133+ hematopoietic stem cells. In an alternative 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 an alternative 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 alternative 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. To describe these embodiments in another way, the sample can comprise a mixture of CD34+ hematopoietic stem cells either with or without one or more further 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. In some embodiments of the invention where there is a mixture of CD34+ hematopoietic stem cells and CD34+ hematopoietic stem cells comprising and / or lacking other specified markers, the daughter CD34+ hematopoietic stem cells of step ii) will comprise and / or lack the same specified markers as the parent CD34+ hematopoietic stem cells; for example, the daughter hematopoietic stem cells derived from a parent CD34+ CD133+ hematopoietic stem cell will also comprises the markers CD34 and CD133, and / or the daughter hematopoietic stem cells derived from a parent CD34+ CD133+ CD38- hematopoietic stem cell will also comprises the markers CD34 and CD133 and lack the CD38 marker. In some other embodiments, the daughter CD34+ hematopoietic stem cells of step ii) will comprise and / or lack different specified markers as the parent CD34+ hematopoietic stem cells due to variations in the conditions; for example, the daughter hematopoietic stem cells derived from a parent CD34+ CD133+ CD38- could lack the CD133 marker and / or will additionally comprise the CD38 marker. In one embodiment, the CD34+ hematopoietic stem cells comprise a sustained stemness phenotype. In particular, the CD34+ CD133+ hematopoietic stem cells comprise a sustained stemness phenotype In general terms, HSC stemness is the ability of a HSC to self-renew and produce two identical progeny, though, unless sustained, daughter cells may fail to produce two identical progeny further but one of them retains the same phenotype and the other commits differentiation to progenitor of specific blood cell. Therefore, a sustained stemness phenotype of a HSC comprises self-renewal with limited differentiation, whilst retaining the ability to differentiate to all type of blood cells when the relevant inductive signals are provided. Sustained stemness may also be referred to as retained stemness. In one embodiment, the sustained stemness phenotype is characterised by the expanded CD34+ hematopoietic stem cells comprising the same specified markers as the CD34+ hematopoietic stem cells from which they were derived (for example, 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 lack of one or more markers selected from the list consisting of: CD38, HLA DR and / or Lin). In a further embodiment, the sustained stemness phenotype is characterised by the expanded CD34+ hematopoietic stem cells being able to sustain long-term cultures (for example, long-term cultures on marrow feeder cells). In a further embodiment, the sustained stemness phenotype is characterised by the expanded CD34+ hematopoietic stem cells being capable of differentiating into progenitor cells (preferably wherein the progenitor cells are capable of differentiating into all type of mature blood cells). How to identify if the HSCs are capable of differentiating into progenitor cells (and ultimately mature blood cells) would be known to one skilled in the art of cell biology. For example, this can be assessed using the methods described in the Examples, such as the expanded HSCs being able to produce colonies of different progenitors. In one embodiment, step ii) comprises culturing the hematopoietic stem cells in a media comprising the estrogen receptor agonist. As would be known to one skilled in cell biology, a media (generally referred to as a cell media) is generally a liquid containing components to allow cells to grow and be maintained. In one embodiment, the media is a defined media. As would be known to one skilled in cell biology, a defined media is a media in which all of the ingredients, and their relative concentrations and / or quantities, are known. This is as opposed to a media which contains ingredients with unknown or undefined contents, concentrations and / or quantities. Generally, defined media does not contain ingredients derived from animals, but instead includes ingredients that are chemically- synthesised and / or not derived from animals, which allows for a greater control of the media’s contents. In one embodiment, the media is a serum-free media. In a particular embodiment, the serum-free media is an animal serum-free media. As would be known to one skilled in cell biology, serum is an example of a media ingredient which does not have well-defined contents, concentrations and / or quantities. Therefore, the inclusion of serum in a media would lead to it not being a defined media. Additionally, it can be preferable not to use media including serum in order to avoid contamination with infectious agents (such as viruses) and / or the immunization of any recipient patient with foreign proteins. In one embodiment, the media is a xeno-free media. As would be known to one skilled in cell biology, a xeno-free media is a media which does not include components not derived from the same species as the cells to be cultured; for example, if human cells are to be cultured, the corresponding xeno-free media would not include any non-human biological components. In one embodiment, step ii) comprises culturing the cells of step i) in the absence of feeder cells. As would be known to one skilled in cell biology, feeder cells can be co-cultured with HSCs, and provide an undefined mixture of ingredients to the HSCs. In one embodiment, step ii) further comprises culturing the cells of step i) in the presence of one or more growth factors and / or cytokines. A growth factor is a biological substance that can regulate a cellular process, such as stimulate cell proliferation. Growth factors are often cell signalling substances and / or hormones which are produced by cells; however, in some cases growth factors can be artificially synthesised. A cytokine is also a biological substance that can mediate a cellular process such as hematopoiesis. Cytokines are signaling molecules which are also produced by cells and can be synthesized artificially. It would be known to the person skilled in cell biology how to identify growth factors and cytokines which could be ultilised as part of the present invention. Accordingly, by growth factor and cytokine we include a biological substance that can stimulate cell proliferation. In one embodiment, 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) IGF binding protein 2 (IGFBP2) (preferably, recombinant human Insulin-like growth factor (IGF) binding protein 2 (rhIGFBP2)); a 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 an alternative preferred embodiment, the one or more growth factors are interleukin-3 (IL-3) and interleukin-6 (IL-6). In a particular embodiment, 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) – these are the growth factors added to a basal media in the “FKT36 media” referred to herein. In an alternative embodiment, a notch ligand (such as notch ligand sDLL1) and / or IGFBP2 are not used in the method of the present invention. In a particular embodiment, 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 notch ligand sDLL1) is not used in the method of the present invention. In a particular embodiment, the growth factors and / or cytokines can be at the following amounts: about 1 ng / mL to about 20 ng / mL (preferably, about 10 ng / mL) Interleukin- 3 (IL-3); about 50 ng / mL to about 150 ng / mL (preferably, about 100 ng / mL) Interleukin-6 (IL-6); about 50 ng / mL to about 150 ng / mL (preferably, about 100 ng / mL) Thrombopoietin (TPO); about 50 ng / mL to about 150 ng / mL (preferably, about 100 ng / mL) Flt-3 Ligand (Flt-3L); about 50 ng / mL to about 150 ng / mL (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). The cytokines and growth factors described herein can be procured commercially, and are generally chemically synthesized or produced as recombinant products. Preferably, the growth factors / cytokines are derived from the same species as the starting sample cell population (preferably, human) and recombinantly expressed for which 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). The term “basal medium” relates to a medium which can support growth of hematopoietic cells, such as CD34+ hematopoietic cells or other cells of the present invention, when supplemented either with serum, or with the serum-free ingredients of the present invention. The basal medium should be able to provide inorganic salts, e.g. sodium, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, an energy source such as glucose, a pH buffer system, and essential amino acids that are necessary for standard cell growth. Basal media which can be used as part of the present invention include but are not limited to Iscove's Modified Dulbecco's Medium, RPMI 1640, Minimal Essential Medium-a (MEM-a), and other related media. In one embodiment, the basal media used in the present invention (for example, in step i) and / or step ii) of the present invention) is Iscove's Modified Dulbecco's Medium (IMDM) (ThermoFisher Scientific, Merck, KGaA / Sigmaaldrich), RPMI 1640, or Minimal Essential Medium-a (MEM-a). In one embodiment, the basal media (such as Iscove's Modified Dulbecco's Medium (IMDM)) is further supplemented with one or more of the components selected from the list of components comprising of: human serum albumin; ethanolamine; sodium selenite; hydrocortisone; D,L-tocopherol; Human transferrin (holo); human insulin (zinc); N-acteyl-L cysteine; 2-mercaptoethanol; and / or cholesterol lipid concentrate (such as obtainable from ThermoFisher Scientific or others). In a particular embodiment, the components can be at 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; 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- acteyl-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, ThermoFisher Scientific). In a particularly preferred embodiment, the basal media (such as Iscove's Modified Dulbecco's Medium (IMDM)) comprises 5% human serum albumin, 10 mg / L ethanolamine, 5μg / 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 commonly-available mixture of insulin and zinc), 160 mg / L N-acteyl-L cysteine, 4 mg / L 2-mercaptoethanol and cholesterol lipid concentrate (250X, ThermoFisher Scientific). In one embodiment, the one or more CD34+ hematopoietic stem cells are cultured in the one or more growth factors before and / or at the same time and / or after the cells are cultured in the one or more estrogen receptor agonists. In one embodiment, 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 media 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 an alternative preferred embodiment, the bone marrow sample comprises CD34+ hematopoietic stem cells. In an embodiment wherein the sample is a media comprising CD34+ hematopoietic stem cells, step ii) comprises adding the estrogen receptor agonist to the media. In one embodiment, the blood sample is one or more selected from the list consisting of: peripheral blood; mobilised peripheral blood; placenta blood; and / or umbilical cord blood. Preferably, the sample is a blood sample, which is umbilical cord blood and / or placenta blood. Preferably, the sample is a human sample and / or is a sample of human origin. The blood samples described herein would be well known to one skilled in medicine and / or hematology, who would know how to identify and obtain such samples. The blood samples can be obtained from a subject, or can be obtained from a commercial source. For example, umbilical cord blood can be obtained from Zen Bio, USA. Peripheral blood is found circulating within a subject, and is not sequestered in, for example, the lymphatic system, spleen, liver, or bone marrow. Accordingly, by “peripheral blood” we include that the blood is obtained, or obtainable, from blood circulating in a subject. Exemplarily methods of obtaining peripheral blood includes venipuncture and / or lymphapheresis. Mobilised peripheral blood is peripheral blood that has been treated with a so-called “mobilising agent”, which is an agent that increases the number of stem cells (particularly HSCs) within the peripheral blood. Examples of mobilising agents are Granulocyte-Colony Stimulating Factor and Plerixafor. The subject is provided with a mobilising agent prior to the peripheral blood being obtained. Accordingly, by “mobilised peripheral blood” we include blood that is obtained, or obtainable, from blood circulating in a subject, wherein the subject has first been provided and / or administered with a mobilising agent. Umbilical cord blood is blood that is obtained from an umbilical cord and placenta blood is obtained from the placenta, and are often a rich source of stem cells and, in particular, HSCs. These blood samples are usually collected soon after the birth of a baby. Accordingly, by “umbilical cord blood” we include a blood sample obtained, or obtainable, from an umbilical cord. Accordingly, by “placenta blood” we include a blood sample obtained, or obtainable, from a placenta. A bone marrow sample would be well known to one skilled in medicine and / or hematology, who would know how to identify and obtain such a sample. Bone marrow is the tissue found within some bone within a subject, and is often a rich source of stem cells and, in particular, HSCs. An example of how bone marrow can be obtained is via a bone marrow aspiration, which involves inserting a hollow needle through the bone into the bone marrow, which can then be withdrawn into an attached syringe. Bone marrow can also be obtained from commercial sources. By “bone marrow sample”, we include a sample obtained, or obtainable, from bone marrow. As described herein, preferable blood samples are umbilical cord blood and / or placenta blood. Such blood samples can be obtained by any procedure known in the art. There are several advantages of using cord / placental blood as stem cell source, including that the blood sample can be acquired readily without any trauma to the donor. See, e.g., U.S. Patent No. 5004681 for an overview of cord and placental blood collection at birth. Collection of a blood sample (such as umbilical cord blood and / or placenta blood) is performed under sterile conditions, and the blood is instantly mixed with an anticoagulant. Accordingly, in one embodiment the sample of step i) of the method of the invention comprises an anticoagulant and / or step i) comprises contacting the sample with an anticoagulant, such as adding an anticoagulant to the sample. Anticoagulants for use in the present invention can be any known in the art (such as those described herein, or in 1968, Storage of Blood, Academic Press, New York, pp. 26-160 which is incorporated herein by reference). In one embodiment, the anticoagulant is one or more anticoagulant selected from the list comprising: ACD (acid citrate-dextrose); CPD (citrate-phosphate-dextrose); De Gowin’s Solution (De Gowin, et al., 1940, J. Am. Med. Ass.114:850); Alsever’s solution (Alsever et al., 1941, N. Y. St. J. Med. 41:126); Rous-Turner Solution (Rous and Turner, 1916, J. Exp. Med. 23:219); Edglugate-Mg (Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573); other glucose mixtures; heparin; and / or ethyl biscoumacetate. In one embodiment, the sample (such as the blood sample) does not comprise contamination, such as contamination with an infectious agent. In one embodiment, the infectious agent is one or more infectious agent 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. In one embodiment, the method of the invention comprises a step (preferably preceding step i)) wherein the sample is tested for contamination, such as contamination with an infectious agent. Tests for contamination would be known to one skilled in the art, particularly one skilled in microbiology. In a particular embodiment, the test for contamination comprises bacterial culture and / or pathogenic microorganism screening. By “bacterial culture” we include culturing for bacteria under aerobic and anaerobic environments. By “pathogenic microorganisms screening” we include various diagnostic tests that can be performed to ensure the absence of specific pathogenic microorganisms transmissible through blood. For example, the sample (such as the blood sample) can be screened for the presence of Human Immunodeficiency Virus-1 or 2 (HIV-1 or HIV- 2). In another example, the collected sample can also be screened for other transmissible diseases, including but not limited to human Hepatitis B, Hepatitis C, T- Cell lymphotropic virus I and II (HTLV-I and HTLV-II), West Nile Virus, Cytomegalovirus, and Syphilis. In particular where the blood sample is umbilical cord blood and / or placenta blood, before collecting the blood maternal health history can be assessed to identify risks of transmitting infectious or genetic diseases, such as leukemia, immune disorders, cancer, hepatitis, neurological disorders, viral disease or AIDS. The collected blood samples can be screened for one or more of total nucleated cell count, cell viability, CD34+ cell count, HLA typing, and ABO / Rh typing. As will be appreciated, if the sample comprises contamination then it may not be used in the method of the invention and / or the method will include a step (preferably preceding step i)) of removing the contamination. Following the collection of the sample (such as a blood sample; in particular, umbilical cord blood and / or placental blood), the sample may be processed for enrichment of the hematopoietic stem / progenitor cells (in particular, CD34+ hematopoietic stem cells), as described herein. In one embodiment, the estrogen receptor agonist is one or more molecule selected from the list consisting of: an endoestrogen; a phytoestrogen; a steroidal ligand; a tamoxifen analogue; a synthetic estrogen; a xenoestrogen; and / or a non-steroidal ligand. An endoestrogen is a molecule that can bind to, and preferably activate, an estrogen receptor and is produced by a subject, such as endogenously by a human subject. In one embodiment, the endoestrogen is one or more molecule selected from the list consisting of: estrogen E2 (also known as E2, estradiol and / or oestradiol), and derivates thereof; estrogen E3 (also known as E3, estriol and / or oestriol) , and derivates thereof; estrogen E1 (also known as E1, estrone and / or oestrone) , and derivates thereof; estrogen E4 (also known as E4, estetrol and / or oestetrol) , and derivates thereof^^Ʃ^^^-androstenediol, and derivates thereof^^^Į-androstanediol, and derivates thereof^^^ǃ-androstanediol (^ǃ-Adiol) , and derivates thereof; and / or 27- hydroxycholesterol, and derivates thereof. Most preferably, the estrogen receptor agonist is an endoestrogen, which is estrogen 2. Estrogens 1-4, described herein, are all steroid hormones that are metabolically derived from cholesterol through a series of catalytic reactions. The chemical structure of estrogens is known in the art and is further described in 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. Estrogens, in general, are hormones that are metabolically derived from cholesterol through a series of catalytic reactions involving several enzymes including Cytochrome P450 aromatase (CYP19A1), of which nucleic and amino acid sequences are known. Further information on the chemical structures of estrogens can be found at 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. A phytoestrogen is a molecule that can bind to, and preferably activate, an estrogen receptor and is a non-steroidal polyphenolic compounds naturally produced by plants. In one embodiment, the phytoestrogen is one or more molecule selected from the list consisting of: an isoflavone; a coumestan; a lignan; a genistein; a S-equol; and / or a liquiritigenin. Phytoestrogens, also called ‘dietary estrogens’ are present in certain edible plants including but not limited to soy, linseed, sesame, licorice, hops and ginseng, being most abundant in soy (isoflavone). Phytoestrogens are analogous to the endoestrogens and exhibit both genomic and non-genomic action of phytoestrogens including selective but weak binding to the estrogen receptors. Phytoestrogens can have beneficial effects on menopausal symptoms, bone health, and cardiovascular health (North American Menopause Society (2011), NAMS 2011 Flavones Report, Menopause, 18:732–753; Cederroth & Nef (2009), Molecular and Cellular Endocrinology, 304: 30– 42. A steroidal ligand is a molecule that can bind to, and preferably activate, an estrogen receptor and is a steroidal molecule. In one embodiment, the steroidal ligand is one or more molecule selected from a list FRQVLVWLQJ^RI^^D^^^ǃ-ether-^^Į-ethinyl-3,17ß-estradiol; a combretastatin A4 analogue (preferably combretastatin A4 analogue on a steroidal framework); and / or a C6- piperazine-substituted purine steroid–nucleoside. A tamoxifen analogue is a molecule that can bind to, and preferably activate, an estrogen receptor, which is tamoxifen or is structurally and / or functionally similar to tamoxifen. In one embodiment, the tamoxifen analogue is one or more molecule selected from a list consisting of: 4-hydroxytamoxifen (4-HT); tamoxifen; and / or raloxifene; and / or toremifene. Tamoxifen, raloxifene, and toremifene that have been used to treat estrogen-sensitive breast cancer and prevent osteoporosis in postmenopausal women (Jordan (2003), Nature Reviews Drug Discovery, 2:205–213; Barrett-Connor et al. (2006), New England Journal of Medicine, 355:125–137). A synthetic estrogen is a molecule that can bind to, and preferably activate, an estrogen receptor and is a chemically modified form of the naturally-occurring hormone estradiol. Synthetic estrogens are used in various medical applications, such as hormone replacement therapy (HRT) and oral contraceptives, and have been shown to relieve menopausal symptoms, prevent osteoporosis, and treat certain reproductive disorders (Lobo (2017) Nature Reviews Endocrinology, 13:220–231). In one embodiment, the synthetic estrogen is one or more molecule selected from a list consisting of: ethinylestradiol; mestranol; a conjugated equine estrogen. In one embodiment, the estrogen receptor agonist is a xenoestrogen. Xenoestrogens are synthetic or naturally-occurring compounds that mimic the actions of endogenous estrogens and that are found in some industrial chemicals, pesticides, and plastics, such as bisphenol A (BPA). Xenoestrogens can affect human health due to their estrogenic activity (Gore et al. (2015), Endocrine Reviews, 36: E1–E150). A non-steroidal ligand is a molecule that can bind to, and preferably activate, an estrogen receptor and is a non-steroidal molecule. In one embodiment, the steroidal ligand is one or more molecule selected from a list consisting of: a benzopyran derivative (for example, LY-500307 (also known as Erteberel); 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)) a 2,3-diaryl isoquinolinone derivative; a diphenylmethane skeleton derivative; a deoxybenzoin analogue; a hydrazide derivative; a luoren-3-one derivative; a triphenylethylene–coumarin hybrid derivative; fulvestrant; a 1,1,2-WULDU\OROHILQH^GHULYDWLYH^^D^^^Į-(carboranylalkyl) estradiol; and / or a fluorinated carbon aryl phenol. LY500307 is a synthetic ligand that selectively binds to and activates the estrogen UHFHSWRU^ EHWD^ ^(5ǃ^^ VXEW\SH^^ LY500307 has neuroprotective effects and reduces accumulation of amyloid-beta, a protein associated with Alzheimer’s Disease (Zhao et al. (2011), Neurobiology of Aging, 32:1949-1963), and reduces inflammation and improves cognitive function in rat models of traumatic brain injury (Barreto et al. (2009), European Journal of Neuroscience, 29:1997-2006). LY3201 is a synthetic ligand that selectively binds to and activates the estrogen UHFHSWRU^EHWD^ ^(5ǃ^^ VXEW\SH^^ $FWLYDWLRQ^RI^(5ǃ^KDV^EHHQ^DVVRFLDWHG^ZLWK^potential beneficial effects on several conditions, such as neurodegenerative diseases, inflammation, and certain types of cancer (Paterni et al. (2014), Expert Opinion on Drug Discovery, 9:467-481). PPT is a synthetic ligand that selectively binds to and activates the estrogen receptor DOSKD^^(5Į^^VXEW\SH^ PPT has been shown to have agonistic effects on bone and uterus WLVVXHV^^ZKLFK^DUH^PRVWO\^PHGLDWHG^E\^(5Į^^Stauffer et al. (2000), Journal of Medicinal Chemistry, 43:4934-4947). DPN is a synthetic ligand that selectively binds to and activates the estrogen receptor EHWD^^(5ǃ^^VXEW\SH^^ DPN has been shown to have anti-inflammatory, neuroprotective, and anti-tumorigenic effects in various experimental models (Meyers et al. (2001), Journal of Medicinal Chemistry, 44:4230-4251). In one embodiment, the estrogen receptor agonist is a nuclear estrogen receptor agonist and / or a membrane bound estrogen receptor agonist. The terms nuclear estrogen receptor and membrane bound estrogen receptor would be known to one skilled in cell biology, who would be able to identify such receptors, as well as identify the molecules that can act as their agonists. In general terms, a nuclear estrogen receptor is an intracellular receptor that can be bound by, and 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 gene activity. Accordingly, by “nuclear estrogen receptor agonist” we include a molecule that is capable of binding to, and preferably activating, nuclear estrogen receptor, which is an intracellular estrogen receptor. In general terms, a membrane bound estrogen receptor is an estrogen receptor located at, or embedded in, the membrane of a cell that can be bound by, and preferably activated by, estrogen. Accordingly, by “membrane bound estrogen receptor agonist” we include a molecule that is capable of binding to, and preferably activating, a membrane bound estrogen receptor, which is an estrogen receptor located at, or embedded in, the membrane of a cell. A common type of membrane bound estrogen receptor is an estrogen G protein- coupled receptor. Particular examples of membrane bound estrogen receptors are GPRC6A (also known as G protein-coupled receptor family C group 6 member A, which is encoded for in a human subject by the GPRC6A gene), ER-X and / or Gq-mER (also known as Gq-coupled membrane estrogen receptor). In one embodiment, the nuclear estrogen receptor agonist is an estrogen receptor DOSKD^^(5Į^^DJRQLVW^DQG^RU^DQ^HVWURJHQ^UHFHSWRU^EHWD^^(5ǃ^^DJRQLVW^ (VWURJHQ^UHFHSWRU^DOSKD^^(5Į^^LV^DOVR^NQRZQ^DV^QXFOHDU^UHFHSWRU^VXEIDPLO\^^^^JURXS^$^^ member 1 (NR3A1) which, in a human subject, is encoded for by the gene ESR1 (Estrogen Receptor 1^^^^(VWURJHQ^UHFHSWRU^EHWD^^(5ǃ^^LV^DOVR^NQRZQ^DV^QXFOHDU^UHFHSWRU^ subfamily 3, group A, member 2 (NR3A2) which, in a human subject, is encoded for by the gene ESR2 (Estrogen Receptor 2). In one embodiment, WKH^(5Į^DJRQLVW^LV^one or more molecule selected from the group consisting of: estrogen E2; estrogen E1; estrogen E3; estrogen E4; and / or propyl pyrazole triol (PPT). In one embodiment, WKH^(5ǃ^DJRQLVW^LV^one or more molecule selected from the group consisting of: estrogen E2; estrogen E1; estrogen E3; estrogen E4; Diarylpropionitrile (DPN); LY3201; and / or LY-500307. ,Q^VRPH^HPERGLPHQWV^^WKH^DJRQLVWV^GHVFULEHG^DERYH^DV^(5Į^DJRQLVWV^RU^(5ǃ^DJRQLVWV^ PD\^ELQG^ERWK^ WKH^(5Į^DQG^(5ǃ^^ ^$^SDUWLFXODU^DJRQLVW^ LV^ then GHVFULEHG^DV^DQ^(5Į^ agonist LI^LW^KDV^D^VWURQJHU^ELQGLQJ^DIILQLW\^IRU^(5Į^WKDQ^(5ǃ^^L^H^^LW^ELQGV^(5Į^ZLWK^D^ highHU^SRWHQF\^^^^ / LNHZLVH^^D^SDUWLFXODU^DJRQLVW^LV^GHVFULEHG^DV^DQ^(5ǃ^DJRQLVW^LI^LW^KDV^ D^ VWURQJHU^ ELQGLQJ^ DIILQLW\^ IRU^ (5ǃ^ WKDQ^ (5Į^ The skilled person will be aware of standard techniques in the art for measuring binding affinity of particular compounds for a receptor. In one embodiment, the membrane bound estrogen receptor agonist is one or more molecule selected from the group consisting of: a GPRC6A agonist; an ER-X agonist; and / or a Gq-mER agonist. In one embodiment, the estrogen receptor agonist is present at a low dose / concentration, such as those described below. The inventors have surprisingly found that low doses / concentrations of estrogen receptor agonists lead to surprisingly high levels of CD34+ hematopoietic stem cells expansion; in particular expansion of CD34+ CD133+ hematopoietic stem cells with sustained stemness. In one embodiment, the estrogen receptor agonist is present at a concentration of about 0.5 nM to about 25 nM, preferably about 1nM to about 10 nM. As described in the Examples, the inventors have identified that these concentrations are particularly advantageous in the methods of the present invention. In a further embodiment, the estrogen receptor agonist is present at a concentration of about 0.5 nM or more; for example, 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; 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. In some other embodiments, the estrogen receptor agonist is present at a concentration of about 100 nM or more; for example 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 (10μM). In one embodiment, the method further comprises the step of isolating and / or enriching the CD34+ hematopoietic stem cells. In one embodiment, the step of isolating the CD34+ hematopoietic stem cells comprises the step of isolating the CD34+ hematopoietic stem cells from the sample, preferably following step i) and before step ii). In a particular embodiment, the step of isolating the CD34+ hematopoietic stem cells from the sample comprises isolating a CD34+ hematopoietic stem cell subpopulation (for example, CD34+ CD133+ hematopoietic stem cells and / or a CD34+ CD133+ CD38- hematopoietic stem cellsand / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cells), asdescribed herein. The hematopoietic stem cells (in particular, CD34+ hematopoietic stem cells) can be enriched for higher expression of a specific marker on the hematopoietic stem cells or hematopoietic stem and progenitor cells, comparatively to other hematopoietic cell types. The hematopoietic stem cells are identified as being CD34+ that can also be used to identify the HSCs as having these further markers (for example: CD133; CD45; CD43; CD45RO; CD45RA; CD59; CD90; CD109; CD117; CD166; and / or lack one or more markers selected from the list consisting of: CD38, HLA DR and / or Lin). Preferably, the enriched hematopoietic cells are CD34+ CD45+(low / intermediate)CD38- or CD34+ CD45+(low / intermediate)CD133+, as described herein. In one embodiment, the method of the invention comprises a step (preferably preceding step i)) of enriching the CD34+ hematopoietic stem cells in the sample and / or of isolating the CD34+ hematopoietic stem cells (for example, isolating the CD34+ hematopoietic stem cells from the sample, such as the blood sample). In an embodiment in which the CD34+ hematopoietic stem cells are isolated, step i) may comprise providing one or more isolated CD34+ hematopoietic stem cells. The isolation and / or enrichment of the CD34+ hematopoietic stem cells can be undertaken using flow cytometry with fluorescent dye conjugated monoclonal antibodies and / or immunomagnetic cell separation with magnetic bead conjugated monoclonal antibody and / or immunopanning with antibody attached to the solid matrix of culture plate or bag and / or affinity chromatography, wherein the isolation method is not excessively damaging to the cells and method should also maximize the viability of isolated cells. A particular isolation technique will depend on the availability of the equipment, separation efficacy, ease of using the method, time taken to isolate the cells and damaging effect of the method, if any, on the isolated cell population. Preferably, the isolation and / or enrichment of CD34+ hematopoietic stem cells is attained by using a magnetic bead conjugated CD34 monoclonal antibody and a magnetic cell separation device. In another embodiment, subpopulations of CD34+ hematopoietic stem cells, as described above, can be isolated by using one or more magnetic bead conjugated monoclonal antibodies of other surface markers alone or in combination thereof, e.g. CD45, CD133 by retaining the positive population. For isolating the subpopulation of CD34+ cells lacking CD38, isolated cells can further be subjected to magnetic bead conjugated CD38 monoclonal antibody and negative population is retained. Accordingly, in one embodiment the step of enriching and / or isolating comprises one or more method selected from the list consisting of: flow cytometry, such as described herein; immunomagnetic cell separation, such as described herein; immunopanning, such as described herein; and / or affinity chromatography, such as described herein. In another embodiment, the sample is a formerly cryopreserved sample (such as a blood sample; in particular, umbilical cord blood and / or placental blood), a frozen sample (such as a blood sample; in particular, umbilical cord blood and / or placental blood), or a newly-collected sample (such as a blood sample; in particular, umbilical cord blood and / or placental blood). Preferably, the sample is a newly-collected sample. In a preferred embodiment, a cryopreserved (such as cryopreserved in vapour nitrogen) umbilical cord blood sample is processed to isolate / enrich for CD34+ hematopoietic stem cells using magnetic beads conjugated to anti-CD34 antibodies and a magnetic cell separator; for example, the MiniMACS or MidiMACS or OctoMACS or CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). This method utilizes nano-sized super-paramagnetic particles that are composed of iron oxide and dextran which is coupled to specific monoclonal antibodies. In another embodiment, the method can comprise a frozen umbilical cord blood sample, wherein the method further comprises a step of thawing the umbilical cord blood sample (preferably preceding step i)). Accordingly, the frozen umbilical cord blood sample can be provided in a sample bag, in which an exemplary method of thawing includes removing the sample bag from vapour nitrogen storage and inspecting for any breakage in the bag. The sample bag is quickly placed in a sterile plastic zipper bag and thawed in a 37°C water bath as rapidly within 5 min. Alternatively, any thawing method known in the art can be utilized including, but not limited to, blood thawing systems, plasma thawing system, microcontroller based thawing system, dry rapid plasma thawing device, ThawStar cryobag thawing system. After thawing the sample, bag is gently kneaded and removed from the zipper bag and the infusion ports are disinfected with 70% alcohol wipes or any disinfectant known in the art, such as 70% isopropanol, desidos, 2% chlorhexidine, and / or betadine. The blood is drawn in a 50 ml sterile tube using a spike connector of suitable size according to the blood bag, e.g. Braun blood spike adapter. Thawed blood sample is diluted with thawing solution made of any volume expander solution of crystalloids or colloids known in the art including, but not limited to, 5-10% Dextran-40, 3-6% Hydroxyethyl starch (HES), Hespan, Voluven, Volulyte and / or PlasmaLyte. Optionally, the thawing solution is supplemented with a natural or synthetic albumin solution of non-animal origin known in the art including, but not limited to, human serum albumin, recombinant human albumin, synthetic serum substitute, and / or knockout serum replacement. Preferably, human serum albumin is added to the thawing solution. Alternatively, knockout serum replacement, xeno-free can be added to the thawing solution. Preferably a thawing solution of isotonic 10% Dextran-40 and 5% human serum albumin is used based on the prior information known in the art. One-part thawed blood sample can be diluted with one or more part of any of the above-mentioned thawing solutions, e.g., 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 and then 1:3 by adding two more volumes of same solution and then 1:5 by adding two more volumes of same solution. The sample is stabilized for 15 min each after addition of thawing solution at every dilution step. The sample can be stabilized after dilution with thawing solution either at each dilution step or at once after final dilution for any length of time known in the art, such as for 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 a particular embodiment, the sample can be stabilized after dilution with thawing solution either at each dilution step or at once after final dilution for about 1 minute to about 1 hour. In a particular embodiment, the sample can be stabilized after dilution with thawing solution either at each dilution step or at once after final dilution for no more than 1 hour in total. In a preferred embodiment, the sample is stabilized 15 minutes after each dilution step. The sample obtained via this exemplary thawing method can be used for isolation and / or enrichment of hematopoietic stem cells In a particular embodiment, the method comprises enrichment and / or isolation of the CD34+ hematopoietic stem cells by retaining a CD34 positive hematopoietic stem cell population (for example, using an anti-CD34 antibody; preferably an anti-CD34 antibody conjugated to a magnetic bead); optionally, further comprising a step of isolating the CD38- hematopoietic stem cells from the CD34+ hematopoietic stem cells (for example, using an anti-CD38 antibody; preferably an anti-CD38 antibody conjugated to magnetic beads, wherein the 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 isolated; for example, using anti-CD133 antibodies conjugated with magnetic beads, with CD133+ cells being retained. Also, cells expressing CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD166, HLA DR, or a combination of one or more markers, can be selected by using antigen specific antibodies. Preferably, CD34+ CD38- hematopoietic stem cells are enriched and / or isolated. In one embodiment, one or more suitable samples (such as a blood sample; in particular, umbilical cord blood and / or placental blood) can be combined before hematopoietic stem cells enrichment and / or isolation (preferably preceding step i)). In another embodiment, individually enriched cord blood hematopoietic stem cells samples can be combined that are from the donors of same race e.g., African- American, Caucasian, Asian, Hispanic, Native-American, Australian Aboriginal, Inuit, Pacific Islander, and / or from the donors of the same ethnicity, e.g., Irish, Italian, Indian, Japanese, Chinese, Russian, etc. The selection of suitable cord blood samples for pooling can be based on partial or complete HLA matching, as known in the art. In one embodiment wherein the sample is a blood sample, it can be depleted of red blood cells (RBC) and / or plasma; for example, before isolation / enrichment of hematopoietic stem cells and / or before cryopreservation and / or after thawing of a cryopreserved blood sample. In a further embodiment, following depletion of RBC the white blood cell fraction (WBC) can be isolated; for example, for hematopoietic stem cells isolation and / or enrichment. In another embodiment, a thawed and diluted blood sample can be centrifuged at any of the g Force or Relative Centrifugal Force (RCF) e.g., 400 xg, 450 xg, 500 xg, 550xg, 600 xg, 650 xg, 700 xg, 750 xg, 800 xg or more. Preferably the g Force or Relative Centrifugal Force (RCF) used is 650 xg. The supernatant (thawing solution) with plasma is removed and obtained cell pellet is reconstituted to original pre-thaw sample volume with any isotonic solution known in the art than can include, but not limited to, phosphate buffered saline, plasmaLyte, Dulbecco’s modified phosphate buffered saline (DPBS) or Hanks' Balanced Salt Solution (HBSS) or Ringer’s solution. Preferably, Dulbecco’s modified phosphate buffered saline (DPBS) is used for reconstitution of the cell pellet. Depletion of RBCs can be performed by separation of the white and red blood cell fractions by any method known in the art, including, but not limited to, centrifugation techniques or use of density gradient medium e.g., FICOLL™, FICOLL-PAQUE™, PERCOLL™ (GE Healthcare, Piscataway, New Jersey), LYMPHOPREP™ (Alere Technologies) combined with centrifugation or use of erythrocyte aggregation agents e.g. 6% Hydroxyethyl starch (HES), Hespan (B. Braun Medical, Inc.), Hextend(Hospira, Lake Forest, IL), HetasepTM(Stemcell Technologies Inc) or use of RBCspecific antibodies conjugated to magnetic beads and selection with a magnetic device e.g., EasySep (Stemcell Technologies Inc). HetasepTMis used for fresh blood for erythrocyte (RBC) aggregation and sedimentation. However, LYMPHOPREP™ is used for thawed blood as a density gradient medium combined with centrifugation. LYMPHOPREP™ is layered at the bottom of a sterile conical centrifugation tube and the cord blood sample is carefully layered over LYMPHOPREP™. Optionally, special separation tubes e.g., SEPMATE™ (Stemcell Technologies Inc) can be used together with LYMPHOPREP™ for easy handling and shorter centrifugation time. Preferably, SEPMATE™ tubes were used for density gradient-based centrifugation. Following the centrifugation, several layers are formed in the tube that can be identified from top to bottom as plasma and electrolytes and proteins, buffy coat i.e. a dense and opaque band of mono-nuclear cells containing white blood cells, and finally erythrocytes (RBC) and granulocytes as a thick pellet in LYMPHOPREP™. The uppermost layer is reduced in volume by 4-5 ml by aspirating the plasma carefully that also helps in reducing platelets. Finally, the upper layer and buffy coat content of the SEPMATE tube is poured in another conical tube and centrifuged to obtain a cell pellet that is further washed with Dulbecco’s modified phosphate buffered saline (DPBS) supplemented with 1% human serum albumin and cell pellet is resuspended in the same solution. Optionally, a small representative aliquot of fresh or thawed blood can be checked for total nucleated cell count, cell viability and CD34+ frequency / content; for example. prior to CD34+ cell isolation / enrichment. In one embodiment, either full or half volume of fresh blood can be processed for CD34+ or CD34+CD38- or CD34+CD133+ cell isolation and CD34+ positive enriched hematopoietic stem cell fraction and CD34- cell fractions are recovered. When half of the fresh blood is used, CD34- cell fraction can be mixed with untouched half of fresh cord blood that can be used for immediate transplantation or cryopreserved. In another embodiment, the CD34+ or CD34+CD38- or CD34+CD133+ hematopoietic stem cell enriched fraction from a fresh or thawed full blood sample can be successively processed, prior to expansion, such as the hematopoietic stem cells can be resuspended in a suitable cell growth or culture medium for transport or storage. In one embodiment, the cell culture medium can be any suitable medium known in the art, for in vitro growth of cells, 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 Medium / Nutrient Mixture F-12 (DMEM / F-12) or STEMSPAN™ Serum Free Expansion Medium (StemCell Technologies, Vancouver, British Columbia) or StemMACS HSC Expansion Media (Miltenyi Biotec GmbH, Germany), or Gibco™ StemPro™-34 SFM (ThermoFisher Scientific). By using one of the basal media, it is necessary to supplement either with Fetal Bovine serum (FBS) or Knockout serum supplement (ThermoFisher Scientific) or Human serum albumin. Additionally, these basal media can also be supplemented with several other nutrients, lipids, proteins and anti-oxidants to make it serum free media, such as known in the prior art including, but not limited to, ethanolamine, sodium selenite, hydrocortisone, D,L-Tocopherol, Human transferrin (holo), human insulin (zinc), N- acteyl-L cysteine, 2-mercaptoethanol, monothioglycerol, L-ascorbic acid-2-phosphate magnesium. By using one of the basal media or serum free media, it is necessary to supplement with one or more hematopoietic stem cells growth factors and cytokines known in the prior art, including but 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)); and 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 basal media used for 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, 5μg / 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-acteyl- L cysteine, 4 mg / L 2-mercaptoethanol and cholesterol lipid concentrate (250X, ThermoFisher Scientific). Additionally, one or more of the following growth factors and cytokines are added to the above culture media, 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). In a further embodiment, the step of isolating and / or enriching the CD34+ hematopoietic stem cells comprises the step of isolating the CD34+ hematopoietic stem cells from the expanded stem cells of step ii), preferably following step ii) and / or during step ii). In a particular embodiment, the step of isolating and / or enriching the CD34+ hematopoietic stem cells from the expanded stem cells of step ii) comprises isolating a CD34+ hematopoietic stem cell subpopulation (for example, CD34+ CD133+ hematopoietic stem cells and / or a CD34+ CD133+ CD38- hematopoietic stem cells and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cells). In a further particular embodiment, the step of isolating and / or enriching the CD34+ hematopoietic stem cells from the expanded stem cells of step ii) comprises isolating a CD34+ hematopoietic stem cell subpopulation (for example, CD34+ CD133+ hematopoietic stem cells and / or a CD34+ CD133+ CD38- hematopoietic stem cells and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cells) from the CD34+ hematopoietic stem cells. In a particular embodiment, the step of isolating and / or enriching the CD34+ hematopoietic stem cells comprises the step of isolating the CD34+ hematopoietic stem cells from the sample and isolating the CD34+ hematopoietic stem cells from the expanded stem cells of step ii). By “isolating the CD34+ hematopoietic stem cells”, we include separating the CD34+ hematopoietic stem cells from other cells and / or media and / or components; for example, other cells and / or media and / or components in the sample. The resultant isolated CD34+ hematopoietic stem cells can result in a solution substantially consisting of the CD34+ hematopoietic stem cells, or a solution wherein the only cells present are CD34+ hematopoietic stem cells. In one embodiment, the isolating and / or enriching of the CD34+ hematopoietic stem cells comprises the use of flow cytometry and / or immunomagnetic cell separation and / or immunopanning, as described further herein. In one embodiment, step ii) is undertaken for a period of about one hour or more; for example, about two hours or more; or about three hours or more; or about four hours or more; or about five hours or more; or about six hours or more; or about seven hours or more; or about eight hours or more; or about nine 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 one day or more; or about two days or more; or about three days or more; or about four days or more; or about five days or more; or about six days or more; or about seven days or more; or about eight days or more; or about nine 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 a particular embodiment, step ii) is undertaken 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 undertaken for a period of about seven days or more. In an alternative preferred embodiment, step ii) is undertaken for a period of about eight days or more. In a preferred embodiment, step ii) is undertaken for a period of about 14 days or more. In a preferred embodiment, step ii) is undertaken for a period of about 21 days or more, most preferably about 21 days. In one embodiment, step ii) is undertaken 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 nine days or less; about eight days or less; about seven days or less. In a preferred embodiment, step ii) is undertaken for a period of about seven days or less. In an alternative preferred embodiment, step ii) is undertaken for a period of about eight days or less. In a preferred embodiment, step ii) is undertaken for a period of about 14 days or less. In a preferred embodiment, step ii) is undertaken for a period of about 21 days or less. In one embodiment, step ii) is undertaken for a period of about seven days to about 21 days; for example, about eight days to about 21 days; about eight days to about 14 days; or about 14 days to about 21 days. In a preferred embodiment, step ii) is undertaken for a period of about seven days to about 21 days. In one embodiment, during step ii) of the method of the present invention a media comprising one or more estrogen receptor agonist (and optionally one or more growth factor and / or cytokine) can be replaced or added to at least once with a comparable (or identical) media comprising one or more estrogen receptor agonist (and optionally one or more growth factor and / or cytokine); for example: the media is replaced or added to at least two times; the media is replaced or added to at least three times; the media is replaced or added to at least four times; the media is replaced or added to at least five times; and / or the media is replaced or added to at least six times. Preferably, about a half of the volume of the media is replaced or added with an equal amount of media. In a particular embodiment, the volume of the media replaced or added is about 50ml to about 100ml, preferably about 70ml. In a particular embodiment in which step ii) is undertaken for a period of about 21 days, a media comprising one or more estrogen receptor agonist (and optionally one or more growth factor and / or cytokine) is replaced or added to on days 4, 8, 11, 14, 17 and 19, preferably day 7 and / or day 14. In one embodiment, one or each step of the present invention (preferably step ii)) is undertaken in a sealed container, such as a sealed cell culture bag. In one embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one or more CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about two to about 40 CD34+ hematopoietic stem cells; for example, one CD34+ hematopoietic stem cell of the sub- population (preferably and / or CD34+ CD133+ CD38- hematopoietic stem cell and / or CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about two; or about three; or about four; or about five; or about six; or about seven; or about eight; or about nine; or about ten; 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) there is a ratio of one or more CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about two to about 20 CD34+ hematopoietic stem cells. In a further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one or more CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about four CD34+ hematopoietic stem cells. In an alternative further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one or more CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about 15 CD34+ hematopoietic stem cells. In an alternative further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one or more CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about 20 CD34+ hematopoietic stem cells. It will be appreciated by one skilled in cell biology that the ratio of a CD34+ hematopoietic stem cells to a particular CD34+ hematopoietic stem cells sub- population can be further defined by reference to the period over which step ii) of the present invention has been undertaken. Therefore, the ratios described herein can each be combined with the periods over which step ii) is undertaken, as also described herein. It will also be appreciated by one skilled in cell biology that as the period increases the HSC population at a higher starting proportion will expand at a faster rate than a HSC population at a lower starting proportion, so the ratio can increase over time. In a further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about four CD34+ hematopoietic stem cells, wherein step ii) has been undertaken for a period of about 8 days. In an alternative further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about 15 CD34+ hematopoietic stem cells, wherein step ii) has been undertaken for a period of about 14 days. In an alternative further preferred embodiment, in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about 20 CD34+ hematopoietic stem cells, wherein step ii) has been undertaken for a period of about 21 days. In one embodiment, the number of the expanded CD34+ hematopoietic stem cells of step ii) is increased by a fold change of about 88 or more; for example, a fold change of about 90 or more; a fold change of about 95 or more; a fold change of about 100 or more; a fold change of about 110 or more; a fold change of about 120 or more; a fold change of about 130 or more; a fold change of about 140 or more; a fold change of about 150 or more; a fold change of about 160 or more; a fold change of about 170 or more; a fold change of about 180 or more; a fold change of about 190 or more; a fold change of about 200 or more; a fold change of about 300 or more; a fold change of about 400 or more; a fold change of about 500 or more; a fold change of about 600 or more; a fold change of about 700 or more; a fold change of about 800 or more; a fold change of about 900 or more; 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. It will be appreciated by one skilled in cell biology that the fold change of CD34+ hematopoietic stem cells can be further defined by reference to the period over which step ii) of the present invention has been undertaken. Therefore, the fold changes described herein can each be combined with the periods over which step ii) is undertaken, as also described herein. In one embodiment, the number of the expanded CD34+ hematopoietic stem cells (in particular, long-term repopulating hematopoietic stem cells) of step ii) is increased by a fold change of about 90 or more, wherein step ii) has been undertaken for a period of about 8 days. In a further embodiment, the number of the expanded CD34+ hematopoietic stem cells of step ii) is increased by a fold change of about 1,500 or more, wherein step ii) has been undertaken for a period of about 21 days. In one embodiment, the number of the expanded CD34+ hematopoietic stem cells of step ii) is increased by about 8,800% or more; for example, 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. It will be appreciated by one skilled in cell biology that the percentage change of CD34+ hematopoietic stem cells can be further defined by reference to the period over which step ii) of the present invention has been undertaken. Therefore, the percentage changes described herein can each be combined with the periods over which step ii) is undertaken, as also described herein. The quality, purity and / or success of the CD34+ hematopoietic stem cell expansion could be assessed by one skilled in cell biology. In one embodiment, 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 analysed after expansion of hematopoietic stem cells, to determine the potency of expanded cell population; for example, in providing hematopoietic effects. In a further embodiment, the viable cells, as described above, can additionally, and / or alternatively, be assessed during step ii) of the present invention. In one exemplary embodiment, a representative sample can be taken at day 7, 14 and 21 during step ii) from the expansion culture for quantification of total viable nucleated cells and total CD34+ cells which can be enumerated (optionally using flow cytometry), thus providing percentage of CD34+ hematopoietic stem / progenitor cells. Similarly, a representative small volume of the expanded hematopoietic stem cells can be taken, prior to cryopreservation or after thawing, as described further herein, for counting of total nucleated cells and percentage of CD34+ cells. In a specific embodiment, total viable CD34+ hematopoietic stem cells or CD34+CD38- hematopoietic stem cells or CD34+CD133+ hematopoietic stem cells can be assessed by any method known in the art, for example by exclusion of trypan blue or 7-AAD. The viable CD34+ hematopoietic stem cell percentage can be determined by use of a dye that is excluded from viable cells in a flow cytometry assay. The relative increase in viable CD34+ cells can be assessed as Total viable CD34+ cells post-expansion / Total viable CD34+ cells seeded at start of expansion. This ratio is generally referred to as fold expansion. As evident from methods known in the art, CD34 antigen alone or in combination with other antigens can be used for such analysis. As discussed herein, the inventors have identified that their method of expanding CD34+ hematopoietic stem cells leads to a higher quality resultant population of CD34+ hematopoietic stem cells, and CD34+ hematopoietic stem cells in a higher amount, when compared to the prior art methods. This is relevant therapeutically as several studies have indicated that total nucleated and CD34+ cell dose used in therapy (for example, transplant grafts) is positively correlated to a better outcome (for example, better platelet and neutrophil engraftment, reduced incidences of graft failure and graft-versus-host disease following umbilical cord blood transplantation). Possible examples of how to assess quality are the outcome of the transplantation and / or engraftment into a subject – the subject could be a human or, more preferably, an animal model. Animal models for assessing purity of hematopoietic stem cells are known, and include immunodeficient humanised mouse (from which species-specific immune cells are absent). In such animal models, serial transplantation of expanded human HSCs at different doses is undertaken, and immune reconstitution is assessed by flow cytometry of blood and / or bone marrow. An alternative method of assessing the quality, purity and / or success of the CD34+ hematopoietic stem cell expansion is to study the percentage of the CD34+ hematopoietic stem cells (or CD34+ hematopoietic stem cell subpopulation) compared to the total number of cells, resulting from step ii) of the methods of the present invention. The potency of expanded hematopoietic stem cells may also be assessed by their capacity to differentiate into myeloid and lymphoid lineages. Colony forming unit (CFU) assay is one such method used for studying the proliferation and differentiation potential of hematopoietic stem / progenitor cells by their ability to form colonies in a semisolid medium. The numbers and morphology of produced colonies by a fixed number of hematopoietic stem / progenitor cells can provide information about the potency of expanded cell population. A relatively higher number of CFU from a fixed number of input hematopoietic stem cells is considered a predictive of better engraftment potential. 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 the one or more CD34+ hematopoietic stem cells in the sample to differentiate. In an alternative embodiment, the ability of the expanded CD34+ hematopoietic stem cells of step ii) to successfully be transplanted and / or engrafted into a subject is substantially the same as the ability of the CD34+ hematopoietic stem cells in the sample to differentiate. In one embodiment, the percentage of CD34+ hematopoietic stem cells when compared to the total number of cells resulting from step ii) of the present 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. In some embodiments, the other cell types included in the total number of cells resulting 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. Such cells can be identified by expression of one or more mature cell markers including but not limited to the following: CD4; CD7; CD8; CD14; CD15; CD16; CD56. Testing the functionality of the CD34+ hematopoietic stem cells resulting from step ii) of the present invention can be achieved using an animal model, for example a mouse model. An example of such a mouse model is described in 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. In one embodiment, the method is an ex vivo method and / or an in vitro method. In one embodiment, 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. In one method, the method further comprises the step of iii) differentiating the expanded CD34+ hematopoietic stem cells of step ii) into one or more cells derived from the CD34+ hematopoietic stem cells. In a particular embodiment, the method further comprises the step of administering the 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. In one embodiment, the one or more cells derived from the CD34+ hematopoietic stem cells are cells selected from the list consisting of: myeloid progenitor cells; lymphoid progenitor cells; erythroid cells; erythrocyte cells; mast cells; megakaryocyte cells; thrombocyte cells; 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. In an additional version of the first aspect of the invention, step ii) of the method of the first aspect of the invention can comprise culturing the cells of step i) in the presence of an agent capable of increasing the expression of an estrogen receptor and / or an agent capable of increasing downstream effector of a estrogen receptor signaling pathway, in addition to or instead of one or more estrogen receptor agonist. 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. In particular, for later use in transplantation / infusion to human patients. Accordingly, the methods described herein can comprise the step of cryopreserving or freezing the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells (preferably following step ii)). In one embodiment, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells can be distributed in one or more bags or cryogenic tubes or units and kept frozen in at ultra-low 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 stored frozen. Cryopreservation can be any method known in the art that is able to freeze the cells in living / viable state at ultra-low temperatures. Generally, the freezing of the cells is damaging as water inside the cells makes ice crystal on cooling and osmotic effect on cellular membrane induces injury together with dehydration and increased solute concentration. Please refer to Mazur, P., 1977, Cryobiology 14:251-272 for a detailed discussion, as incorporated herein by reference. Generally, these deleterious effects can be avoided by using a cryoprotectant, controlling the rate / speed of freezing and storing the frozen cells at ultra-low temperatures to minimize further injuries. Accordingly, in one embodiment, cryopreserving comprising contacting the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells with one or more cryoprotectant. Cryoprotectants that can be used include, but are not limited to, polyvinylpyrrolidine (Rinfret, 1960, Ann. N.Y. 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, iinositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), amino acids (Phan The Tranz and Bender, 1960, Exp. Cell Res. 20:651), and inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388). Preferably, DMSO, non-toxic at low concentration, is used as cryoprotectant that easily penetrates the cell membrane and modifies the freezing pattern of water molecules and protects intracellular organelles by preventing their damage from ice crystal formation. Additionally, supplementing the cryoprotective medium with serum albumin or plasma, e.g. upto a concentration of 20- 40%, can further enhance the cryoprotective effect of DMSO. After adding the cryoprotective medium, in one embodiment, cells can be placed at 0°C until frozen because DMSO concentrations of 1% or more are toxic above 4°C. Controlled slow cooling of the cells is critical and the process can be carried out in an insulated box that ensures controlled rate -1°C / minute freezing or by using a programmed freezing device. A controlled rate freezing box can be alcohol-free cell freezing container that has highly-insulative outer material to control the rate of heat removal and provide reproducible cell cryopreservation. Controlled rate freezing box such as CoolCell (Corning Inc, Arizona, USA) provides reproducible cell cryopreservation. For example, for most stem cells in a cryoprotective medium comprised of 10% DMSO and 25% serum albumin, the optimal rate of cooling is -1° to -3°C per minute from 0° C to -80° C. 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 smaller aliquots and in Ethyl vinyl acetate (e.g. Pall Corporation CA, USA; Thermogenesis holdings Inc. CA, USA) bags can be frozen by holding in between metal plates for larger volumes. After the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells have been frozen at -80°C, cells can be transferred to a cryogenic tank for long-term storage. Preferably, cells can be stored frozen in liquid phase (-190°C) or vapour phase (-170°C) of nitrogen. After expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells are cryopreserved as described herein, frozen samples can be deposited in a cryostorage facility. This facility can be a bank that can exist in one or more physical locations having one or more temperature-controlled freezers using liquid nitrogen for maintaining ultra-low temperatures. Thus, in one embodiment, the method comprises depositing expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells in a cryostorage bank or cryostorage repository, following cryopreserving or freezing. The cryopreserved collection can comprise any number of samples of expanded hematopoietic stem cells, where each sample can be derived from single blood sample or multiple samples can belong to a single blood sample from a human. In another embodiment, the cryostorage bank can comprise of each frozen expanded cell sample of two or more independent cord blood or blood samples, e.g. pooled samples as described in the invention above. The samples of expanded hematopoietic stem cells are stored at temperatures that are below -20°C, at -80°C or preferably at ultra-low temperature obtained with liquid nitrogen (-190°C) or with vapours of nitrogen (- 170°C). Cryopreserved and frozen expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells can be thawed rapidly on requirement, e.g. in a water bath maintained at 35°-37°C or in a Vial Thawing Systems (e.g. ThawStar, BIOLIFE Solutions, WA, USA) or in a water free cryobag thawing device (e.g. Thaw star CB, BIOLIFE Solutions, WA, USA). In a specific embodiment, the cryovial of frozen cells can be submerged 3 / 4thin the warm water of the bath keeping the neck area of the tube outside the water and vial is constantly and gently rotated to ensure proper heat transfer from the surface to the internal ice mass and mixing of cell suspension. Thawing is done rapidly to make sure there is small pellet of ice left in the vial in order to not bring the temperature of the cell suspension above 4°C since DMSO in the cryoprotective medium can damage the cells above this temperature. As soon as the thawing is complete, the vial can be immediately placed on ice for further use, as thawed or a part thereof, for infusion in a human patient for delivering hematopoietic function. There are several methods available to the person known in the art for further processing of thawed hematopoietic stem cells that can be utilized if considered suitable. It may be necessary to remove cryoprotective agent such as DMSO, if toxic, from thawed hematopoietic stem cells. It may also be desirable to prevent the cells from clumping with each other by utilizing any procedure known in the art, including but not limited to, the addition of hydroxyethyl starch (HES, Zhu et al., 2015, Cytotherapy 17(12): 1813-1819, doi: 10.1016 / j.jcyt.2015.08.007), low molecular weight Dextran and HSA (Berz et al., 2007, Am J Hematol 82(6): 463-472, doi:10.1002 / ajh.20707), DNase (García-Piñeres et. al., 2006, J Immunol methods 30;313(1-2):209-213, doi: 10.1016 / j.jim.2006.04.004), etc. It may also be desirable to wash the cells before infusion to the human patient in need thereof, by addition of any suitable isotonic medium known in the art, including but not limited to, 5-10% Dextran-40, 3-6% Hydroxyethyl starch (HES), Hespan, Voluven, Volulyte or PlasmaLyte, followed by one or more steps of centrifugation to concentrate the cells in bottom of the tube, removal of cryoprotectant and diluents as supernatant and resuspending the cells in above mentioned isotonic medium to get final cell product for infusion in human patient to provide hematopoietic function. This procedure should be done gradually to minimize abrupt changes in osmotic gradients and prevent cell damage due to DMSO removal. Optionally, a small representative aliquot of thawed hematopoietic stem cells can be taken and checked for total nucleated cell count, cell viability and CD34+ hematopoietic stem cell percentage prior to therapeutic use. The procedure used to assess such parameters can be any method known in the art depending on the suitability. Preferably, the similar procedure can be used that as described for the sample of step i) of the method of the present inventions. In a third aspect, the invention provides a population of CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein. As will be appreciated, it may be possible to distinguish the expanded CD34+ hematopoietic stem cells obtained from, or obtainable from, the present invention 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 will be appreciated by one skilled in cell biology, such gene expression can be identified by measuring messenger RNA (mRNA) and / or any resultant protein and / or micro RNA (miRNA). Genes associated with estrogen receptor activity may be identified using publicly available databases (for example as described in 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). It may also be possible to identify the expanded CD34+ hematopoietic stem cells obtained from, or obtainable from, the present invention due to the DNA within those cells being physically-associated with nuclear estrogen receptors. In one embodiment, the expanded CD34+ hematopoietic stem cells of step ii) comprise gene expression associated with estrogen receptor activity. In a further embodiment, the expanded CD34+ hematopoietic stem cells of step ii) comprise nuclear estrogen receptor(s) physically associated with their DNA. In some embodiments, one or more of the following genes associated with estrogen receptor activity may be expressed or repressed in the CD34+ hematopoietic cells obtained from, or 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. In some additional or alternative embodiments, it may be possible to distinguish the expanded CD34+ hematopoietic stem cells obtained from, or obtainable from, the present invention from hematopoietic stem cells obtained by different methods, based on the populations of CD34+ hematopoietic stem cells obtained by the methods of the present invention having a higher proportion of CD34+ CD133+ cells compared to populations of CD34+ hematopoietic stem cells obtained by other methods. In some embodiments, the cells obtainable by the methods of the present invention are CD34+ CD133+ CD38-. In some embodiments, the higher proportion of CD34+ CD133+ and / or CD34+ CD133+ CD38- cells in the populations obtained, or obtainable by, the methods of the present invention are associated with a higher quality stem cell population and / or greater clinical potential, such cells being of higher quality would be understood by a person skilled in medicine (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; Ishikawa F, et al., Human cord blood long- term engrafting cells are CD34+ CD38-. Leukemia. 2003 May;17(5):960-4; and Andre Görgens, 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). In one embodiment, the expanded CD34+ hematopoietic stem cells of the present invention do not have any significant difference in their surface phenotype when 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 present invention do not have any difference in their surface phenotype when compared to the CD34+ hematopoietic stem cells provided in step i) of the method described herein. The inventors have identified that this feature of their invention demonstrates the superiority of their method in providing expanded CD34+ hematopoietic stem cells, particularly with sustained stemness. In a fourth aspect, the invention provides a one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein. In one embodiment of the fourth aspect of the invention, the one or more cells derived from the CD34+ hematopoietic stem cells are cells selected from the list consisting of: myeloid progenitor cells; lymphoid progenitor cells; erythroid cells; erythrocyte cells; mast cells; megakaryocyte cells; thrombocyte cells; 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. In one embodiment, the T lymphocytes are chimeric antigen receptor (CAR)-T cells. In a fifth aspect, the invention provides a composition comprising CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein and / or a population of CD34+ hematopoietic stem cells as described herein. HSCs for transplantation can be mixed with 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 Ringers solution (LR). Therefore, in some embodiments, the composition comprising the CD34+ hematopoietic cells obtained by the methods described herein comprises an isotonic, buffered intravenous crystalloid solution such as those described above. The preparation and components of such solutions for inclusion in the composition are well known in the art. In a sixth aspect, the invention provides a kit of parts comprising CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein and / or a population of CD34+ hematopoietic stem cells as described herein. In an alternative sixth aspect, the invention provides a kit of parts for expanding CD34+ hematopoietic stem cells comprising one or more estrogen receptor agonist, and / or 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. The kit of parts of the alternative sixth aspect of the invention can comprise instructions explaining the steps 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. The instructions may include details of how to expand the cells in a cord blood bank setting using Good Manufacturing Practice (GMP) using a tissue culture incubator (5% CO2, 98% humidity), cell culture bags and a centrifuge. In some embodiments, the kit further comprises the growth factors and / or cytokines described herein (either provided as a mixture with the one or more estrogen receptor agonist or as a separate component). The kit may also further comprise serum free media that is suitable for expansion of the hematopoietic stem cells. The composition of such media is well known in the art. In a seventh aspect, the invention provides CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein and / or a population of CD34+ hematopoietic stem cells as described herein for use in medicine. In an alternative seventh aspect, the invention provides one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses and / or from a population of CD34+ hematopoietic stem cells as described herein for use in medicine. In an eighth aspect, the invention provides CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein and / or a population of CD34+ hematopoietic stem cells as described herein for use in treating and / or preventing a condition in a subject in need thereof. In an alternative eighth aspect, the invention provides one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses and / or from a population of CD34+ hematopoietic stem cells as described herein for use in treating and / or preventing a condition in a subject in need thereof. In a ninth aspect, the invention provides a use of CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein and / or a population of CD34+ hematopoietic stem cells as described herein for the manufacture of a medicament for treating and / or preventing a condition in a subject in need thereof. In an alternative ninth aspect, the invention provides a use of one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses and / or from a population of CD34+ hematopoietic stem cells as described herein for the manufacture of a medicament for treating and / or preventing a condition in a subject in need thereof. In a tenth aspect, the invention provides a method for treating and / or preventing a condition in a subject in need thereof, comprising administering CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses as described herein and / or a population of CD34+ hematopoietic stem cells as described herein to the subject in need thereof. In an alternative tenth aspect, the invention provides a method for treating and / or preventing condition in a subject in need thereof, comprising one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the methods and / or uses and / or from a population of CD34+ hematopoietic stem cells as described herein to the subject in need thereof. In a further alternative tenth aspect, the invention provides a method for 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; optionally differentiating the expanded CD34+ hematopoietic stem cells into one or more cells derived from the CD34+ hematopoietic stem cells; and c) administering the cells of step b) to the subject in need thereof. The method of the further alternative tenth aspect of the invention can comprise the steps 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. As will be appreciated, step a) of the further alternative tenth aspect of the invention corresponds to step i) of the first aspect, and step b) of the further alternative tenth aspect of the invention corresponds to step ii) of the first aspect. In one embodiment, the condition is one or more condition selected from list consisting of: a condition requiring transplantation; a hematologic condition and / or a hematologic malignancy; a condition resulting from a failure or dysfunction of normal blood cell production and maturation; immunosuppression a subject with a malignant, and / or solid, tumor; an autoimmune disease and / or an immune condition; a genetic disorder; cancer; a tumor; osteopetrosis; myelosclerosis; acquired hemolytic anemias; infectious disorders causing primary or secondary immunodeficiencies; acquired immunodeficiencies; neutrophil actin deficiency; neutrophil membrane GP-180 deficiency; bacterial infections (for example Brucellosis infections, Listerosis infection, tuberculosis infection, and / or leprosy); parasitic infections (for example, malaria and / or Leishmaniasis); fungal infections; disorders involving disproportions in lymphoid cell sets and / or impaired immune functions due to aging; phagocyte disorders; Kostmann’s agranulocytosis; chronic granulomatous disease; Chediak- Higachi syndrome; Wiskott-Aldrich Syndrome; metabolic storage diseases; PXFRSRO\VDFFKDULGRVHV^^ PXFROLSLGRVHV^^ Į^-antitrypsin deficiency; neurologic disorders; and / or miscellaneous disorders involving immune mechanisms. In one embodiment, the transplantation is an allogenic transplantation or an autologous transplantation. In one embodiment, the condition resulting from a failure or dysfunction of normal blood cell production and maturation is one or more condition selected from list consisting of: pancytopenia (such as radiation and / or chemotherapy induced pancytopenia); aplastic anemia; thrombocytopenia; agranulocytosis; red cell aplasia; a hyperproliferative stem cell disorder; Blackfan-Diamond syndrome; Blackfan- Diamond syndrome resulting from drug use; Blackfan-Diamond syndrome resulting from radiation; Blackfan-Diamond syndrome resulting from infection; and / or Idiopathic Blackfan-Diamond syndrome. In one embodiment, the hematologic condition and / or hematopoietic malignancy is one or more condition selected from list consisting of: Hodgkin’s lymphoma; non-Hodgkin’s lymphoma; acute lymphoblastic (lymphocytic) leukemia; acute myelogenous leukemia; acute malignant myelosclerosis; chronic lymphocytic leukemia; chronic myelogenous leukemia; multiple myeloma; agnogenic myelometaplasia; Waldenstrom’s macroglobulinemia; and / or polycythemia vera. In one embodiment, the immunosuppression in a subject with a malignant, and / or solid, tumor is one or more condition selected from list consisting of: ovarian carcinoma; breast carcinoma; malignant melanoma; testicular carcinoma; carcinoma of the stomach; small cell lung carcinoma; rhabdomyosarcoma; Ewing’s sarcoma; retinoblastoma; glioblastoma; neuroblastoma; and / or lymphoma. In one embodiment, the autoimmune disease and / or an immune condition is one or more condition selected from list consisting of: diabetes type I; rheumatoid arthritis; systemic lupus erythematosus; chronic hepatitis; and / or multiple sclerosis. In one embodiment, the genetic disorder is one or more condition selected from list consisting of: anemia; familial aplastic; Bloom’s syndrome; Fanconi’s syndrome; pure red cell aplasia (PRCA); dyskeratosis congenital; Blackfan-Diamond syndrome; congenital dyserythropoietic syndromes I-IV; Chwachmann-Diamond syndrome; dihydrofolate reductase deficiency; formamino transferase deficiency; pyruvate kinase deficiency; sickle cell disease; thalassemia alpha; thalassemia beta; thalassemia gamma; met-hemoglobinemia; Lesch-Nyhan syndrome; congenital spherocytosis; congenital elliptocytosis; congenital stomatocytosis; congenital Rh null disease; congenital erythropoietin sensitivity deficiency; paroxysmal nocturnal hemoglobinuria G6PD (glucose-6-phosphate dehydrogenase); paroxysmal nocturnal hemoglobinuria G6PD (glucose-6-phosphate dehydrogenase) variant 1; paroxysmal nocturnal hemoglobinuria G6PD (glucose-6-phosphate dehydrogenase) variant 2; paroxysmal nocturnal hemoglobinuria G6PD (glucose-6-phosphate dehydrogenase) variant 3; congenital disorders of immunity; severe combined immunodeficiency disease (SCID) bare lymphocyte syndrome; ionophore-responsive combined immunodeficiency; combined immunodeficiency with a capping abnormality; nucleoside phosphorylase deficiency; granulocyte actin deficiency; infantile agranulocytosis; Gaucher’s disease; adenosine deaminase deficiency; Kostmann’s syndrome; reticular dysgenesis; and / or congenital leukocyte dysfunction syndrome. In one embodiment, the genetic disorder is one or more condition selected from list comprising or consisting of: cerebral palsy; autism spectrum disorders; Alzheimer’s disease; multiple sclerosis, Parkinson’s disease; Huntington’s disease; stroke; motor neuron disease; traumatic brain injury; spinal cord injury; amytrophic lateral sclerosis; Hypoxic-ischemic brain injury; eye damage; and / or epilepsy. The term "treating" includes the meaning of administering CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells described herein in an amount, manner, and / or mode effective to improve a condition, symptom, or parameter associated with a condition or to prevent progression or exacerbation of the condition (including secondary damage caused by the condition) to either a statistically significant degree or to a degree detectable to one skilled in medicine. The term "preventing" includes the meaning of administering CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells described herein in an amount, manner, and / or mode effective to avoid the development of a condition, symptom, or parameter associated with a condition to either a statistically significant degree or to a degree detectable to one skilled in medicine. The expanded CD34+ stem cell populations produced as described herein can be administered by any convenient route known in the art, for example systemic or local, by infusion or bolus injection, and may be administered together with other biologically active agents, for example, 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 Ringers solution (LR). Preferably, the expanded CD34+ stem cell populations are administered by an infusion. Subjects can be administered alone (monotherapy) or in combination with other agents (combination therapy), either in admixture or by separate, simultaneous or sequential administration. In the case of a combination therapy, the amounts and times of administration can be those that provide, e.g., an additive or a synergistic therapeutic effect. Further, the administration can be used as a primary (for example, first line treatment) or as a secondary treatment (for example, for subjects who have an inadequate response to a previously administered therapy). In one embodiment, the one or more estrogen receptor agonist (and / or other growth factors or cytokines, if used) are removed from, or diluted in, the expanded CD34+ hematopoietic stem cells and / or the one or more cells derived from the CD34+ hematopoietic stem cells (such as by washing or dilution) prior to being administered to a subject. Figure 1: Fold expansion of UCB-HSPCs with 0, 1, and 10 nM E2 treatment for 8 days (p<0.05). Figure 2: Flow cytometric analysis of expanded cells for. No changes are observed in surface phenotype of CD34+ and CD34+CD133+ cell population. Figure 3: Fold expansion of CD34+ cells with 0, 1, and 10 nM E2 treatment for 8 days (p<0.05). Figure 4: Flow cytometry analysis of expanded UCB-HSCs. The cells retained their phenotype and stemness in control and all treatment conditions. Figure 5: Expansion of CD34+CD38- HSCs in various growth factors and test compound combinations. A) Cell number (in millions) and, b) Fold expansion of cells (compared to the control) after expansion for 14 days (left) and 21 days (right) (p<0.05). Figure 6: Phenotype of cells expanded from CD34+CD38- HSCs in various growth factors and test compound combinations. a) Fold expansion of CD34+ (HSPCs) and, b) percentage of CD34+ cells after expansion for 14 days (left) and 21 days (right) (p<0.05). Figure 7: Fold expansion of CD34+CD133+ cells (LT-HSC) expanded from CD34+CD38- HSCs in various growth factors and test compound combinations for 14 days (left) and 21 days (right) (p<0.05). Figure 8: Assessment of hematopoietic multilineage potential of ex vivo expanded UCB- HSCs after 21 days culture in various growth factors and test compound combinations. Figure 9: Expansion of CD34+CD38- selected HSCs in various serum containing (FBS) and serum free media supplemented with growth factors and estrogenic ligand combinations. a) Cell numbers (in millions) at day 14 (left) and day 21 (right) and, b) Fold expansion of cells after 14 days (left) and 21 days culture (right) (p<0.05). Figure 10: Phenotype of cells expanded from CD34+CD38- selected HSCs in various serum containing (FBS) and serum free media supplemented with growth factors and estrogenic ligand combinations. a) Fold expansion of CD34+ (HSPCs) at day 14 (left) and day 21 (right) and ,b ) Fold expansion of CD34+CD38- (LT-IC HSCs) at day 14 (left and day 21 (right) (p<0.05). Figure 11: Assessment of hematopoietic multilineage potential of ex vivo expanded CD34+ HSPCs after 21 days culture in various serum containing (FBS) and serum fee media supplemented with growth factors and estrogenic ligand combinations. Example The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Summary Transplantation of Hematopoietic stem cells (HSCs) to treat various hematologic, oncological and immune disorders is very promising but often limited due to availability of matched donors. Umbilical cord blood (UCB) contains less mature HSCs with greater telomere length and thus offer a better alternative. However, the clinical use in adults is often limited due to low stem cell counts resulting in slow engraftment. Ex vivo expansion of UCB-HSCs to clinically relevant doses may overcome this hurdle and benefit patients. Several approaches and methods have been employed by research groups worldwide which include the use of hematopoietic growth factors, cytokines and blockers of differentiation such as Notch ligands, copper chelators, nicotinamide and aryl hydrocarbon receptor antagonist. Estrogen (E2) and estrogen receptors (ERs) have been implicated in the proliferation of multipotent and committed progenitor cells but has not been tested yet in the proliferation of UCB-HSCs. Interestingly, E2, which is readily available in placenta and normal cord blood, has been implicated in the growth of lineage committed progenitors and differentiation in rodents. To date, comparatively less is known about the role of E2 in expansion and self-renewal of HSCs with long-term repopulating potential. E2 has not yet been tested in the expansion of human UCB derived hematopoeitic stem cells in completely xeno- free environment using defined serum free growth media. A reliable and clinically useful UCB-HSC expansion will not only be beneficial for allogenic transplants but also provide a suitable platform to derive effector immune cells for ex vivo expansion and / or gene modifications to improve cellular immunotherapies, e.g. UCB derived chimeric antigen receptor (CAR)-T and NK cells. These enhanced anti-tumorgenic cells could thus be offered as an off-the-shelf product, minimizing the time to therapy by circumventing the need for modifying the patient’s own cells. The inventors have developed a new xeno-free method to efficiently expand UCB-HSPCs ex vivo by using a growth supplement containing estrogenic ligands. In this way, we can uniquely achieve relevant therapeutic doses and thereby facilitate the clinical use of UCB-HSC for both autologous and allogenic transplants, as well as opening up the potential for the use of UCB CAR-T cells as effective adoptive cellular immunotherapy. The present method is robust and unique in terms of reliable expansion of UCB-HSCs in limited time span to provide greater number of therapeutic cells, compared to other reports, with sustained stemness phenotype. Interestingly, the method is efficient to expand CD34+ HSPCs and long term repopulating HSC (LT- HSC) upto 1500- and 90-fold respectively in 21 days, without affecting their multilineage differentiation potential, which is not observed by others to date and these cells will exemplify the advantage of expanded UCB-HSCs in clinical settings. Background Hematologic and non-hematologic disorders can be treated with autologous or allogeneic hematopoietic stem cell (HSC) transplantation. However, the application is limited by the identification of a suitable donor. Umbilical cord blood (UCB) contains less mature HSCs with greater telomere length and thus offers a better alternative source for HSC transplantation. However, UCB transplantation in adults is often clinically limited due to the low number of total nucleated (~1 billion, TNC) and CD34+ progenitor (~1.2 million) cells, ensuing in slow / delayed engraftment, compared to bone marrow or mobilized peripheral blood stem cell grafts. Most of the healthy full- term deliveries provide just over a million blood-forming stem cells (HSCs) in their umbilical cord blood. Consequently, a significant number of UCB units are not considered optimal in most public cord blood banks if the donations are below a threshold. The KLJKHU^QXPEHU^RI^QXFOHDWHG^^^^^^-50 million cells / kg) and CD34+(1- 1.5x105 / kg) cell doses per kg of weight correlates with better outcomes (1) of cord blood transplantation and overcoming of the delayed engraftment. Thus, ex vivo expansion of cord blood hematopoietic stem cells (UCB-HSC) may serve to increase the available cell dose for transplants. Ex vivo expansion of stem cells requires a symmetric division and both daughter cells should maintain properties of stem cells. Precise doses of cytokines, medium, serum and other small molecules used for expansion are also extremely important. The final aim of cord blood expansion should be targeted to generate clinically safe, transplantable HSCs, which can reliably repopulate recipient. Another acceptable alternative could be ex vivo generation of more mature progenitors, which are able to engraft rapidly. The initial efforts on HSC expansion were started in the 1970s and since then many efforts have been made to expand cells in order to retain stemness and restricting differentiation with use of either cytokines or stromal cells, or their combination resulting in a mixed success and the quest is still continued worldwide to stably expand reliable population of HSC from cord blood. 1. Hematopoietic stem cell (HSC) Phenotype and self-renewal pathways HSCs represent a rare sub-population in the hematopoietic system which are able to produce all the mature blood cells throughout life (2). Thus, the peculiar feature of HSC is the dynamic balance between quiescence, self-renewal and differentiation governed by various mechanisms, such as genetic and epigenetic regulation and microenvironmental (niche) factors. Flow cytometric routine CD34 analysis enumerates hematopoietic stem and progenitor cells irrespective of their subpopulations although this might not predict true engraftment dynamics and immune reconstitution. During the past two decades, CD34 antigen has been widely used to define human hematopoietic stem cells. Experiments using the umbilical cord blood to identify human HSCs have produced varied results using CD34 as a sole marker for long-term repopulating HSCs (LT-HSC). Ishikawa et. al. (3) have strongly indicated that the phenotype of human CB stem cells is CD34+CD38- while CD34+only positive cells represent not only LT-HSC but also Short- term hematopoietic stem cells (ST-HSC) and progenitor subpopulations (HSPCs) in hematopoeitic system hierarchy. Thus, primitive progenitors (LT-HSC) lack the expression or only marginally express the cell surface antigen CD38, which, in contrast, is abundantly expressed on more mature progenitors. Moreover, Görgens et.al. have meticulously shown that all HSCs with long-term potentials, including those with capabilities to engraft into NOD / SCID or NSG mice, were found to segregate into the CD133+CD34+CD38ífraction (4). Thus long-term potential UCB-HSCs are phenotypically characterized as CD133+CD34+CD38-. Further, monoclonal antibody recognition have been used to identify the presence or absence of cell surface protein markers for recognizing and isolating hematopoietic stem cells. Such markers comprise, but are not limited to, Lin, CD34, CD38, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and HLA DR, and combinations thereof. More information on this can be found in Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006 (http: / / stemcells.nih.gov / info / scireport / 2006report.htm). Functional readouts of HSCs and HSPCs have been linked to cell survival, self- renewal, proliferation, differentiation, and migration (homing, mobilization, and chemotaxis) (10). These processes involve several intracellular signaling molecules LQFOXGLQJ^^ EXW^ QRW^ OLPLWHG^ WR^^ S^^FLS^^ZDI^^^ S^^NLS^^^ 6WDW^^^ 6WDW^^^ 1RWFK^^:QW^^ ǃ- catenin, GSK-3, sonic hedgehog, bone morphogenic protein, members of the Hox family (such as Hoxb4), FoxO, Pu.1, GATA-1, Sirt1 (and other members of the sirtuin family of deacetylases), HIF-^Į^^5KHE^^^DQG^WKH^UDSDP\FLQ^VHQVLWLYH^P-TOR pathway (reviewed in (17)). . Hematopoietic growth factors in self-renewal and proliferation Hematopoietic growth factors are released under inflammatory or cytopenic conditions and induce mobilization and proliferation of HSCs in vivo. These growth factors are also known as colony stimulating factors (CSF) 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). Later in 1990s, many of these cytokines were introduced to cell culture media to ex vivo expand the UCB-HSCs for elucidation of its capacities to self-renewal and reconstitution in mice. Most experimental evidence indicated that a combination of several cytokines such as stem cell factor (SCF), FLT- 3 / FLK-2 Ligand (Flt3-ligand), thrombopoietin (TPO) seems to be essential for progenitor amplification. Among these growth factors, SCF is unanimously agreed to be indispensable for stem and progenitor expansion and even shows to be a key factor for hematopoietic progenitor cell survival. With this cytokine cocktail, CD34+ cells can be expanded ex vivo about several fold over pre-expanded values. At the crux of ex vivo HSC expansion is the increase in undifferentiated stem cells rather than expansion of both differentiated and undifferentiated cells. Mohamed et.al. (6) used SCF, GM- CSF, IL-3 and TPO under defined conditions for ex vivo expansion of CB stem cells for 7 and 11 days. They reported better expansion of CD34+ cells in 7 days and addition of more cytokines (IL-6 and FLT-3) did not affect CD34+ cell numbers but resulted in appearance of apoptotic cells. Yao et. al. used a statistically formulated cytokine- limited serum-free medium and reported 20-fold expansion of CD34+CD133+ cells and 723-fold expansion of CD34+CD38+ cells and 8-fold long-term culture-initiating cells after 7 days culture. Madkaikar et. al. (7) combined different cytokines (SCF, IL-3, GM- CSF) with stromal cell support for expansion of HSC and achieved maximum 21-fold CD34+ cell and nucleated cell expansion. Another group (Wei et. al.) defined 7 groups of culture conditions for the amplification of CD34+ cells containing basic combination of SCF, IL-3 and IL-6 for 3, 7 and 14 days. The fold expansion rates of CD34+ cells ranged from 10 to 50 fold as compared to fresh UCB. Despite the abundance of recombinant cytokines that are used for the expansion of primitive hematopoietic progenitors, no optimal combination has yet been approved for use in clinical practice. The most commonly used factors are the stem cell factor (SCF), IL-3 and -6, G-CSF, thrombopoietin (TPO), and Flt-3 ligand (8, 9) It should be noted that in addition to the set of factors, their concentration and the sequence of their use are also important. Fetal calf serum (FCS), which contains a natural cocktail of growth factors, adhesion mediators, minerals, lipids and hormones, is the standard component for cultivation of most human cell types, including HSCs. However, there is no consensus on the possibility of using cells after FCS-supported expansion in clinical settings. The disadvantages of a serum include difficulties in standardization of its composition, potential viral contamination, and high risk of immunization of a recipient with foreign proteins (10, 11). Therefore, excluding FCS and using a defined serum-free media for ex vivo expansion of HSPCs will greatly help in optimizing cytokine combinations and acceptability of expanded cells for transplant use (12, 13). . Ex vivo expansion strategies for cord blood HSCs A multitude of ex vivo expansion methods have been developed to increase UCB cell dose and the results of traditional UCB expansion methods using cytokines alone have been disappointing due to variability of concentration used for each cytokine. Another approach is to use blockers of HSC differentiation, such as nicotinamide analogs, copper chelators, induction of constitutive Notch signaling or using an aryl hydrocarbon receptor antagonist (StemReginin1). Many of these methods lead to substantial expansions of total nucleated cells and CD34+ cells, and significantly improved time to neutrophil or platelet engraftment in patients transplanted with the expanded products compared to the recipients of unmanipulated CBT. These studies differ not only in the expansion method but also with regards to the cytokines used, patient population, conditioning regimens, and transplantation practices, to name a few. Some of these methods start with unselected cell population from cord blood, while others use a selected subset, such as CD34+ or CD133+. Another UCB cell expansion technique utilised computer automated ‘continuous perfusion’ culture for 12 days and resulted in 2.4 fold expansion of TNC and 0.5 fold expansion of CD34+ cells. 3.1 Copper Chelator Tetraethylenepentamine (TEPA), A polyamine copper chelator, together with cytokines FLt-3, IL-6, TPO and SCF for 3 weeks has been reported to expand CD133+ selected HSCs by blocking cytokine-induced differentiation without affecting subsequent differentiation and proliferation of mature committed cells (14). This resulted in expansion of CD34+ cells by 89-IROG^^&'^^^^&'^^í^E\^^^-fold and colony forming units (CFU) by 172-fold. 3.2 Notch Signaling Another method of blocking EPC differentiation is the induction of constitutive Notch signaling, which can establish immortalized cell lines that can be cultured in liquid medium with cytokines. Interestingly, cell fate choices in both invertebrates and vertebrates are controlled by Notch signaling (15, 16) which inhibits certain differentiation pathways and thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Hematopoietic precursors express Notch receptors and Notch signaling augments the in vitro generation of human and mouse hematopoietic precursors (17-19) and also governs T- or B-cell lineage specification from a common lymphoid precursor (20). Delaney et al. (21) used CD34+ selected CB cells and transduced them with an engineered Notch ligand (Delta1ext-IgG) and cultured for 16 days in the presence of IL-3, IL-6, TPO, SCF, and flt-3 ligand, which led to 222-fold average expansion of CD34+ cells. 3.3 Nicotinamide Another technique of inhibiting the differentiation of HSCs involves the use of NAM, a sirutin family of protein deacetylases (SIRT1), which plays an important role in determining lifespan of a number of lower organisms (22) and self-renewal in mouse embryonic stem cells. it is known to inhibit differentiation of HPCs and plays an important role in adhesion, migration, and proliferation of stem cells (23). CD34+Lin- selected CB cells were expanded in media containing SCF, FLT-3, IL-6 and TPO with NAM. After 3 weeks of culture, TNCs expanded by a median of 486-fold and CD34+ cells expanded by a median of 72-fold (24). 3.4 Aryl Hydrocarbon Receptor Antagonism The University of Minnesota group presented the results of a phase I / II study of CB expansion using StemReginin1 (SR1), an aryl hydrocarbon receptor antagonist, which helps in the proliferation of CD34+ cells without differentiation in the presence of SCF, flt-3 ligand, TPO and IL-6. The culture for 21 days led to 328-fold median expansion of CD34+ cells (25). 3.5 Estrogen The female sex hormone Estrogen (E2) plays a crucial role in female and male sexual differentiation and reproduction. An important question is whether stem cells are targets of sex hormonal regulation in tissues that do not have sex-specific morphological differences. The terminal differentiation and function of some committed hematopoietic cells are regulated by sex hormones (26, 27) but hematopoietic stem cell (HSC) function is mostly similar in both sexes. Apart from its importance in reproduction, E2 has been shown to regulate proliferation and differentiation of multipotent and pluripotent stem cells (28, 29). In mice, E2 has been implicated in promoting cycling of bone marrow hematopoietic multipotent progenitors (MPPs) and their differentiation into megakaryocyte-erythroid progenitors (MEPs) (30). E2 has also been reported to enhance the proliferation and osteogenic differentiation of bone marrow mesenchymal stromal cells (BM-MSC) by activating Notch signaling and upregulation of estrogen receptors (ERs) (31). Interestingly, E2, which is readily available in placenta and normal cord blood, has been implicated in the growth and differentiation of lineage committed progenitors in rodents. E2 acts on target cells by binding nuclear receptors, of which two nuclear UHFHSWRUV^KDYH^EHHQ^ LGHQWLILHG^^HVWURJHQ^UHFHSWRU^DOSKD^^(5Į^^DQG^EHWD^^(5ǃ^^^^^^^^ The E2-ER complex binds to specific promoter regions on the DNA to modulate transcription of hundreds of target genes. This is known as genomic action of E2. E2 can also mediate rapid non-genomic actions by interacting with signaling cascades in WKH^F\WRSODVP^^(5Į^KDV^EHHQ^GHWHFWHG^LQ^QRQ-hematopoietic cells in bone marrow and in B lymphocyte precursors in mouse (33) and in human hematopoietic multipotent SURJHQLWRU^FHOOV^^033V^^^^^^^^(5ǃ^KDV^DOVR^EHHQ^IRXQG^LQ^QRQ-hematopoietic cells in mouse bone marrow and human spleen (33) but there are no reports on the status of (5ǃ^LQ^8&%-HSCs. We have recently sKRZQ^WKDW^ORVV^RI^(5ǃ^SHUWXUEV^WKH^SUROLIHUDWLRQ^ of neural stem cells. These effects were likely propagated through Notch–Hes signaling, ZKLFK^ZDV^GHUHJXODWHG^E\^WKH^ ORVV^RI^(5ǃ^^^^^^^ ,QWHUHVWLQJO\^^(5ǃ^FDQ^PHGLDWH^ WKH^ expression of p21, p27, and Notch1 in both a ligand-dependent and -independent manner (36, 37) and has been shown to control NOTCH1 transcription in squamous FHOO^ FDUFLQRPD^ FHOOV^ ^^^^^^ / LJDQG^PHGLDWHG^ DFWLRQV^ RI^ (5Į^ DUH^ RIWHQ^ UHODWHG^ WR^ FHOO^ SUROLIHUDWLRQ^DQG^WKRVH^RI^(5ǃ^DUH^DVVRFLDWHG^ZLWK^multiple cell-cycle regulatory anti- proliferative events and often oppose excessive proliferation (anti-tumorigenic effects). Illing et. al. (39) have reported that estradiol treatment of mice increases progenitor cell numbers but not long-term (LT) repopulating HSCs in the bone marrow vascular compartment, independent of estrogenic bone growth. Interestingly, the study showed that estradiol action on stromal cells potently favours HSPC cell frequency. Moreover, these proliferative effects of E2 on BM HSC were due to enhanced cycling (divisions) resulting in more short-term (ST) HSCs and exhaustion of LT-HSCs. This was further evidenced by a significant decrease in LT-HSCs derived from an E2 treated donor when given to a tertiary recipient mouse. However, the effects of E2 on the proliferation of these cells were likely not mediated through the classical ERs. During pregnancy, placental trophoblast cells increase maternal E2 levels upto 1000-fold (40) and this could be attributed to maternal adaptation for increased blood demand associated with fetal nutrition and physiological blood loss during childbirth. Based on these findings, Nakada et. al. (41) have observed that replicative activity of the HSCs is elevated in pregnant female mice. They have reported that E2 promotes enhanced cell cycle entry of HSCs and multipotent progenitors (MPPs) and increases differentiation into megakaryocyte erythrocyte progenitors (MEPs). The number of HSCs in the pregnant mice did not differ from control mice or from male mice, despite increased proliferation of HSCs and production of red blood cells and platelets. These HIIHFWV^RI^HVWURJHQ^ZHUH^DVFULEHG^WR^EH^PHGLDWHG^WKURXJK^(5Į^DQG^WKDW^HVWURJHQ^ODUJHO\^ promotes asymmetric division of HSCs rather than symmetric self-renewal in bone marrow. However, it is noteworthy to mention that the inventors have broadly ascribed this as HSC self-renewal despite they have not observed differences in HSC numbers in both male or female as well as after ovariectomy. BrdU incorporation of HSCs in their experiment does not reflect self-renewal (producing identical HSCs) rather enhanced cell division, which could lead to more committed progenitors. Anguilera et. al. (42) have attempted to determine the effects of E2 on specific HSPC subpopulaWLRQV^LQ^PLFH^DQG^IRXQG^WKDW^(5Į^DQG^(5ǃ^DUH^GLIIHUHQWLDOO\^H[SUHVVHG^ in LT-HSC, MPP and committed progenitors which might suggest different roles of E2 VLJQDOLQJ^ LQ^ GLIIHUHQW^ FHOO^ W\SHV^^ 7KH^ VWXG\^ VXJJHVWHG^ WKDW^ (5Į^ DFWLYDWLRQ^ WKURXJK^ selective ER modulators induces apoptosis, partly through Myc induction in ST-HSC and MPPs. In contrast, E2-mediated proliferation of quiescent LT-HSCs was associated with a loss of self-renewal activity and thereby loss of ability to reconstitute haematopoiesis, resulting in increased differentiation. Kim et. al. (43) have recently shown that supplementation of E2 in the growth media improved hematopoietic differentiation of human pluripotent stem cells (hPSCs) in vitro YLD^ (5Į^ PHGLDWHG^ SDWKZD\V^^ 7KH^ VWXG\^ UHSRUWHG^ WKDW^ (5Į^ is persistently expressed during the differentiation process and was exclusive to hematopoietic phenotype. However, E2 signaling did not have critical influence on the functional capacity of the differentiated hematopoietic cells. Further, the authors suggested that E2 signaling is involved in the fate decision process during early haematopoiesis as E2 treatment produced large number of hemangioblasts. Moreover, E2 alone had no effect on generation of hematopoietic cells from hPSCs but improved the generation of the cells by two fold in presence of hematopoietic growth factors. Contradictory, the authors notably reported an increase in erythropoietic differentiation from hPSCs after E2 supplementation. Interestingly, they also reported that the number of CD34+ blood cells from human umbilical cord could be increased by two fold when cultured in the presence of E2 for 10 days. The conclusion of the authors were that E2 augmented the proliferation of hematopoietic progenitors by maintaining their ER positivity and their functional capacity. However, the study reported by Kim et. al. is mostly focused on the development of hematopoietic cells from human pluripotent stem cells, and there are many pitfalls which do not clearly support their claim that E2 acts proliferative on HSCs rather than on later stage progenitors. Some of the questionable findings are given below: 1. The HSC markers used in the study, such as CD34+CD45+ does not truly reflect LT-HSC with long-term repopulation capacity and thus, may fail to reconstitute the haematopoiesis for long-term. (See 1. Hematopoietic stem cell (HSC) Phenotype and self-renewal pathways) 2. Even if the use of identifying markers in the study is accepted, simple E2 supplementation has only achieved upto 8% cells expressing hematopoietic progenitor markers in their study, which is not feasible for therapeutic applications due to low yield of cells. 3. Authors have interchangeably used the term hematopoietic stem cells and progenitors for CD34+CD45+ cells in their experiment while the true hematopoietic stem cell fraction is only CD133+CD34+CD38- and the rest are multipotent or committed progenitors. The CD133+CD34+CD38- has not been investigated in their study. 4. The study mostly focuses on the hematopoietic fate specification from pluripotent stem cells and it did not provide any experimental proof of further expansion of differentiated HSCs in the presence of E2. 5. The present study is in agreement with others performed in mouse models where E2 has been reported to enhance proliferation of progenitors but not HSC and subsequent increased differentiation to erythrocytes. This observed bias of progenitors under above mentioned experimental conditions is not beneficial for HSC expansion and use in transplant as these expanded fractions will not be able to reconstitute the recipient properly. 6. The authors claim to have proliferation of UCB hematopoietic progenitors upto 2-fold in 10 days which is meagre in terms of clinical usability. Moreover, there is no information provided on the other growth factors used in combination with E2, and the assessment of multilineage potential of E2 expanded progenitors is not accurate since there is no mention of number of progenitors used for the assay, and the number of CFUs shown is therefore technically not possible to reproduce. 7. The study has widely used fetal bovine serum as media supplement to culture cord blood stem and progenitor cells which may carry several unknown growth factors, hormones, matrix proteins and other factors which might have contributed to noted increased differentiation of hematopoietic progenitor cells despite a 2-fold expansion. Moreover, use of animal components for ex vivo expansion is not suitable for therapeutic purposes since they pose a risk of exposure to non-human molecules and pathogens that may elicit a strong immune reaction in humans. Thus, the above study has mostly focused on the effects of E2 on proliferation of hematopoietic progenitors and their enhanced differentiation but completely lacks to show that HSCs can be significantly expanded while retaining their self-renewal. Moreover, the study has used the opportunity to define hematopoietic development under the influence of E2 rather than expansion of hematopoietic stem cells per se. In light of the above mentioned in vivo rodent studies, estrogens have been shown to increase, decrease, or not affect HSC numbers, depending on the methodology / markers used to define the cell populations. Animal based studies do not translate well into human settings since differences exist in cell surface marker phenotype and growth factor requirement, as well as in telomere activity which may lead to more frequent cell cycle and cell divisions in the mouse. Although E2’s proliferative capacity is well described for several mature tissues, very little is known about the role of E2 in expansion and self-renewal of human HSCs with retained long-term repopulating potential. In addition, E2 has not been tested yet in the expansion of human UCB derived hematopoeitic stem cells in completely xeno- free environment using therapeutically relevant defined serum-free growth media. Results The few studies mentioned above that suggest a possibility of estrogens to mediate proliferative actions on cord blood derived HSCs is of high clinical relevance since great numbers of HSCs are needed for therapeutic efficacy. However, these studies do not fully address this question since they did not discriminate between HSCs and the more differentiated progenitors, leaving a large gap in interpretation. Moreover, these studies have mostly used rodent models that may not represent ideal conditions for human HSCs least the differences in cell surface markers. To fully determine the capacity of E2 on therapeutic HSC expansion, we explored the effect of E2 on the primitive (long-term) HSC population, i.e. the CD34+CD133+cells. In addition we used more therapeutically relevant serum free media with a carefully selected chemically defined additives and unique combination of growth factors, cytokines and ER selective ligands. Surpassing our expectations, we observed that low doses of E2 led to remarkably higher number of CD34+cells with retained stemness (CD34+CD133+or CD34+CD38-). We have examined the dose response of E2 and / or of ER subtype specific ligands in combination with known cytokines and growth factors at specific concentrations in defined serum free media. Experiment 1 UCB HSCs were outsourced from a commercial supplier (Zen-Bio Inc., USA) and the cells were 80-95% CD34+CD133+HSC. The HSCs were cultured feeder cell-free in a defined serum-free media with an optimized cocktail of growth factors and cytokines. ^^ǃ-estradiol (E2) and ER specific estrogenic ligands were added at different concentrations ranging from 0.5 nM to 25 nM. Cells were grown for 8 days and then counted with trypan blue for the exclusion of dead cells. Cell numbers were quantified and calculated for fold expansion in comparison to control group. Fold expansion of HSCs cultured in the presence of E2 at 1 nm and 10 nm doses for 8 days was significantly higher, 129±0.79 and 121.1±3.98 respectively, in comparison to untreated cells 94.23±6.97 (Fig.1). Interestingly, the expansion of UCB-HSCs was also satisfactorily good even in the absence of E2 in comparison to methods reported by others for ex vivo expansion, suggesting that our specific formulation of growth factors may robustly promote HSC expansion. Nevertheless, addition of E2 in this media led to even higher expansion of cells. Subsequently, HSC stemness of the ex vivo expanded cells was assessed using flow cytometry analyses, following ISHAGE recommendations by gating cells for CD45 expression, and confirmed that all the treatment and control conditions contained 70-80% CD34+cells and 24-26% CD34+CD133+cells (Fig.2). The number of CD34+CD133+is surprisingly high, which further gives credit to our expansion method. Quantification of CD34+ cells in expanded cultures revealed that E2 treatment at 1 nM and 10 nM doses resulted in significantly higher fold expansion of 91.94±3.11 and 95.07±1.47 respectively, compared to 74.66±6.68 fold in control group (Fig.3). This is the highest ever CD34+ cell expansion in shortest time period (8 days) compared to previously reported expansion by others (186-fold / 14 days, 222-fold / 16 days, 118-fold / 18 days, and 425- fold / 21 days). Importantly, the inventors did not find any significant difference in surface phenotype between untreated and E2 treated populations indicating that expanded cells retained their stemness and treatment conditions did not affect their phenotype. This supports our hypothesis that estrogenic ligands can be successfully used to promote robust ex vivo expansion of HSC population from cord blood without affecting the HSC phenotype (Fig.4) (see 1. Hematopoietic stem cell (HSC) Phenotype and self- renewal pathways). However, further studies on their capability of differentiating into myeloid and lymphoid lineage in vitro, as well as repopulating the recipients are warranted. Experiment 2 In another experiment, to fully assess the estrogenic effects on the proliferation of UCB-HSCs in the absence of Notch ligand, we expanded CD34+CD38- magnetically selected cells (80-90%) from Cord blood (Zen Bio, USA) in different growth factor, cytokines, estrogen and ER subtype specific ligands for 14 and 21 days respectively. Cells were analysed by flow cytometry as mentioned above in experiment 1. Results from this experiment showed that addition of GM-CSF and G-CSF in the culture media increases the expanded cell numbers (in millions) significantly compared to control group (7.26±0.11 vs 3.7±0.17) (Fig. 5a). Addition of E2 to this cytokine cocktail also resulted in significant expansion of cells (5.86±0.11) compared to the control. Interestingly, addition of notch ligand (sDLL1) and IGF binding protein 2 (IGFBP2), at different concentrations, to the standard media recipe (FKT36, i.e. Flt-3, SCF, TPO, IL- 3 and Il-6) did not have significant effect on expansion of cells (Fig. 5a). However, addition of both the ligands in the media containing GM-CSF and G-CSF increased the number of cells at day 14 (5.6±0.72) but not on day 21 (Fig. 5a) compared to control. SurprisingO\^^DGGLWLRQ^RI^(^^DW^^^^^QP^GRVH^DV^ZHOO^DV^(5ǃ^VHOHFWLYH^ / <^^^^^^^^^^QP^^ DQG^(5Į^VHOHFWLYH^337^^^^QP^^OLJDQGV^VLJQLILFDQWO\^LQFUHDVHG^WKH^QXPEHU^RI^H[SDQGHG^ cells in both the culture periods (day 14 and day 21). Similar effects were observed in terms of fold expansion of cells where addition of estrogen and its ligands in the absence of notch ligand significantly increased the fold expansion at both 14 and 21 days of culture period. Moreover, addition of E2 and its ligands in the presence of Notch ligand also resulted in an increased fold expansion of cells compared to control group (Fig. 5b). Expanded cells were assessed by flow cytometry for phenotype characterization. As expected from the above observation, fold expansion of CD34+ HSPCs was significantly higher in GG supplemented group compared to control group (Fig. 6a) at day 14. However, there was no significant difference in fold expansion of CD34+ cells between all the treatment groups at day 21 which could be due to exhaustion of HSC self-renewal capacity at this point and thus reached a plateau. Interestingly, the percentage of CD34+ cells amongst the total viable expanded cells did not change at both culture periods (Fig. 6b). These findings indicate that 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 which are often termed as LT-HSC and are also defined as CD38- which is not very different from our starting cell population (CD34+CD38- HSCs). Surprisingly, fold expansion of CD34+CD133+ cells was higher in GM-CSF and G-CSF supplemented media while addition of notch ligand did not have significant effect at both 14 and 21 days culture period (Fig.7). As expected from the observations above, addition of estrogen, in the absence or presence of notch ligand, significantly increased the fold expansion of CD34+CD133+ cells at both culture periods. Moreover, addition of ER selective ligands in the media containing notch ligand also had a significant expansion at day 21 but not at day 14 and this could partly be attributed to the highly selective nature of these ligands that may take time to reach an optimal effect in vitro. Functional assessment and characterization of ex vivo expanded HSPCs for repopulating capacity and long-term engraftment Ex vivo expanded HSCs have to be characterized and quantified for their stem cell activity. For a single HSC to repopulate the entire hematopoietic system requires self-renewal as well as capability to efficiently differentiate into every type of hematopoietic cell (37). However direct evaluation of HSC functions in humans is very limited and thus alternative in vitro assays have been developed for functional evaluation of immature HSCs (38). Flow cytometric analysis, is widely accepted method that can prospectively identify and isolate HSCs. Although, it does not provide functional data, it can be used to assess repopulating capability (39). Colony Forming Cell assay (CFC) is a good in vitro method of identifying hematopoietic progenitors, exploiting their ability to form a multi-lineage colony, which requires both the ability to differentiate as well as some self-renewal. Functional assessment of the most primitive HSC population can also be done with cobblestone area-forming cells (CAFC) and long-term culture initiating cells (LTC-IC) assays in vitro (40) and both of these assays entail more extensive self-renewal capacity than the CFC assay. However, the best assessments of HSCs require engraftment in vivo into immunodeficient mice, such as NOD-SCID mice, and assaying for long-term multi-lineage reconstitution and serial reconstitution (41). We have expand cord blood HSCs and assessed the cell surface phenotype during the course of the culture and the stem cell activity of resulting cells through in vitro CFC assay. CFC assay from the above experiment (#2), showed that cells from all the experimental group (growth factor / ligand combinations) have functional capacity of hematopoietic progenitors in addition to proliferation and thus retain their differentiation potential. Moreover, all the estrogenic ligand treated groups showed the potential of HSPCs to differentiate to myeloid (CFU-GEMM, CFU-GM) and erythroid (CFU-E and BFU-E) lineage. Interestingly, we did not observe any enhanced bias of expanded HSCs, in estrogenic ligand supplemented media, towards megakaryocyte- erythroid lineage. Experiment 3 In another experiment, to fully assess the estrogenic effects on the proliferation of UCB-HSCs from cryopreserved cord blood unit, we magnetically selected CD34+CD38- cells from cryopreserved Cord blood unit (Nationella navelsträngsblodbanken, Sahlgrenska Universitetssjukhuset Sweden) in different commercial and lab made culture media with combinations of growth factors, cytokines, estrogen and ER subtype specific ligands for 14 and 21 days respectively. Cells were analysed by flow cytometry as mentioned above in experiment 1 and 2. Results from this experiment showed that both serum free media (SFM / SPro34) and serum containing media (FBS media) without standard growth factors, known for HSC expansion (FKT), does not promote cell expansion at all. Surprisingly all the viable cells died in the absence of FKT factors. However, addition of these factors with or without addition of E2 promoted cell expansion significantly in both serum free and serum containing media. Further addition of cytokines IL3 and IL6 (marked as 36) almost doubled the cell expansion compared to FKT alone media. Further addition of GM-CSF and G-CSF in different serum free media (SFM / SPro34, Invitrogen Corp.; StemSpanII, StemCell Technologies Inc.; StemMACS HSC media, Miltenyi Biotec; KISCO-H1, in- house; KISCO-H2, in-house), with or without estrogenic ligands, significantly increased the expanded cell numbers (in millions) compared to control group (SFM / SPro34+FKT36, 3.7±0.1106) (Fig. 9a). Surprisingly, addition of E2 (at 10 nm dose) to different serum free media, with FKT36GG cocktail, resulted in significant expansion of cells compared to control group (SFM / SPro34+FKT36, 3.7±0.1106). 0RUHRYHU^^DGGLWLRQ^RI^(5ǃ^VHOHFWLYH^OLJDQG^ / <^^^^^^^^QP^^DQG^(5Į^VHOHFWLYH^OLJDQG^337^ (1 nm) also increased the number of expanded cells during 14 and 21 days culture period (Fig. 9a). Similar effects were observed in terms of fold expansion of cells where addition of estrogen and its ligands together with our optimized growth factor / cytokine cocktail FKT36GG significantly increased the fold expansion at both 14 and 21 days culture period (Fig. 9b). Expanded cells were assessed by flow cytometry for phenotype characterization. As expected from the above observations, fold expansion of CD34+ HSPCs was significantly higher in all the serum free media supplemented with FKT36GG compared with FBS media or SFM / SPro34 media with standard FKT supplementation (Fig. 10a) at day 14 and 21. Different serum free media performed comparatively well with our optimized growth factor cocktail (FFKT36GG) supplemented with estrogenic ligands. We further analyzed the fold expansion of CD34+CD38-cells which are often termed as long-term culture initiating cells (LTC-IC) and are also considered as primitive hematopoietic stem cells with long-term reconstitution capability. Considering this fact, our starting population for this expansion experiment was also CD34+CD38- HSCs. Surprisingly, fold expansion of CD34+CD38- cells was almost two times or more higher (41.31 fold to 110.37 fold) in FKT36GG+E2 supplemented serum free media (except for StemMACS media) at day 14 compared to FKT36GG alone SFM / SPro34 media (21.3 fold) while this effect was not apparent at day 21, except for our in-house serum free media which showed 223.79 fold (KISCO-H2) or 173.50 fold (KISCO-H1) expansion of CD34+CD38- LTC-IC HSC compared to control media (65.02 fold, SFM / SPro34+FKT36GG) (Fig. 10b). Functional assessment and characterization of ex vivo expanded HSPCs for repopulating capacity and long-term engraftment We assessed the cell surface phenotype during the course of the culture and the stem cell activity of resulting cells through in vitro CFC assay as mentioned in experiment 2 above. CFC assay from the experiment (#3), showed that cells from all the experimental group (growth factor / ligand combinations) have functional capacity of hematopoietic progenitors in addition to proliferation and thus retain their differentiation potential. Moreover, all the estrogenic ligand treated groups showed higher total colony forming units (CFU) of myeloid (CFU-GEMM, CFU-GM) and erythroid (CFU-E and BFU-E) lineage compared to the source cord blood unit after thaw (dashed line, Fig. 11). Conclusions Ex vivo expansion of UCB derived HSCs to therapeutic relevant numbers is promising to treat malignant and non-malignant blood disorders. However, many approaches to expand the cells have been reported but have not satisfactorily showed reliable expansion in shorter as well as longer time periods. Here, we have shown that UCB-HSCs can be satisfactorily expanded, with a unique and optimal growth factors and ligand composition, in shorter (8 days) as well as longer periods (14 and 21 days) without affecting their multipotency. Moreover, the presented media supplement is significantly effective in expansion of pluripotent LT-HSC with the addition of estrogenic compounds which enhances their cycling as well as helps in maintaining self-renewal. Utilizing this method results in more than 500- and 30- fold expansion of CD34+ HSPCs and CD34+CD133+ LT-HSCs in 14 days. Interestingly, this expansion is significantly increased when cells are cultivated for 21 days and results in more than 1500- and 90- fold expansion of CD34+ HSPCs and CD34+CD133+ LT-HSCs. Thus, the present study offers a reliable method of expansion of cord blood HSCs in both shorter and longer periods ex vivo (1-3 weeks) without affecting their differentiation potential. This method far exceeds the number of expanded cells than any other method presented elsewhere that will exemplify the advantage of expanded UCB-HSCs in clinical settings. Table 1 – Select abbreviations used in the Example IMDMIscove's Modified Dulbecco's MediumFBSF l B i References 1. Barker JN, Byam CE, Kernan NA, Lee SS, Hawke RM, Doshi KA, Wells DS, Heller G, Papadopoulos EB, Scaradavou A, Young JW, van den Brink MR. Availability of cord blood extends allogeneic hematopoietic stem cell transplant access to racial and ethnic minorities. Biol Blood Marrow Transplant. 2010 Nov;16(11):1541-8. doi: 10.1016 / j.bbmt.2010.08.011. Epub 2010 Aug 25. PMID: 20800103; PMCID: PMC3685152. 2. Zon LI. Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal. Nature 2008;453:306-313 3. Ishikawa, F., Livingston, A., Minamiguchi, H. et al. Human cord blood long-term engrafting cells are CD34+CD38í. Leukemia 17, 960–964 (2003). https: / / doi.org / 10.1038 / sj.leu.2402878 4. 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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):2309-2318. 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. 30. Nakada D, et al. (2014) / / Nature 505(7484):555-558. 31. Fan JZ, Yang L, Meng GL, Lin YS, Wei BY, Fan J, Hu HM, Liu YW, Chen S, Zhang JK, He QZ, Luo ZJ, Liu J. Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway. 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Improved hematopoietic differentiation of human pluripotent stem cells via estrogen receptor signaling pathway. Cell Biosci. 2016 Aug 30;6(1):50. doi: 10.1186 / s13578-016-0111-9. PMID: 27583127; PMCID: PMC5006567. 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 for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, any defined estrogen receptor agonist can be combined with any defined sample. The invention is also described as outlined in the following 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; and ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonist, 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). 5. The method of any one of Paragraphs 1-4, 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 1-5, 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 1-6, 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 wherein 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 are long-term repopulating hematopoietic stem cells (LT-HSCs). 9. The method of any one of Paragraphs 1-8, wherein the CD34+ hematopoietic stem cells comprise a sustained stemness phenotype. 10. The method of any one of Paragraphs 1-9, wherein the media is a serum-free media. 11. The method of any one of Paragraphs 1-10, wherein step ii) comprises culturing the cells of step i) in the absence of feeder cells. 12. The method of any one of Paragraphs 1-11, 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); a 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 1-13, 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 including CD34+ hematopoietic stem cells; and / or a blood sample; and / or a bone marrow sample. 15. The method of Paragraph 14, wherein the blood sample is one or more selected from the list consisting of: peripheral blood; mobilised peripheral blood; or umbilical cord blood. 16. The method of any one of Paragraphs 1-15, 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 HVWURJHQ^ UHFHSWRU^ DOSKD^ ^(5Į^ agonist and / or an estrogen receptor beta ^(5ǃ^ agonist. 18. The method of Paragraph 17^^ZKHUHLQ^WKH^(5Į^DJRQLVW^LV^one or more molecule selected from the group consisting of: estrogen E2; estrogen E1; estrogen E3; estrogen E4; and / or propyl pyrazole triol (PPT). 19. The method of Paragraph 17^^ZKHUHLQ^WKH^(5ǃ^DJRQLVW^LV^one or more molecule selected from the group consisting of: estrogen E2; estrogen E1; estrogen E3; estrogen E4; Diarylpropionitrile (DPN); LY3201; and / or LY-500307. 20. The method of Paragraph 16, wherein the membrane bound estrogen receptor agonist is one or more molecule 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 1-20, wherein the estrogen receptor agonist is present at a concentration of about 0.5 nM to about 25 nM, preferably about 1nM to about 10 nM. 22. The method of any one of Paragraphs 1-21, further comprising the step of isolating the CD34+ hematopoietic stem cells. 23. The method of Paragraph 22, wherein the isolating of the CD34+ hematopoietic stem cells comprises the use of flow cytometry and / or immunomagnetic cell separation and / or immunopanning. 24. The method of any one of Paragraphs 1-23, wherein step ii) is undertaken for a period of about 8 days or more; for example, about 14 days or more; or about 21 days or more. 25. The method of any one of Paragraphs 1-24, wherein in the expanded CD34+ hematopoietic stem cells of step ii) there is a ratio of one or more CD34+ hematopoietic stem cell of a CD34+ hematopoietic stem cell subpopulation (preferably a CD34+ CD133+ hematopoietic stem cell and / or a CD34+ CD133+ CD38- hematopoietic stem cell and / or a CD34+ CD45+(low / intermediate)CD133+ CD38- hematopoietic stem cell) to every about two to about 20 CD34+ hematopoietic stem cells. 26. The method of any one of Paragraphs 1-25, wherein the number of the expanded CD34+ hematopoietic stem cells of step ii) is increased by a fold change of about 88 or more. 27. The method of any one of Paragraphs 1-26, wherein the number of the expanded CD34+ hematopoietic stem cells of step ii) is increased by about 8,800% or more. 28. The method of any one of Paragraphs 1-27, further comprising the step of administering the expanded CD34+ hematopoietic stem cells of step ii) to a subject in need thereof. 29. The method of any one of Paragraphs 1-27, further comprising the step of iii) differentiating the expanded CD34+ hematopoietic stem cells of step ii) into one or more cells derived from the CD34+ hematopoietic stem cells. 30. The method of Paragraph 29, further comprising the step of administering the one or more cells derived from the CD34+ hematopoietic stem cells of step iii) to a subject in need thereof. 31. The method of any one of Paragraphs 1-30, wherein the method is an ex vivo method and / or an in vitro method. 32. Use of an estrogen receptor agonist for expanding one or more CD34+ hematopoietic stem cells. 33. A population of CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 and / or the use as defined in Paragraph 32. 34. One or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31. 35. One or more cells of Paragraph 34 or the method of any one of Paragraphs 1- 31, wherein the one or more cells derived from the CD34+ hematopoietic stem cells are cells selected from the list consisting of: myeloid progenitor cells; lymphoid progenitor cells; erythroid cells; erythrocyte cells; mast cells; megakaryocyte cells; thrombocyte cells; 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. 36. One or more cells of Paragraph 35 or the method of Paragraph 35, wherein the T lymphocytes are CAR-T cells. 37. A composition comprising CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or a population of CD34+ hematopoietic stem cells as defined in Paragraph 33. 38. A kit of parts comprising CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 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 agonist, 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. 40. CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or a population of CD34+ hematopoietic stem cells as defined in Paragraph 33 for use in medicine. 41. One or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or from 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 as defined in any one of Paragraphs 1-31 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 derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or from 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. 44. Use of CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or a population of CD34+ hematopoietic stem cells as defined in Paragraph 33, for the manufacture of a medicament for treating and / or preventing a condition in a subject in need thereof. 45. Use of one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or from a population of CD34+ hematopoietic stem cells as defined in Paragraph 33, for 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 one or more cells derived from the CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or from a population of CD34+ hematopoietic stem cells as defined in Paragraph 33 to the subject in need thereof. 47. A method for treating and / or preventing a condition in a subject in need thereof, comprising administering CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Paragraphs 1-31 or a population of CD34+ hematopoietic stem cells as defined in Paragraph 33 to the subject in need thereof. 48. A method for 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; optionally differentiating the expanded CD34+ hematopoietic stem cells into one or more cells derived from the CD34+ hematopoietic stem cells; and c) administering the cells of step b) to the subject in need thereof. The CD34+ hematopoietic stem cells for the use of any one of Paragraphs 40- 43, the use of any one of Paragraphs 44-45, or the method of any one of Paragraphs 46-48, wherein the condition is one or more condition selected from list consisting of: a condition requiring transplantation; a hematologic condition and / or a hematopoietic malignancy; a condition resulting from a failure or dysfunction of normal blood cell production and maturation; immunosuppression a subject with a malignant, and / or solid, tumor; an autoimmune disease and / or an immune condition; a genetic disorder; cancer; a tumor; osteopetrosis; myelosclerosis; acquired hemolytic anemias; infectious disorders causing primary or secondary immunodeficiencies; acquired immunodeficiencies; neutrophil actin deficiency; neutrophil membrane GP-180 deficiency; bacterial infections (for example Brucellosis infections, Listerosis infection, tuberculosis infection, and / or leprosy); parasitic infections (for example, malaria and / or Leishmaniasis); fungal infections; disorders involving disproportions in lymphoid cell sets and / or impaired immune functions due to aging; phagocyte disorders; Kostmann’s agranulocytosis; chronic granulomatous disease; Chediak-Higachi syndrome; Wiskott-Aldrich Syndrome; metabolic storage diseases; mucopolysaccharidoses; PXFROLSLGRVHV^^ Į^-antitrypsin deficiency; and / or miscellaneous disorders involving immune mechanisms. A method, use, population of CD34+ hematopoietic stem cells, one or more cells derived from the CD34+ hematopoietic stem cells, composition, kit of parts or method for treating and / or preventing a condition, substantially described herein with reference to the description and / or figures.

Claims

Claims 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; and ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonist, to expand the CD34+ hematopoietic stem cells.

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 of any one of Claims 1-3, 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 1-4, 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 1-5, wherein step ii) further comprises culturing the cells of step i) in the presence of one or more growth factors and / or cytokines.

7. The method of any one of Claim 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); a 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 of any one of Claims 1-7, 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 including CD34+ hematopoietic stem cells; and / or a blood sample; and / or a bone marrow sample.

9. The method of Claim 8, wherein the blood sample is one or more selected from the list consisting of: peripheral blood; mobilised peripheral blood; or umbilical cord blood.

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

11. The method of Claim 10, wherein the nuclear estrogen receptor agonist is an HVWURJHQ^UHFHSWRU^DOSKD^^(5Į^^DJRQLVW^DQG^RU DQ^HVWURJHQ^UHFHSWRU^EHWD^^(5ǃ^^ agonist.

12. The method of Claim 11^^ZKHUHLQ^ WKH^ (5Į^ DJRQLVW^ LV^ RQH^ RU^PRUH^PROHFXOH^ selected from the group consisting of: estrogen E2; estrogen E1; estrogen E3; estrogen E4; and / or propyl pyrazole triol (PPT).

13. The method of Claim 11^^ZKHUHLQ^ WKH^ (5ǃ^ DJRQLVW^ LV^ RQH^ RU^PRUH^PROHFXOH^ 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 1-13, wherein the estrogen receptor agonist is present at a concentration of about 0.5 nM to about 25 nM, preferably about 1nM to about 10 nM.

15. The method of any one of Claims 1-14, wherein step ii) is undertaken for a period of about 8 days or more; for example, about 14 days or more; or about 21 days or more.

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

17. The method of any one of Claims 1-16, wherein the number of the expanded CD34+ hematopoietic stem cells of step ii) is increased by a fold change of about 88 or more.

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

19. Use of an estrogen receptor agonist for expanding 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 1-18 and / or the use as defined in Claim 19.

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

22. A composition comprising CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Claims 1-18 or a population of CD34+ hematopoietic stem cells as defined in Claim 20.

23. CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Claims 1-18 or a population of CD34+ hematopoietic stem cells as defined in Claim 20 for use in medicine.

24. CD34+ hematopoietic stem cells obtainable, and / or obtained, by the method as defined in any one of Claims 1-18 or a population of CD34+ hematopoietic stem cells as defined in Claim 20 for use in treating and / or preventing a condition in a subject in need thereof.

25. The CD34+ hematopoietic stem cells for the use of Claim 23 or 24, wherein the condition is one or more condition selected from list consisting of: a condition requiring transplantation; a hematologic condition and / or a hematopoieticmalignancy; a condition resulting from a failure or dysfunction of normal blood cell production and maturation; immunosuppression a subject with a malignant, and / or solid, tumor; an autoimmune disease and / or an immune condition; a genetic disorder; cancer; a tumor; osteopetrosis; myelosclerosis; acquired hemolytic anemias; infectious disorders causing primary or secondary immunodeficiencies; acquired immunodeficiencies; neutrophil actin deficiency; neutrophil membrane GP-180 deficiency; bacterial infections (for example Brucellosis infections, Listerosis infection, tuberculosis infection, and / or leprosy); parasitic infections (for example, malaria and / or Leishmaniasis); fungal infections; disorders involving disproportions in lymphoid cell sets and / or impaired immune functions due to aging; phagocyte disorders; Kostmann’s agranulocytosis; chronic granulomatous disease; Chediak-Higachi syndrome; Wiskott-Aldrich Syndrome; metabolic storage diseases; PXFRSRO\VDFFKDULGRVHV^^ PXFROLSLGRVHV^^ Į^-antitrypsin deficiency; and / or miscellaneous disorders involving immune mechanisms.

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