Process

Abstract

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

Description

[0001] The present invention provides methods for expanding CD34+ hematopoietic stem cells (HSCs), as well as associated uses, cells produced by such methods, and medical treatments.

[0002] The transplantation of HSC has great potential in the medical treatment of a variety of diseases, including various hematological, oncological and immune disorders. However, usually due to the lack of HSC donors that match potential patients, the practical application of this type of treatment is limited due to the low availability of HSC. The resulting low dose of HSC that can be used for transplantation leads to slow implantation, which may lead to treatment failure.

[0003] To overcome the low availability of HSCs, ex vivo expansion of HSCs, particularly HSCs from umbilical cord blood, shows great potential by allowing the preparation of these cells in more clinically relevant quantities and is more likely to allow successful treatment.

[0004] Although several methods have been developed for ex vivo expansion of HSCs using molecules such as hematopoietic growth factors, cytokines, and differentiation blockers such as Notch ligands, copper chelators, nicotinamide, and aryl hydrocarbon receptor antagonists, there is a need for additional and improved expansion methods.

[0005] For example, Liedtke et al., 2019, Stem Cell Translational Medicine 8: S20-S20, doi: 10.1002 / sctm.12563, described in the expansion of hematopoietic stem cells / progenitor cells. In a specific example, hematopoietic stem cells / progenitor cells can be thawed, enriched and supplemented with 100ng / mL interleukin-3 (IL-3), 100ng / mL interleukin-6 (IL-6), 100ng / mL thrombopoietin (TPO), 100ng / mL Flt-3 ligand (Flt-3L), 100ng / mL stem cell factor or KIT ligand or steel factor (SCF) in serum-free medium for 3-4 days.

[0006] In another example, the expansion of hematopoietic stem / progenitor cells is as described 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 a serum-free medium containing IMDM, 1% bovine serum albumin, 10 mg / mL bovine pancreatic insulin, 200 mg / mL human transferrin, 10-4 M β-mercaptoethanol, 2 mM L-glutamine, 40 mg / mL low-density lipoprotein and growth factors; 25 ng / mL FGF-4, 25 ng / mL IL-3, 25 ng / mL SCF, 25 ng / mL FLT3 ligand, plus 25 ng / mL heparin. The cells are grown for 4 to 28 days, and the culture medium and growth factors are replaced 3 times per week. All cells were grown in tissue culture treated 6-, 12-, or 24-well plates.

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

[0008] Estrogen has been used as a media supplement for lineage committed progenitor cell growth and stem cell differentiation. However, the use of estrogen has not been considered or identified as a molecule that can be used for the expansion of HSCs.

[0009] Against this background, the inventors have identified for the first time the use of estrogen receptor agonists (including estrogen) for amplifying highly medically relevant HSCs (e.g., CD34+HSCs) expressing CD34 markers and subpopulations of CD34+HSCs such as those subpopulations (e.g., CD34+CD133+HSCs) that also express CD133 markers. Surprisingly, the method of using estrogen receptor agonists to amplify CD34+HSCs and CD34+CD133+HSCs results in high-quality amplified cell populations, which makes them very valuable for medical treatment.

[0010] In a first aspect, the present invention provides a method for expanding CD34+ hematopoietic stem cells, comprising the following steps:

[0011] i) providing a sample comprising one or more CD34+ hematopoietic stem cells; and

[0012] ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonists to expand CD34+ hematopoietic stem cells.

[0013] In a second aspect, the present invention provides the use of an estrogen receptor agonist for expanding one or more CD34+ hematopoietic stem cells. The use of the second aspect of the present invention may include the steps described herein with respect to the method of the first aspect of the present invention.

[0014] Hematopoietic stem cells (HSCs) are multipotent stem cells 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 immune system cells (such as T lymphocytes and B lymphocytes). HSCs can be found in the bone marrow and umbilical cord blood in vivo.

[0015] HSCs expressing the cell surface marker CD34 are referred to as CD34+HSCs. CD34 is a transmembrane phosphoglycoprotein protein. The nucleic acid sequence of CD34 is known and described, for example, in Satterthwaite AB et al., Structure of the gene encoding CD34, a human hematopoietic stem cell antigen, Genomics, 1992 Apr; 12(4): 788-794. The nucleic acid sequence of CD34 is known and described, for example, 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.

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

[0017] Therefore, "one or more CD34+ hematopoietic stem cells" include one or more hematopoietic stem cells that contain the CD34 marker and / or express the CD34 gene.

[0018] By "one or more" CD34+ hematopoietic stem cells, we include about two or more CD34+ hematopoietic stem cells, which may also be referred to as a CD34+ hematopoietic stem cell population. In one embodiment, about two or more CD34+ hematopoietic stem cells and / or a CD34+ hematopoietic stem cell population are 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. 0 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.

[0019] 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.

[0020] In some other embodiments, the sample contains up to about 3,000,000 CD34+ hematopoietic stem cells. For example, "one or more" CD34+ hematopoietic stem cells and / or CD34+ hematopoietic stem cell populations are 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 contains about 3,000,000 CD34+ hematopoietic stem cells. In some embodiments, the sample contains between about 100 and about 3,000,000 CD34+ hematopoietic stem cells.

[0021] The number of CD34+ hematopoietic stem cells contained in the sample of step (i) can depend on the time that the culture step of (ii) is to be performed to ensure that the desired level of expansion is achieved. For example, if step (ii) is to be performed for 8 days, the number of CD34+ hematopoietic stem cells contained in the sample of step (i) can be 30,000-40,000, preferably in the region of 35,000-36,000 cells. In another example, if step (ii) is to be performed for 14 to 21 days, the number of CD34+ hematopoietic stem cells contained in the sample of step (i) can be 5,000 to 15,000 cells, preferably in the region of 9,000 to 10,000 cells. In a further example, if step (ii) is to be performed for 14 to 21 days, the number of CD34+ hematopoietic stem cells contained in the sample of step (i) can be 1000-1500.

[0022] Stem cell expansion (or amplification of stem cells, as mentioned in the present invention) is the following process, by which stem cells divide to form two daughter cells, both of which maintain the self-renewal stem cell properties of the parental cell, or produce two daughter cells, one of which retains the self-renewal properties of the parental stem cell, and the other daughter cell differentiates into a more specialized type of cell that may not have self-renewal potential or have limited self-renewal potential. The cell division in the former case is sometimes referred to as symmetrical stem cell division, while the latter case is referred to as asymmetric cell division. Both types of cell divisions that cause the proliferation of stem cells with self-renewal capacity are included in the definition of "amplification" as used herein. By stem cell expansion, we further include culturing CD34+ hematopoietic stem cells under specific conditions, as described herein, which promote cell growth (e.g., mitosis) so that the cells will multiply and divide, thereby producing amplified hematopoietic stem cell / progenitor cell populations.

[0023] Stem cell expansion is different from the process of stem cell differentiation. Differentiation involves 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, while the other daughter cell differentiates into a more specialized type of cell, ultimately resulting in the terminal differentiation of both progeny. Cell division in stem cell differentiation can sometimes be referred to as asymmetric stem cell division; however, it does not result in the propagation of stem cells with self-renewal capabilities.

[0024] Self-renewal is a cell division that is unique to stem cells. When self-renewal occurs, stem cells split into two cells, one of which is still a stem cell (it can show the potential for differentiation into one or more types of differentiated cells, and almost infinite division), and the other is a differentiated cell, which may or may not divide, but its division (if any) is not self-renewal because: (1) stem cells (i.e., cells with developmental potential) are not produced, and; (2) these divisions are limited (therefore they are not self-renewal, because self-renewal is unlimited but controlled proliferation). In short, self-renewal is the "unlimited" division of stem cells while retaining its developmental potential. In other words, "stem cell expansion" as mentioned herein refers to stem cell self-renewal, i.e., any process that causes unlimited division and proliferation while retaining developmental potential.

[0025] Using HSC as an example, expansion would be the division of a parent HSC forming two identical HSCs or at least one progeny retaining the same stem cell properties (where another progeny forms a progenitor cell), while differentiation would be the division of a parent HSC forming two progenitor cells (such as myeloid progenitors or lymphoid progenitors).

[0026] Therefore, by "expanding CD34+ hematopoietic stem cells" (as described in step ii of the present invention), we include the result of the method that one or more CD34+ hematopoietic stem cells divide to form one or two daughter CD34+ hematopoietic stem cells, wherein at least one daughter cell maintains the stem cell properties of the parent cell. As will be understood, any daughter CD34+ hematopoietic stem cell can itself become expanded to generate further daughter CD34+ hematopoietic stem cells, and so on.

[0027] When amplifying HSC, it is most preferred to avoid HSC differentiation, because the differentiation will cause a variety of cell types to be present in the resulting cell population. When compared with a cell population with a very high proportion of HSC or a cell population comprising only HSC, a heterogeneous population of cells comprising a mixture of HSC and differentiated cells provides a lower quality therapeutic effect. The advantage of the inventor's method is that, when compared with other amplification methods known in the art, it greatly limits and / or avoids HSC differentiation, thereby producing a resulting cell population that will provide high-quality therapy. Therefore, by the so-called amplification of CD34+HSC by the method of the present invention, we include HSC that is not significantly differentiated or HSC that is not differentiated.

[0028] Estrogen receptor is a group of proteins that are bound by estrogen and activated by estrogen. As further described herein, estrogen receptor can be mainly divided into two categories-nuclear estrogen receptor and membrane estrogen receptor. So-called "estrogen receptor agonist", we include molecules that can bind to and preferably activate estrogen receptor. As described herein, estrogen receptor agonist can be a naturally occurring molecule or a synthetic molecule. In one embodiment, estrogen receptor agonist is a molecule that simulates estrogen in function, and in a specific embodiment, estrogen receptor agonist is a molecule that simulates estrogen in function completely or in part. In a specific embodiment, estrogen receptor agonist is an estrogen, as described herein.

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

[0030] In general, culturing cells includes growing and maintaining cells in an in vitro environment. Therefore, the so-called "culturing HSC in the presence of one or more estrogen receptor agonists" (as described in step ii) of the present invention), we include growing and / or maintaining HSC in the presence of one or more estrogen receptor agonists, preferably in an in vitro environment. Cultivating HSC in the presence of an estrogen receptor agonist includes allowing the estrogen receptor agonist to contact the HSC in a manner that will allow the estrogen receptor agonist to activate the estrogen receptor of the HSC. We further include that "culturing HSC in the presence of one or more estrogen receptor agonists" can include adding one or more estrogen receptor agonists to the sample.

[0031] 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 (e.g., CD34+CD133+ hematopoietic stem cells). In an alternative preferred embodiment, the CD34+ hematopoietic stem cells further comprise the marker CD45 (e.g., CD34+CD45+ hematopoietic stem cells). In a further preferred embodiment, the CD34+ hematopoietic stem cells further comprise the markers CD133 and CD45 (e.g., CD34+CD45+CD133+ hematopoietic stem cells).

[0032] CD133 (also known as prominin-1) is a five-transmembrane glycoprotein and is expressed on a subset of CD34+ cells obtained from different sources, including fetal liver and bone marrow, adult bone marrow, umbilical cord blood, and mobilized peripheral blood. CD133+ stem cells can reconstitute myelopoiesis and lymphopoiesis in mice (see, e.g., Handgretinger R, 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。

[0033] CD45 is a receptor-linked protein tyrosine phosphatase that is present in cells of all hematopoietic lineages except erythrocytes and plasma cells. It was originally called 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, all of which are products of a single complex gene. CD45RA contains only the A protein region, while CD45RO (the shortest CD45 isoform) lacks all three 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.2006Feb;117(2):145-55.

[0034] CD43, also known as white sialoprotein or sialoglycoprotein, has been shown to be expressed on early hematopoietic cells derived from hESC. Comparable to CD34, CD43 is a strongly glycosylated transmembrane sialomucin that is expressed on all hematopoietic cells except mature erythrocytes. CD43 is further described 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 mouseleukosialin (CD43), a T lymphocyte mucin without introns in the coding sequence. Eur J Immunol. 1990 Apr; 20 (4): 875-81.

[0035] CD59 is a phosphoinositol glycan-linked membrane protein that is consistently expressed by most nucleated cells of the hematopoietic system, including all BM-derived CD34+ cells. However, CD59 is differentially expressed in CD34+ progenitor cells, with the highest levels present in a subset of highly enriched 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.

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

[0037] CD90, or thymocyte differentiation antigen (Thy-1), is a small 25-35 kDa GPI-anchored glycoprotein that is an important regulator of cell-cell and cell-matrix interactions. It is involved in tumor suppression, cell adhesion, migration, and apoptotic signaling. CD90 is highly expressed on highly proliferative umbilical cord blood primitive cells and is involved in the development of HSCs. 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 May1; 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.

[0038] CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein that is localized on 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 β (TGF-β) signaling associated with HSC differentiation. CD109 is further discussed in Martin Lin et al.; Cell surface antigen CD109 is a novel member of the α2 macroglobulin / C3, C4, C5 family of thioester-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.

[0039] CD117 or c-KIT is a type III receptor tyrosine kinase that binds to 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 (HSCs), multipotent progenitor cells (MPPs) and common myeloid progenitor cells (CMPs) and promotes cell proliferation. CD117 is further discussed in Edling CE, Hallberg Bc-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 2007Jan 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.

[0040] CD166, also known as activated leukocyte cell adhesion molecule (Alcam), is a member of the immunoglobulin superfamily, which mediates homophilic (CD166-CD166) and heterophilic (CD166-CD6) interactions. CD166 is expressed on stem cells and progenitor cells of all three embryonic lineages, and CD166 interactions lead to the activation of key molecular pathways, which are involved in enhancing HSC / HPC functions related to maintaining 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), aCD6 ligand. J Exp Med. 1995 Jun 1; 181(6): 2213-20.

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

[0042] By “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 one or more hematopoietic stem cells expressing CD133, CD45, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117 and / or CD166 genes.

[0043] As will be understood by those 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.

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

[0045] CD38, also known as cyclic ADP ribose hydrolase, is a glycoprotein found on the surface of leukocytes and plays a role in cell adhesion, signal transduction and calcium signaling. The CD38 protein, which is rearranged at high levels by activated lymphocytes, is a marker of cell activation. Expression of the cell surface antigen CD38 in hematopoietic cells is activated by differentiation, and therefore, exclusion of CD38 expression on CD34+ cells is associated with long-term HSC activity. CD38 is further discussed 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.

[0046] HLA-DR is a major histocompatibility complex class II (MHC class II) cell surface receptor encoded by several loci and genes of different functions of the human leukocyte antigen complex on chromosome 6 region 6p21.31. The receptor / peptide complex is a ligand for the T cell receptor (TCR) and mediates the acceptability / rejection of organ / blood transplantation in the recipient. HLA-DR is also involved in several autoimmune conditions, disease susceptibility, and disease resistance. HLA-DR is expressed in umbilical cord and peripheral blood cells. The most primitive HSCs co-express c-kit, FLT-3, and Thy-1, and are negative for HLA-DR, CD38, and lineage markers. HLA-DR is further discussed 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 are 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.

[0047] Lineage (Lin) marker refers to the expression of various lineage markers associated with a certain cell type. Hematopoietic stem cells lead to different types of blood cells, called myeloid and lymphoid. Myeloid and lymphoid lineages both involve 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). Flow cytometry can be used to identify or separate hematopoietic stem cells from peripheral blood cells, wherein one or more different cell surface markers (as described above) are used in combination with a combination of CD34. Lack of mature blood cell markers (including but not limited to CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 and CD235a) expression of HSC, therefore referred to as lineage negative (Lin-). The lack of lineage marker expression is combined with the detection of several positive cell surface markers to separate hematopoietic stem cells. Therefore, in the context of the present invention, the hematopoietic stem cells lacking lineage markers (Lin) lack the expression of one or more markers associated with mature blood cells. In some embodiments, hematopoietic cells therefore lack the expression of one or more markers selected from the following: CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 and CD235a. The separation and / or enrichment of hematopoietic stem cells containing and / or lacking specific markers are further described herein.

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

[0049] By “hematopoietic stem cells lacking one or more markers selected from the list consisting of CD38, HLA DR and / or Lin”, we include one or more hematopoietic stem cells not containing CD38, HLA DR and / or Lin markers and / or not expressing the above-mentioned CD38, HLA DR and / or Lin genes.

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

[0051] "Low / medium" CD45 is related to CD45 expression, and those skilled in molecular biology will understand those relative levels. By low / medium CD45 expression, we include low CD45 expression and / or medium CD45 expression. A method of identifying such expression is to use flow cytometry. Relative CD45 expression is relevant to the present invention because high CD45 expression is associated with differentiated lymphocytes. Typically, in flow cytometry or other fluorescence-based separation methods, cells are gated based on the relative intensity of a fluorescent marker (e.g., CD45). For HSC selection, high intensity CD45 expressing cells are excluded to select only HSC. As will be understood by the skilled person, the intensity scale depends on the sensitivity of the machine (flow cytometer) and may differ between different devices, and for this reason, cells are gated based on their relative intensity within the analyzer / device.

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

[0053] In one embodiment, the CD34+ hematopoietic stem cell is a long-term repopulating hematopoietic stem cell (LT-HSC).

[0054] Long-term repopulating hematopoietic stem cells (LT-HSCs) are generally understood to be CD34+CD133+CD38- hematopoietic stem cells. The inventors have surprisingly determined that their method is effective in expanding this subset of hematopoietic stem cells as well as CD34+CD133+CD45+ (低 / 中) CD38- hematopoietic stem cells are particularly effective, as described in the Examples herein.

[0055] In a specific embodiment, the CD34+ hematopoietic stem cells include one or more CD34+ hematopoietic stem cells and one or more CD34+ hematopoietic stem cells as indicated below: the CD34+ hematopoietic stem cells further include 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 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 include 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 include one or more CD34+ hematopoietic stem cells and one or more CD34+CD45+ (低 / 中) In a further preferred embodiment, the CD34+ hematopoietic stem cells include one or more CD34+ hematopoietic stem cells and one or more CD34+CD45+ (低 / 中) Hematopoietic stem cells and / or one or more CD34+CD133+ hematopoietic stem cells and / or one or more CD34+CD45+ (低 / 中) CD133+ hematopoietic stem cells. In an alternative preferred embodiment, the CD34+ hematopoietic stem cells include 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 include one or more CD34+ hematopoietic stem cells and one or more CD34+CD133+CD45+ (低 / 中) CD38-hematopoietic stem cells.

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

[0057] In some embodiments of the present invention in which there is a mixture of CD34+ hematopoietic stem cells and CD34+ hematopoietic stem cells containing and / or lacking other specialized markers, the daughter CD34+ hematopoietic stem cells of step ii) will contain and / or lack the same specialized markers as the parent CD34+ hematopoietic stem cells; for example, the daughter hematopoietic stem cells derived from the parent CD34+CD133+ hematopoietic stem cells will also contain markers CD34 and CD133, and / or the daughter hematopoietic stem cells derived from the parent CD34+CD133+CD38- hematopoietic stem cells will also contain markers CD34 and CD133 and lack the CD38 marker. In some other embodiments, due to changes in conditions, the daughter CD34+ hematopoietic stem cells of step ii) will contain and / or lack specialized markers different from the parent CD34+ hematopoietic stem cells; for example, the daughter hematopoietic stem cells derived from the parent CD34+CD133+CD38- can lack the CD133 marker and / or additionally contain the CD38 marker.

[0058] In one embodiment, the CD34+ hematopoietic stem cell comprises a persistent stem phenotype. In particular, the CD34+CD133+ hematopoietic stem cell comprises a persistent stem phenotype.

[0059] Generally speaking, HSC stemness is the ability of HSC to self-renew and produce two identical offspring, but, unless it is sustained, the daughter cells may not be able to further produce two identical offspring, but one of them retains the same phenotype and the other is committed to differentiate into a progenitor cell of a specific blood cell. Therefore, the sustained stemness phenotype of HSC includes self-renewal and restricted differentiation, while retaining the ability to differentiate into all types of blood cells when relevant induction signals are provided. Sustained stemness can also be called retained stemness.

[0060] In one embodiment, the persistent stemness phenotype is characterized in that the expanded CD34+ hematopoietic stem cells comprise specific markers in common with the CD34+ hematopoietic stem cells from which they were derived (e.g., CD34+ and / or one or more markers selected from the list consisting of: CD133; CD45; CD43; CD45RO; CD45RA; CD59; CD90; CD109; CD117; and / or CD166; and / or lack one or more markers selected from the list consisting of: CD38, HLADR and / or Lin).

[0061] In further embodiments, the persistent stemness phenotype is characterized by the ability of the expanded CD34+ hematopoietic stem cells to be maintained in long-term culture (eg, long-term culture on bone marrow feeder cells).

[0062] In a further embodiment, the persistent stemness phenotype is characterized by the expanded CD34+ hematopoietic stem cells being able to differentiate into progenitor cells (preferably wherein the progenitor cells are able to differentiate into all types of mature blood cells).

[0063] How to identify whether HSCs are able to differentiate into progenitor cells (and ultimately mature blood cells) is known to those skilled in the art of cell biology. For example, this can be assessed using the methods described in the Examples, such as whether expanded HSCs are able to generate colonies of different progenitor cells.

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

[0065] As is known to those skilled in cell biology, a culture medium (often referred to as a cell culture medium) is generally a liquid containing components that allow the growth and maintenance of cells.

[0066] In one embodiment, the culture medium is a defined medium.

[0067] As known to those skilled in cell biology, a defined medium is a medium in which all ingredients and their relative concentrations and / or quantities are known. This is in contrast to a medium containing ingredients with unknown or undefined content, concentration, and / or quantity. In general, a defined medium does not contain ingredients derived from animals, but rather contains chemically synthesized and / or non-derived ingredients, which allows for better control of the contents of the medium.

[0068] In one embodiment, the culture medium is a serum-free culture medium. In a specific embodiment, the serum-free culture medium is an animal serum-free culture medium.

[0069] As known to those skilled in the art of cell biology, serum is an example of a culture medium component that does not have a clear content, concentration and / or quantity. Therefore, including serum in the culture medium will result in it not being a defined culture medium. In addition, it may be preferred not to use a culture medium containing serum to avoid contamination by a source of infection (such as a virus) and / or immunization of any recipient patient with foreign proteins.

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

[0071] As will be known to those skilled in cell biology, a xeno-free medium is a medium that does not contain components that are not derived from the same species as the cells to be cultured; for example, if human cells are to be cultured, a corresponding xeno-free medium will not include any non-human biological components.

[0072] In one embodiment, step ii) comprises culturing the cells of step i) in the absence of feeder cells.

[0073] As is known to those skilled in cell biology, feeder cells can be co-cultured with HSCs and provide an undefined mixture of components to the HSCs.

[0074] 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.

[0075] Growth factors are biological substances that can regulate cellular processes (such as stimulating cell proliferation). Growth factors are generally cell signaling substances and / or hormones produced by cells; however, in some cases, growth factors can be synthesized artificially. Cytokines are also biological substances that can mediate cellular processes such as hematopoiesis. Cytokines are signaling molecules that are also produced by cells and can be synthesized artificially. Those skilled in cell biology will know how to identify growth factors and cytokines that can be used as part of the present invention. Therefore, by growth factors and cytokines, we include biological substances that can stimulate cell proliferation.

[0076] 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)); 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 factor (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 specific 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 growth factors added to the basal medium in the "FKT36 medium" described herein.

[0077] In alternative embodiments, notch ligands (eg, notch ligand sDLL1) and / or IGFBP2 are not used in the methods of the invention.

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

[0079] In certain embodiments, the growth factors and / or cytokines may be in the following amounts: 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) 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).

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

[0081] When supplemented with serum or a serum-free component of the present invention, the term "basal medium" refers to a medium that can support the growth of hematopoietic cells (such as CD34+ hematopoietic cells or other cells of the present invention). The basal medium should be able to provide inorganic salts, such as sodium, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, energy sources such as glucose, pH buffer systems and essential amino acids required for standard cell growth. Basal media that can be used as part of the present invention include, but are not limited to, Iscove's Modified Dulbecco's Medium, RPMI 1640, Minimal Essential Medium-a (MEM-a) and other related culture media.

[0082] In one embodiment, the basal medium used in the present invention (e.g., 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).

[0083] In one embodiment, the basal medium (such as Iscove's Modified Dulbecco's Medium (IMDM)) is further supplemented with one or more components selected from the list of components comprising: human serum albumin; ethanolamine; sodium selenite; Cortisol; D,L-tocopherol; human transferrin (holo); human insulin (zinc); N-acetyl-L-cysteine; 2-mercaptoethanol; and / or a cholesterol lipid concentrate (such as available from ThermoFisher Scientific or other companies). In a specific embodiment, the components can be in the following amounts: about 1% to about 10% (preferably about 5%) human serum albumin; about 1 mg / L to about 20 mg / L (preferably about 10 mg / L) ethanolamine; about 1 μg / L to about 10 μg / L (preferably about 5 μg / L) sodium selenite; about 20 μg / L to about 70 μg / L (preferably 50 μg / L) cortisol; 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-acetyl-L-cysteine; about 1 mg / L to about 10 mg / L (preferably about 4 mg / L) 2-mercaptoethanol; and / or cholesterol lipid concentrate (about 200X to about 300X, preferably about 250X, ThermoFisher Scientific). In a particularly preferred embodiment, the basal medium (such as Iscove's Modified Dulbecco's Medium (IMDM)) comprises 5% human serum albumin, 10 mg / L ethanolamine, 5 μg / L sodium selenite, 50 μg / L Cortisol, 20 μg / LD, 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-acetyl-L cysteine, 4 mg / L 2-mercaptoethanol, and cholesterol lipid concentrate (250X, ThermoFisher Scientific).

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

[0085] 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, the isolated hematopoietic stem cells comprising CD34+ hematopoietic stem cells; a culture medium comprising CD34+ hematopoietic stem cells; a blood sample; and / or a bone marrow sample. In a preferred embodiment, the blood sample comprises CD34+ hematopoietic stem cells. In an alternative preferred embodiment, the bone marrow sample comprises CD34+ hematopoietic stem cells.

[0086] In one embodiment, wherein the sample is a culture medium comprising CD34+ hematopoietic stem cells, step ii) comprises adding an estrogen receptor agonist to the culture medium.

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

[0088] The blood samples described herein are well known to medical and / or hematological technicians, who know how to determine and obtain such samples. The blood sample can be obtained from the subject, or can be obtained from a commercial source. For example, umbilical cord blood can be obtained from Zen Bio, USA. It is found that peripheral blood circulates in the subject and is not isolated in, for example, the lymphatic system, spleen, liver or bone marrow. Therefore, by so-called "peripheral blood", we include blood obtained from or available from blood circulating in the subject. Exemplary methods for obtaining peripheral blood include venipuncture and / or lymphocyte apheresis. Mobilized peripheral blood is peripheral blood that has been treated with so-called "mobilizing agents", which are agents that increase the number of stem cells (particularly HSC) in peripheral blood. Examples of mobilizing agents are granulocyte colony stimulating factor and Plerixafor. Mobilizing agents are provided to the subject before peripheral blood is obtained. Therefore, by so-called "mobilized peripheral blood", we include blood obtained from or available from blood circulating in the subject, wherein a mobilizing agent is first provided and / or applied to the subject. Umbilical cord blood is blood obtained from the umbilical cord, and placental blood is obtained from the placenta, and is generally a rich source of stem cells and, in particular, HSCs. These blood samples are generally collected shortly after the baby is born. Thus, by "umbilical cord blood," we include a blood sample obtained or obtainable from the umbilical cord. Thus, by "placental blood," we include a blood sample obtained or obtainable from the placenta.

[0089] Bone marrow samples are well known to those skilled in medicine and / or hematology who know how to determine and obtain such samples. Bone marrow is tissue found in certain bones in a subject's body and is a rich source of stem cells in general and, in particular, HSCs. One example of how bone marrow may be obtained is via bone marrow aspiration, which involves inserting a hollow needle through the bone into the marrow, which may then be withdrawn into an attached syringe. Bone marrow may also be obtained from commercial sources. By "bone marrow sample" we include a sample obtained or obtainable from the bone marrow.

[0090] As described herein, preferred blood samples are umbilical cord blood and / or placental blood. Such blood samples can be obtained by any procedure known in the art. There are several advantages to using umbilical cord blood / placental blood as a source of stem cells, including that blood samples can be easily obtained without causing any trauma to the donor. For example, for an overview of umbilical cord blood and placental blood collection at birth, see US Patent No. 5,004,681.

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

[0092] Thus, in one embodiment, 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.

[0093] The anticoagulant used in the present invention can be any anticoagulant 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).

[0094] In one embodiment, the anticoagulant is one or more anticoagulants 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, NY 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 biscoumarate.

[0095] In one embodiment, the sample (such as a blood sample) contains no contamination, such as contamination with an infectious agent.

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

[0097] In one embodiment, the method of the invention (preferably before step i)) comprises a step wherein the sample is tested for contamination, such as contamination with an infectious agent.

[0098] Tests for contamination will be known to those skilled in the art, particularly those skilled in microbiology.

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

[0100] By "bacterial culture" we include growing bacteria under both aerobic and anaerobic conditions.

[0101] By "pathogen screening," we include various diagnostic tests that can be performed to ensure the absence of specific pathogens that are transmissible through the blood. For example, a sample (e.g., a 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 viruses I and II (HTLV-I and HTLV-II), West Nile virus, cytomegalovirus, and syphilis.

[0102] In particular, where the blood sample is cord blood and / or placental blood, prior to blood collection, the maternal health history may be assessed to determine the risk of transmitting infectious or genetic diseases such as leukemia, immune disorders, cancer, hepatitis, neurological disorders, viral diseases, or AIDS. The collected blood sample may be screened for one or more of total nucleated cell count, cell viability, CD34+ cell count, HLA typing, and ABO / Rh typing.

[0103] As will be appreciated, if the sample contains contaminants, it cannot be used in the method of the invention and / or the method will include a step to remove the contaminants (preferably prior to step i)).

[0104] As described herein, after collecting a sample (such as a blood sample; in particular umbilical cord blood and / or placental blood), the sample can be processed to enrich for hematopoietic stem / progenitor cells (in particular CD34+ hematopoietic stem cells).

[0105] In one embodiment, the estrogen receptor agonist is one or more molecules selected from the list consisting of: endoestrogen; phytoestrogen; steroidal ligand; tamoxifen analog; synthetic estrogen; xenoestrogen; and / or non-steroidal ligand.

[0106] Endogenous estrogens are molecules that can bind to and preferably activate estrogen receptors and are produced endogenously by a subject, such as a human subject.

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

[0108] Estrogens 1-4 described herein are all steroid hormones derived from cholesterol metabolism 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.

[0109] In general, estrogen is a hormone derived from cholesterol metabolism through a series of catalytic reactions involving a variety of enzymes, including cytochrome P450 aromatase (CYP19A1), whose nucleic acid and amino acid sequences are known. Further information about the chemical structure of estrogen can be found 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. doi: 10.2174 / 138955708784223503. PMID: 18473934.

[0110] Phytoestrogens are molecules that can bind to and preferentially activate estrogen receptors and are non-steroidal polyphenolic compounds produced naturally by plants.

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

[0112] Phytoestrogens, also known as "dietary estrogens", are found in certain edible plants, including but not limited to soybeans, flaxseeds, sesame, licorice, hops and ginseng, with soybeans (isoflavonoids) being the most abundant. Phytoestrogens are similar to endogenous estrogens and exhibit both genomic and non-genomic effects of phytoestrogens, including selective but weak binding to 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.

[0113] Steroidal ligands are molecules that can bind to and preferably activate estrogen receptors and are steroidal molecules.

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

[0115] Tamoxifen analogs are molecules that can bind to and preferentially activate the estrogen receptor and are either tamoxifen or are structurally and / or functionally similar to tamoxifen.

[0116] In one embodiment, the tamoxifen analog is one or more molecules selected from the list consisting of: 4-hydroxytamoxifen (4-HT); tamoxifen; and / or raloxifene; and / or toremifene. Tamoxifen, raloxifene and toremifene 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).

[0117] Synthetic estrogen is a molecule that can bind and preferably activate estrogen receptors, and is a chemically modified form of the naturally occurring hormone estradiol. Synthetic estrogen is used in various medical applications, such as hormone replacement therapy (HRT) and oral contraceptives, and it has been shown that synthetic estrogen relieves menopausal symptoms, prevents osteoporosis and treats certain reproductive diseases (Lobo (2017) Nature Reviews Endocrinology, 13: 220-231). In one embodiment, synthetic estrogen is one or more molecules selected from the list consisting of: ethinyl estradiol; mestranol; combined equine estrogen.

[0118] In one embodiment, the estrogen receptor agonist is a xenoestrogens. Xenoestrogens are synthetic or naturally occurring compounds that mimic the effects of endogenous estrogens and 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).

[0119] Nonsteroidal ligands are molecules that can bind to and preferably activate estrogen receptors and are nonsteroidal molecules.

[0120] In one embodiment, the steroidal ligand is one or more molecules selected from the list consisting of: benzopyran derivatives (e.g., 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)chroman-8-ol ((3aS,4R,9bR)-2,2-Difluoro-4-(4-hydroxyphenyl)-3,3a,4,9 b-tetrahydro-1H-cyclopenta(c)chromen-8-ol); diarylpropionitrile (DPN) and / or propylpyrazoletriol (PPT)) 2,3-diarylisoquinolinone derivatives; diphenylmethane skeleton derivatives; diphenylacetophenone analogs; hydrazide derivatives; fluorene-3-one derivatives; triphenylethylene-coumarin hybrid derivatives; fulvestrant; 1,1,2-triarylolefine derivatives; 17α-(carboryl)estradiol; and / or fluorinated carboarylphenol. LY500307 is a synthetic ligand that selectively binds to and activates the estrogen receptor β (ERβ) subtype.

[0121] LY500307 has neuroprotective effects and reduces the accumulation of amyloid-β, 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 a rat model of traumatic brain injury (Barret et al. (2009), European Journal of Neuroscience, 29: 1997-2006).

[0122] LY3201 is a synthetic ligand that selectively binds and activates the estrogen receptor β (ERβ) subtype. Activation of ERβ has been associated with 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).

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

[0124] DPN is a synthetic ligand that selectively binds and activates the estrogen receptor β (ERβ) subtype.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).

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

[0126] The terms "nuclear estrogen receptor" and "membrane-bound estrogen receptor" are known to those skilled in the art of cell biology, who will be able to identify such receptors, as well as molecules that can act as agonists thereof. In general, nuclear estrogen receptors are intracellular receptors that can be bound by estrogen and preferably activated by estrogen. When activated by an estrogen receptor agonist, the nuclear estrogen receptor translocates to the nucleus, where it regulates gene activity. Therefore, by so-called "nuclear estrogen receptor agonists", we include molecules that can bind to and preferably activate nuclear estrogen receptors, which are intracellular estrogen receptors. In general, membrane-bound estrogen receptors are estrogen receptors located or embedded in the cell membrane, which can be bound by estrogen and preferably activated by estrogen. Therefore, by so-called "membrane-bound estrogen receptor agonists", we include molecules that can bind to and preferably activate membrane-bound estrogen receptors, which are estrogen receptors located or embedded in the cell membrane. The common type of membrane-bound estrogen receptor is the estrogen G protein-coupled receptor. Specific examples of membrane-bound estrogen receptors are GPRC6A (also known as G protein-coupled receptor family group C 6 member A, which in human subjects is encoded by the GPRC6A gene), ER-X and / or Gq-mER (also known as Gq-coupled membrane estrogen receptor).

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

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

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

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

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

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

[0133] In one embodiment, the estrogen receptor agonist is present at a low dose / concentration, such as those described below. The inventors surprisingly found that low doses / concentrations of estrogen receptor agonists lead to surprisingly high levels of CD34+ hematopoietic stem cell expansion; in particular, expansion of CD34+CD133+ hematopoietic stem cells with sustained stemness.

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

[0135] In further embodiments, 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 about 12nM or more; about 13nM or more; about 14nM or more; about 15nM or more; about 16nM or more; about 17nM or more; about 18nM or more; about 19nM or more; about 20nM or more; about 21nM or more; about 22nM or more; about 23nM or more; about 24nM or more; about 25nM or more; about 26nM or more; about 27nM or more; about 28nM or more; about 29nM or more; or about 30nM or more.

[0136] 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 3000 nM or more; about 4,000 nM or more; about 5,000 nM or more; about 6,000 nM or more; about 7,000 nM or more; about 8,000 nM or more; about 9,000 nM or more; or about 10,000 nM (10 μM).

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

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

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

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

[0141] The separation and / or enrichment of CD34+ hematopoietic stem cells can be performed using flow cytometry using monoclonal antibodies conjugated to fluorescent dyes and / or immunomagnetic cell separation using monoclonal antibodies conjugated to magnetic beads and / or immunopanning and / or affinity chromatography using antibodies attached to a solid matrix of a culture plate or culture bag, wherein the separation method does not excessively damage the cells, and the method should also maximize the viability of the separated cells. The specific separation technique will depend on the availability of equipment, the efficiency of the separation, the ease of use of the method, the time it takes to separate the cells, and the destructive effect (if any) of the method on the isolated cell population.

[0142] Preferably, the separation and / or enrichment of CD34+ hematopoietic stem cells is obtained by using a magnetic bead-conjugated CD34 monoclonal antibody and a magnetic cell separation device. In another embodiment, as described above, a subpopulation of CD34+ hematopoietic stem cells can be separated by retaining a positive population using one or more magnetic bead-conjugated monoclonal antibodies of other surface markers (e.g., CD45, CD133) alone or in combination. In order to separate a subpopulation of CD34+ cells lacking CD38, the separated cells can be further subjected to a magnetic bead-conjugated CD38 monoclonal antibody, and a negative population is retained.

[0143] Thus, in one embodiment, the step of enriching and / or isolating comprises one or more methods 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.

[0144] In another embodiment, the sample is a previously 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.

[0145] In a preferred embodiment, magnetic beads conjugated to anti-CD34 antibodies and a magnetic cell separator are used; for example, MiniMACS or MidiMACS or OctoMACS or Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany) is used to process cryopreserved (such as cryopreserved in vaporized nitrogen) umbilical cord blood samples to separate / enrich CD34+ hematopoietic stem cells. The method utilizes nanoscale superparamagnetic particles composed of iron oxide and dextran coupled to specific monoclonal antibodies.

[0146] In another embodiment, the method may include freezing the umbilical cord blood sample, wherein the method further includes the step of thawing the umbilical cord blood sample (preferably before step i)). Thus, the frozen umbilical cord blood sample may be provided in a sample bag, wherein an exemplary method of thawing includes removing the sample bag from the vapor nitrogen storage and inspecting any damage in the bag. The sample bag is quickly placed in a sterile plastic zipper bag and rapidly thawed in a 37°C water bath within 5 minutes. Alternatively, any thawing method known in the art may be utilized, including but not limited to a blood thawing system, a plasma thawing system, a microcontroller-based thawing system, a dry rapid plasma thawing device, a ThawStar frozen bag thawing system. After thawing the sample, gently knead the bag and remove it from the zipper bag, and disinfect the infusion port with a 70% alcohol wipe or any disinfectant known in the art (such as 70% isopropyl alcohol, desidos, 2% chlorhexidine and / or betadine). The blood is drawn into a 50 ml sterile tube using a needle connector (e.g., a Braun blood needle adapter) of appropriate size according to the blood bag. The thawed blood sample is diluted with a thawing solution made of any volume expander solution of crystal or colloid 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 substitutes and / or knockout serum substitutes. Preferably, human serum albumin is added to the thawing solution. Alternatively, a knockout serum substitute that does not contain xenobiotics can be added to the thawing solution. Preferably, based on existing information known in the art, an isotonic thawing solution of 10% dextran-40 and 5% human serum albumin is used.

[0147] A thawed blood sample can be diluted with one or more portions of any of the above thawing solutions, for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. Preferably, the thawed blood is first diluted 1:1 with isotonic 10% dextran-40 and 5% human serum albumin, and then diluted 1:3 by adding another two volumes of the same solution, and then diluted 1:5 by adding another two volumes of the same solution. After each addition of the thawing solution at each dilution step, the sample is stabilized for 15 minutes. The sample can be stabilized for any length of time known in the art after dilution with the thawing solution, at each dilution step or immediately after the final dilution, such as 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 specific embodiment, the sample can be stabilized for about 1 minute to about 1 hour at each dilution step or immediately after the final dilution after dilution with a thawing solution. In a specific embodiment, the sample can be stabilized for no more than 1 hour at each dilution step or immediately after the final dilution after dilution with a thawing solution. In a preferred embodiment, the sample stabilizes for 15 minutes after each dilution step. The sample obtained via this exemplary thawing method can be used for separation and / or enrichment of hematopoietic stem cells.

[0148] In a specific embodiment, the method includes enriching and / or isolating CD34+ hematopoietic stem cells by retaining a CD34-positive hematopoietic stem cell population (e.g., using an anti-CD34 antibody; preferably an anti-CD34 antibody conjugated to magnetic beads); optionally, further comprising a step of separating CD38- hematopoietic stem cells from CD34+ hematopoietic stem cells (e.g., using an anti-CD38 antibody; preferably an anti-CD38 antibody conjugated to magnetic beads, wherein cells that do not bind to the anti-CD38 antibody form a CD38-subpopulation of CD34+ hematopoietic stem cells). Similarly, the CD133+ subpopulation of CD34+ hematopoietic stem cells can be isolated; for example, using an anti-CD133 antibody conjugated to magnetic beads, wherein CD133+ cells are retained. In addition, cells expressing CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD166, HLADR, or a combination of one or more markers can be selected using antigen-specific antibodies. Preferably, CD34+CD38- hematopoietic stem cells are enriched and / or isolated.

[0149] In one embodiment, one or more suitable samples (such as blood samples; in particular umbilical cord blood and / or placental blood) can be combined before hematopoietic stem cell enrichment and / or separation (preferably before step i)). In another embodiment, donors from the same race (race) (e.g., African Americans, Caucasians, Asians, Hispanics, Native Americans, Australian Aborigines, Inuit, Pacific Islanders) and / or donors from the same ethnic origin (ethnicity) (e.g., Irish, Italians, Indians, Japanese, Chinese, Russians, etc.) can be combined. As known in the art, the selection of suitable umbilical cord blood samples for merging can be based on partial or complete HLA matching.

[0150] In one embodiment, wherein the sample is a blood sample, it can remove red blood cells (RBC) and / or plasma; For example, before separation / enrichment of hematopoietic stem cells and / or before cryopreservation and / or after the cryopreserved blood sample is thawed. In a further embodiment, after the RBC is eliminated, the white blood cell fraction (WBC) can be separated; For example, for the separation and / or enrichment of hematopoietic stem cells. In another embodiment, the thawed and diluted blood sample can be centrifuged at any g force or relative centrifugal force (RCF) (e.g., 400xg, 450xg, 500xg, 550xg, 600xg, 650xg, 700xg, 750xg, 800xg or more). Preferably, the g force or relative centrifugal force (RCF) used is 650xg. The supernatant with plasma (thawing solution) is removed and the obtained cell pellet is reconstituted to the original pre-thawing sample volume with any isotonic solution known in the art, which may include but is 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 to reconstitute the cell pellet.

[0151] Depletion of RBCs can be performed by separating the leukocyte and erythrocyte fractions by any method known in the art, including but not limited to centrifugation techniques or the use of density gradient media, such as FICOLL TM 、FICOLL-PAQUE TM PERCOLL TM (GE Healthcare, Piscataway, New Jersey), LYMPHOPREP TM(Alere Technologies) combined with centrifugation or the use of red blood cell aggregating agents, such as 6% hydroxyethyl starch (HES), Hespan (B. Braun Medical, Inc.), Hextend (Hospira, Lake Forest, IL), Hetasep TM (Stemcell Technologies Inc) or use RBC-specific antibodies conjugated to magnetic beads and select with a magnetic device such as EasySep (Stemcell Technologies Inc). TM For red blood cell (RBC) aggregation and sedimentation of fresh blood. However, LYMPHOPREP TM As a density gradient medium combined with centrifugation for use with thawed blood. TM The cord blood sample was carefully layered on the bottom of a sterile conical centrifuge tube. TM Alternatively, special separation tubes such as SEPMATE TM (Stemcell Technologies Inc) can be used with LYMPHOPREP TM Used together to facilitate operation and shorten centrifugation time. Preferably, SEPMATE TM The tube is used for density gradient based centrifugation. After centrifugation, multiple layers are formed in the tube, which can be considered from top to bottom as plasma with electrolytes and proteins, buffy coat (i.e., a dense and opaque band of mononuclear cells containing white blood cells), and finally the LYMPHOPREP TM The thick granules of red blood cells (RBC) and granulocytes in the tube were removed. The volume of the uppermost layer was reduced by 4-5 ml by carefully aspirating the plasma, which also helped to reduce platelets. Finally, the upper layer and the buffy coat contents of the SEPMATE tube were poured into another conical tube and centrifuged to obtain a cell pellet, which was further washed with Dulbecco's modified phosphate buffered saline (DPBS) supplemented with 1% human serum albumin and resuspended in the same solution.

[0152] Optionally, a small representative aliquot of fresh or thawed blood can be examined for total nucleated cell count, cell viability, and CD34+ frequency / content; for example, prior to CD34+ cell isolation / enrichment.

[0153] In one embodiment, all or half of the volume of fresh blood can be processed for CD34+ or CD34+CD38- or CD34+CD133+ cell separation, and the CD34+ positive enriched hematopoietic stem cell fraction and the CD34- cell fraction can be recovered. When half of the fresh blood is used, the CD34- cell portion can be mixed with the uncontacted half of the fresh cord blood, which is used for immediate transplantation or cryopreservation.

[0154] In another embodiment, the CD34+ or CD34+CD38- or CD34+CD133+ hematopoietic stem cell enriched fraction from a fresh or thawed whole blood sample can be processed continuously prior to expansion, such as the hematopoietic stem cells can be resuspended in a suitable cell growth medium or cell culture medium for transport or storage.

[0155] 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 basic Iscove's Modified Dulbecco's Medium (IMDM) or basic Roswell Park Memorial Institute Medium (RPMI1640) or basic Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) or STEMSPAN TM Serum Free Expansion Medium (StemCell Technologies, Vancouver, British Columbia) or StemMACS HSC Expansion Media (Miltenyi Biotec GmbH, Germany), or Gibco TM StemPro TM-34 SFM (ThermoFisher Scientific). By using one of the basal media, it is necessary to supplement with fetal bovine serum (FBS) or knockout serum supplement (ThermoFisher Scientific) or human serum albumin. In addition, these basal media can also be supplemented with several other nutrients, lipids, proteins and antioxidants to make them serum-free media, such as those known in the prior art, including but not limited to ethanolamine, sodium selenite, cortisol, D, L-tocopherol, human transferrin (holo), human insulin (zinc), N-acetyl-L-cysteine, 2-mercaptoethanol, monothioglycerol, L-ascorbic acid-2-phosphate magnesium. By using one of the basal medium or serum-free medium, it is necessary to supplement with one or more hematopoietic stem cell growth factors and cytokines known in the 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 medium 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, 5 μg / L sodium selenite, 50 μg / L cortisol, 20 μg / LD, L-tocopherol, 100 mg / L human transferrin (holo), 10 mg / L human insulin (zinc), 160 mg / L N-acetyl-L-cysteine, 4 mg / L 2-mercaptoethanol and cholesterol lipid concentrate (250X, ThermoFisherScientific).In addition, one or more of the following growth factors and cytokines are added to the above culture medium: 10 ng / mL recombinant human interleukin-3 (rhIL-3), 100 ng / mL recombinant human interleukin-6 (rhIL-6), 100 ng / mL recombinant human thrombopoietin (rhTPO), 100 ng / mL recombinant human Flt-3 ligand (rhFlt-3L), 100 ng / mL recombinant human stem cell factor or KIT ligand or steel factor (rhSCF), 10 ng / mL recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF or GMCSF), and / or 10 ng / mL recombinant human granulocyte colony stimulating factor (rhG-CSF or GCSF); and optionally 100 ng / mL recombinant human insulin-like growth factor (IGF) binding protein 2 (rhIGFBP2) and / or 50 ng / mL recombinant human soluble DLL-1 (rhsDLL-1).

[0156] In another embodiment, the step of isolating and / or enriching CD34+ hematopoietic stem cells comprises the step of isolating CD34+ hematopoietic stem cells from the expanded stem cells of step ii) (preferably after step ii) and / or during step ii)). In a specific embodiment, the step of isolating and / or enriching CD34+ hematopoietic stem cells from the expanded sample of step ii) comprises isolating CD34+ hematopoietic stem cell subpopulations (e.g., CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ (低 / 中) In a specific embodiment, the step of isolating and / or enriching CD34+ hematopoietic stem cells from the amplified sample of step ii) comprises isolating CD34+ hematopoietic stem cell subpopulations (e.g., CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ (低 / 中) CD133+CD38- hematopoietic stem cells).

[0157] In a specific embodiment, the step of isolating and / or enriching CD34+ hematopoietic stem cells comprises the steps of isolating CD34+ hematopoietic stem cells from the sample and isolating CD34+ hematopoietic stem cells from the expanded stem cells of step ii).

[0158] By "isolating CD34+ hematopoietic stem cells", we include separating CD34+ hematopoietic stem cells from other cells and / or culture medium and / or components; for example, other cells and / or culture medium and / or components in a sample. The resulting isolated CD34+ hematopoietic stem cells can produce a solution consisting essentially of CD34+ hematopoietic stem cells, or a solution in which the only cells present are CD34+ hematopoietic stem cells.

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

[0160] In one embodiment, step ii) is performed 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 longer; 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 In certain embodiments, step ii) is performed for a period of about 8 days or more; for example, about 14 days or more; or about 21 days or more; or about 22 days or more; or about 23 days or more; or about 24 days or more; or 25 days or more. In certain embodiments, step ii) is performed for a period of about 8 days or more; for example, about 14 days or more; or about 21 days or more. In preferred embodiments, step ii) is performed for a period of about 7 days or more. In preferred embodiments, step ii) is performed for a period of about 8 days or more. In preferred embodiments, step ii) is performed for a period of about 14 days or more. In preferred embodiments, step ii) is performed for a period of about 21 days or more, most preferably for a period of about 21 days.

[0161] In one embodiment, step ii) is performed 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 performed for a period of about seven days or less. In a preferred embodiment, step ii) is performed for a period of about eight days or less. In a preferred embodiment, step ii) is performed for a period of about 14 days or less. In a preferred embodiment, step ii) is performed for a period of about 21 days or less.

[0162] In one embodiment, step ii) is performed over 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 performed over a period of about seven days to about 21 days.

[0163] In one embodiment, during step ii) of the method of the invention, the culture medium comprising one or more estrogen receptor agonists (and optionally one or more growth factors and / or cytokines) may be replaced or added at least once with a comparable (or identical) culture medium comprising one or more estrogen receptor agonists (and optionally one or more growth factors and / or cytokines); for example: replacing the culture medium or adding the substance thereto at least twice; replacing the culture medium or adding the substance thereto at least three times; replacing the culture medium or adding the substance thereto at least four times; replacing the culture medium or adding the substance thereto at least five times; and / or replacing the culture medium or adding the substance thereto at least six times. Preferably, about half the volume of the culture medium is replaced with an equal amount of culture medium or about half the volume of the culture medium is added. In a specific embodiment, the volume of the culture medium replaced or added is from about 50 ml to about 100 ml, preferably about 70 ml.

[0164] In a particular embodiment wherein step ii) is performed for a period of about 21 days, the culture medium comprising one or more estrogen receptor agonists (and optionally one or more growth factors and / or cytokines) is replaced or substances are added thereto on days 4, 8, 11, 14, 17 and 19, preferably on day 7 and / or day 14.

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

[0166] In one embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), there is the following ratio: about two to about 40 CD34+ hematopoietic stem cells to one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+) of the CD34+ hematopoietic stem cell subset. (低 / 中) CD133+CD38- hematopoietic stem cells); for example, 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 are less than one CD34+ hematopoietic stem cell (preferably and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ (低 / 中 )CD133+CD38- hematopoietic stem cells). In a preferred embodiment, among the expanded CD34+ hematopoietic stem cells in step ii), there are one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+) of the CD34+ hematopoietic stem cell subset for every about two to about 20 CD34+ hematopoietic stem cells. (低 / 中) In a further preferred embodiment, among the expanded CD34+ hematopoietic stem cells in step ii), there are about four CD34+ hematopoietic stem cells to one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ (低 / 中) The ratio of CD133+CD38- hematopoietic stem cells).

[0167] In an alternative embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), there are one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+) of the CD34+ hematopoietic stem cell subset for every about 15 CD34+ hematopoietic stem cells. (低 / 中) The ratio of CD133+CD38- hematopoietic stem cells).

[0168] In an alternative embodiment, in the expanded CD34+ hematopoietic stem cells of step ii), there are one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+) of the CD34+ hematopoietic stem cell subset for every about 20 CD34+ hematopoietic stem cells. (低 / 中) The ratio of CD133+CD38- hematopoietic stem cells).

[0169] Those skilled in the art of cell biology will appreciate that the ratio of CD34+ hematopoietic stem cells to a specific CD34+ hematopoietic stem cell subset can be further determined by reference to the time period during which step ii) of the present invention has been performed. Thus, the ratios described herein can each be combined with a time period during which step ii) is performed as described herein. Those skilled in the art of cell biology will also appreciate that as the time period increases, the HSC population at a higher starting ratio will expand at a faster rate than the HSC population at a lower starting ratio, and therefore the ratio can increase over time.

[0170] In a further preferred embodiment, in the expanded CD34+ hematopoietic stem cells in step ii), there is one CD34+ hematopoietic stem cell (preferably CD34+CD133+ hematopoietic stem cell and / or CD34+CD133+CD38- hematopoietic stem cell and / or CD34+CD45+) per about four CD34+ hematopoietic stem cells to one CD34+ hematopoietic stem cell subset. (低 / 中) In an alternative further preferred embodiment, among the expanded CD34+ hematopoietic stem cells in step ii), there are about 15 CD34+ hematopoietic stem cells to one CD34+ hematopoietic stem cell (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ hematopoietic stem cells) of the CD34+ hematopoietic stem cell subset. (低 / 中) In an alternative further preferred embodiment, among the expanded CD34+ hematopoietic stem cells in step ii), there are about 20 CD34+ hematopoietic stem cells to one CD34+ hematopoietic stem cell (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ hematopoietic stem cells) of the CD34+ hematopoietic stem cell subset. (低 / 中) CD133+CD38- hematopoietic stem cells), wherein step ii) has been performed for a period of about 21 days.

[0171] In one embodiment, the number of expanded CD34+ hematopoietic stem cells in step ii) increases by about 88 times or more; for example, about 90 times or more; about 95 times or more; about 100 times or more; about 110 times or more; about 120 times or more; about 130 times or more; about 140 times or more; about 150 times or more; about 160 times or more; about 170 times or more; about 180 times or more; about 190 times or more; about 200 times or more; about 300 times or more; about 400 times or more; about 500 times or more; about 600 times or more; about 700 times or more or more; about 800-fold or more; about 900-fold or more; about 1,000-fold or more; about 1,100-fold or more; about 1,200-fold or more; about 1,300-fold or more; about 1,400-fold or more; about 1,500-fold or more; about 1,600-fold or more; about 1,700-fold or more; about 1,800-fold or more; about 1,900-fold or more; about 2,000-fold or more; about 2,100-fold or more; about 2,200-fold or more; about 2,300-fold or more; about 2,400-fold or more; or about 2,500-fold.

[0172] Those skilled in cell biology will appreciate that the fold change of CD34+ hematopoietic stem cells can be further determined by reference to the period during which step ii) of the invention has been performed. Thus, the fold changes described herein can each be combined with a period during which step ii) is also performed as described herein.

[0173] In one embodiment, the number of CD34+ hematopoietic stem cells (particularly long-term repopulating hematopoietic stem cells) expanded in step ii) increases by about 90 times or more, wherein step ii) has been performed for a period of about 8 days. In a further embodiment, the number of CD34+ hematopoietic stem cells expanded in step ii) increases by about 1,500 times or more, wherein step ii) has been performed for a period of about 21 days.

[0174] In one embodiment, the number of 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.

[0175] Those skilled in cell biology will appreciate that the percentage of CD34+ hematopoietic stem cells can be further determined by reference to the period during which step ii) of the invention has been performed. Thus, the percentages described herein can each be combined with the period during which step ii) is performed as described herein.

[0176] The quality, purity and / or success of CD34+ hematopoietic stem cell expansion can be assessed by a skilled cell biologist.

[0177] In one embodiment, the total number of cells, viable cells, viable CD34+ hematopoietic stem cells, viable CD34+CD38- hematopoietic stem cells and / or CD34+CD133+ hematopoietic stem cells are analyzed after amplifying hematopoietic stem cells to determine the effectiveness of the amplified cell population; for example, to provide a hematopoietic effect. In another embodiment, as described above, viable cells can be additionally and / or alternatively assessed during step ii) of the present invention. In an exemplary embodiment, representative samples can be taken from the amplified culture at days 7, 14, and 21 during step ii) for quantification of total viable nucleated cells and total CD34+ cells that can be enumerated (optionally using flow cytometry), thereby providing the percentage of CD34+ hematopoietic stem cells / progenitor cells. Similarly, as further described herein, before or after cryopreservation or thawing, a representative small volume of amplified hematopoietic stem cells can be taken for counting total nucleated cells, and the percentage of CD34+ cells.

[0178] In a specific embodiment, the 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 excluding trypan blue or 7-AAD. The percentage of live CD34+ hematopoietic stem cells can be determined by using dyes excluded from viable cells in flow cytometry. The relative increase of viable CD34+ cells can be assessed as the total number of viable CD34+ cells after amplification / the total number of viable CD34+ cells seeded at the beginning of amplification. This ratio is generally referred to as multiple amplification. As is apparent from methods known in the art, the CD34 antigen alone or in combination with other antigens can be used for such analysis.

[0179] As discussed herein, the inventors have identified that their method of expanding CD34+ hematopoietic stem cells results in a higher quality population of CD34+ hematopoietic stem cells and a higher amount of CD34+ hematopoietic stem cells when compared to prior art methods. This is therapeutically relevant because several studies have shown that total nucleated cells and CD34+ cell doses used in therapy (e.g., transplants) are positively correlated with better outcomes (e.g., better platelet and neutrophil engraftment, reduced incidence of graft failure and graft-versus-host disease after cord blood transplantation). Possible examples of how to assess quality are the results of transplantation and / or implantation into a subject-the subject can be a human, or more preferably an animal model. Animal models for assessing the purity of hematopoietic stem cells are known and include immunodeficient humanized mice (where species-specific immune cells are absent). In such animal models, amplified human hematopoietic stem cells are serially transplanted at different doses, and immune reconstitution is assessed by flow cytometry of blood and / or bone marrow. An alternative method to assess the quality, purity and / or success of CD34+ hematopoietic stem cell expansion is to investigate the percentage of CD34+ hematopoietic stem cells (or CD34+ hematopoietic stem cell subpopulation) compared to the total number of cells obtained in step ii) of the method of the present invention.

[0180] The effectiveness of the hematopoietic stem cells of amplification can also be evaluated by their ability to differentiate into myeloid and lymphoid lineages. Colony forming unit (CFU) assay is a method of this type, which is used to study its proliferation and differentiation potential by the ability of hematopoietic stem cells / progenitor cells to form colonies in semisolid culture media. The number and morphology of colonies produced by a fixed number of hematopoietic stem cells / progenitor cells can provide information about the effectiveness of amplified cell populations. The relatively high CFU number of the input hematopoietic stem cells from a fixed number is considered to indicate better implantation potential.

[0181] In one embodiment, the differentiation ability of the expanded CD34+ hematopoietic stem cells of step ii) is substantially the same as the differentiation ability of one or more CD34+ hematopoietic stem cells in the sample. In an alternative embodiment, the ability of the expanded CD34+ hematopoietic stem cells of step ii) to be successfully transplanted and / or engrafted into a subject is substantially the same as the differentiation ability of the CD34+ hematopoietic stem cells in the sample.

[0182] In one embodiment, when compared to the total number of cells obtained in step ii) of the present invention, the percentage of CD34+ hematopoietic stem cells 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.

[0183] In some embodiments, other cell types included in the total number of cells obtained in 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 the expression of one or more mature cell markers including, but not limited to, CD4; CD7; CD8; CD14; CD15; CD16; CD56.

[0184] Testing the function of the CD34+ hematopoietic stem cells obtained in step ii) of the present invention can be achieved using an animal model such as a mouse model. Examples of such mouse models are described in Ishikawa et al., Development of functional human blood and immune systems in NOD / SCID / IL2 receptor{gamma}chain(null)mice.Blood.2005Sep 1;106(5):1565-73.

[0185] In one embodiment, the method is an ex vivo method and / or an in vitro method.

[0186] 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 a human subject.

[0187] In one method, the method further comprises the following step: iii) differentiating the expanded CD34+ hematopoietic stem cells of step ii) into one or more cells derived from CD34+ hematopoietic stem cells. In a specific embodiment, the method does not include the step of administering the one or more cells derived from CD34+ hematopoietic stem cells of step iii) to a subject in need thereof, preferably a human subject.

[0188] In one embodiment, the one or more cells derived from CD34+ hematopoietic stem cells are cells selected from the list consisting of: myeloid progenitor cells; lymphoid progenitor cells; erythroid cells; erythrocytes; mast cells; megakaryocytes; platelet cells; mast cells; myeloblasts; basophils; neutrophils; eosinophils; monocytes; macrophages; dendritic cells; natural killer cells; T lymphocytes; and / or B lymphocytes.

[0189] In an additional form of the first aspect of the invention, step ii) of the method of the first aspect of the invention may comprise culturing the cells of step i) in the presence of an agent capable of increasing estrogen receptor expression in addition to or in place of one or more estrogen receptor agonists, and / or an agent capable of increasing downstream effectors of the estrogen receptor signaling pathway.

[0190] The expanded CD34+ hematopoietic stem cells obtained by the methods described herein and / or one or more cells derived from CD34+ hematopoietic stem cells obtained by the methods described herein can be cryopreserved or frozen, in particular, for subsequent transplantation / infusion into a human patient.

[0191] Thus, the methods described herein may comprise the step of cryopreserving or cryo-expanding CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells (preferably after step ii)).

[0192] In one embodiment, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells can be distributed in one or more bags or cryogenic tubes or units and kept frozen in liquid nitrogen at ultra-low temperatures. In another embodiment, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells can be divided into several units and cryopreserved.

[0193] Cryopreservation can be any method known in the art that is capable of freezing cells in a live / viable state at ultra-low temperatures. Generally, cell freezing is harmful because water within the cell forms ice crystals upon cooling, and osmotic damage to the cell membrane is induced, along with dehydration and increased solute concentrations. For a detailed discussion, please refer to Mazur, P., 1977, Cryobiology 14: 251-272, which is incorporated herein by reference.

[0194] Typically, these deleterious effects can be avoided by using cryoprotectants, controlling the freezing rate / speed and storing the frozen cells at ultra-low temperatures to minimize further damage. Therefore, in one embodiment, cryopreservation comprises contacting the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells with one or more cryoprotectants.

[0195] Cryoprotectants that can be used include, but are not limited to, polyvinyl pyrrolidine (Rinfret, 1960, Ann. NY Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548), glycerol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157), methanol, acetamide, monoacetin (Lovelock, 1954, Biochem. J. 56:265), D-sorbitol, i-inositol, 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, low concentrations of non-toxic DMSO are used as cryoprotectants, which easily penetrate cell membranes and modify the freezing pattern of water molecules and protect intracellular organelles by preventing damage to intracellular organelles from ice crystal formation. In addition, supplementing the cryoprotective medium with serum albumin or plasma (e.g., concentrations up to 20-40%) can further enhance the cryoprotective effect of DMSO.

[0196] After adding the cryoprotectant medium, in one embodiment, the cells can be placed at 0°C until frozen, as DMSO concentrations of 1% or higher are toxic above 4°C. Controlled slow cooling of the cells is critical, and this process can be performed in an insulated box that ensures a controlled rate of freezing of -1°C / min, or using a programmed freezing device.

[0197] The rate controlled freezer can be an alcohol-free cell freezing container with a highly insulating outer material to control the rate of heat removal and provide repeatable cell cryopreservation. Rate controlled freezers such as CoolCell (Corning Inc, Arizona, USA) provide repeatable cell cryopreservation. For example, for most stem cells in a cryoprotectant medium containing 10% DMSO and 25% serum albumin, the optimal cooling rate from 0°C to -80°C is -1°C to -3°C per minute.

[0198] In a preferred embodiment, the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells can be cooled at this rate in sealed cryogenic vials / tubes (e.g., Nunc or Corning) for smaller aliquots and can be sealed in ethyl vinyl acetate (e.g., Pall Corporation CA, USA; Thermogenesis holdings Inc. CA, USA) bags that can be frozen by holding between metal plates for larger volumes.

[0199] After the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells have been frozen to -80°C, the cells can be transferred to a cryostat for long-term storage. Preferably, the cells can be cryopreserved in a liquid phase (-190°C) or a gas phase (-170°C) of nitrogen.

[0200] Following cryopreservation of the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells as described herein, the frozen sample can be stored in a cryogenic storage facility.

[0201] The facility can be a bank that can be present in one or more physical locations, which has one or more temperature-controlled freezers that use liquid nitrogen to maintain ultra-low temperatures. Therefore, in one embodiment, the method includes storing the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from the CD34+ hematopoietic stem cells in a cryogenic storage bank or cryogenic storage warehouse after cryopreservation or freezing.

[0202] The cryopreserved collection can include any number of amplified hematopoietic stem cell samples, wherein each sample can be derived from a single blood sample, or multiple samples can belong to a single blood sample from a person. In another embodiment, the cryogenic storage can include each frozen amplified cell sample of two or more independent cord blood samples or blood samples, such as a combined sample as described above in the present invention. The amplified hematopoietic stem cell samples are stored at a temperature lower than -20 ° C, at -80 ° C or preferably at an ultra-low temperature obtained with liquid nitrogen (-190 ° C) or with nitrogen vapor (-170 ° C).

[0203] Cryopreserved and frozen expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells can be thawed quickly as needed, for example, in a water bath maintained at 35°C-37°C or in VialThawing Systems (e.g., ThawStar, BIOLIFE Solutions, WA, USA) or in a waterless freezing bag thawing device (e.g., Thaw star CB, BIOLIFE Solutions, WA, USA). In a specific embodiment, the frozen vial of frozen cells can be immersed 3 / 4 in the warm water of the bath, the neck area of ​​the tube is kept outside the water, and the vial is constantly and gently rotated to ensure proper heat transfer from the surface to the internal ice mass and mixing of the cell suspension. Thawing is performed quickly to ensure that small ice particles are left in the vial, so as not to make the temperature of the cell suspension exceed 4°C, because the DMSO in the cryoprotective medium will damage the cells at temperatures above this temperature. Once thawing is complete, the vial can be immediately placed on ice for further use, such as thawed or a portion thereof, for infusion into a human patient to deliver hematopoiesis. There are several methods known in the art available to the skilled artisan for further processing of thawed hematopoietic stem cells, which can be used if deemed appropriate.

[0204] If the cryoprotectant (such as DMSO) is toxic, it may be necessary to remove the cryoprotectant (such as DMSO) from the thawed hematopoietic stem cells. It may also be desirable to prevent the cells from aggregating with each other by using 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 ( et al., 2006, J Immunol methods 30; 313(1-2): 209-213, doi: 10.1016 / j.jim.2006.04.004) etc.

[0205] It may also be desirable to wash the cells by adding 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) prior to infusion into a human patient in need thereof, followed by one or more centrifugation steps to concentrate the cells at the bottom of the tube, remove the cryoprotectant and diluent as the supernatant, and resuspend the cells in the above isotonic medium to obtain the final cell product for infusion into a human patient to provide hematopoiesis. This process should be performed gradually to minimize abrupt changes in the osmotic gradient and to prevent cell damage due to DMSO removal.

[0206] Optionally, prior to therapeutic use, a small representative aliquot of the thawed hematopoietic stem cells may be taken and examined for total nucleated cell count, cell viability, and CD34+ hematopoietic stem cell percentage. Depending on suitability, the procedure for assessing such parameters may be any method known in the art. Preferably, a similar procedure as described for the sample of step i) of the method of the present invention may be used.

[0207] In a third aspect, the present invention provides a population of CD34+ hematopoietic stem cells obtainable by and / or by the method and / or use as described herein.

[0208] As will be appreciated, based on the CD34+ hematopoietic stem cells of the present invention expressing genes known to be associated with estrogen receptor activity, it is possible to distinguish the expanded CD34+ hematopoietic stem cells obtained or obtainable from the present invention from hematopoietic stem cells obtained by different methods. As will be appreciated by those skilled in cell biology, such gene expression can be determined by measuring messenger RNA (mRNA) and / or any resulting protein and / or microRNA (miRNA). Genes associated with estrogen receptor activity can use publicly available databases (e.g., 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 is also possible to identify the expanded CD34+ hematopoietic stem cells obtained or obtainable from the present invention because the DNA within these cells is physically associated with the nuclear estrogen receptor.

[0209] 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 a nuclear estrogen receptor physically associated with its DNA. In some embodiments, one or more of the following genes associated with estrogen receptor activity may be expressed or inhibited in CD34+ hematopoietic cells obtained 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.

[0210] In some additional or alternative embodiments, based on the CD34+ hematopoietic stem cell population obtained by the method of the present invention having a higher proportion of CD34+CD133+ cells compared to CD34+ hematopoietic stem cell populations obtained by other methods, it is possible to distinguish the expanded CD34+ hematopoietic stem cells obtained or obtainable from the present invention from hematopoietic stem cells obtained by different methods. In some embodiments, the cells obtainable by the method of the present invention are CD34+CD133+CD38-. In some embodiments, a higher proportion of CD34+CD133+ and / or CD34+CD133+CD38- cells in a population obtained or obtainable by the methods of the present invention is associated with a higher quality stem cell population and / or greater clinical potential, and a person skilled in the art of medicine will understand that such cells are of higher quality (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 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).

[0211] In one embodiment, the expanded CD34+ hematopoietic stem cells of the present invention do not have any significant differences in their surface phenotype when compared to the CD34+ hematopoietic stem cells provided in step i) of the methods described herein. In a specific embodiment, the expanded CD34+ hematopoietic stem cells of the present invention do not have any differences in their surface phenotype when compared to the CD34+ hematopoietic stem cells provided in step i) of the methods described herein. The inventors have determined that this feature of their invention demonstrates the superiority of their method in providing expanded CD34+ hematopoietic stem cells, particularly with sustained stemness.

[0212] In a fourth aspect, the present invention provides one or more cells derived from CD34+ hematopoietic stem cells, which may be obtained by and / or through the methods and / or uses described herein.

[0213] In one embodiment of the fourth aspect of the invention, the one or more cells derived from CD34+ hematopoietic stem cells are cells selected from the list consisting of: myeloid progenitor cells; lymphoid progenitor cells; erythroid cells; erythrocytes; mast cells; megakaryocytes; platelet cells; mast cells; myeloblasts; basophils; neutrophils; eosinophils; monocytes; macrophages; dendritic cells; natural killer cells; T lymphocytes; and / or B lymphocytes.

[0214] In one embodiment, the T lymphocyte is a chimeric antigen receptor (CAR)-T cell.

[0215] In a fifth aspect, the present invention provides a composition comprising CD34+ hematopoietic stem cells obtainable by and / or through the methods and / or uses described herein and / or the CD34+ hematopoietic stem cell population described herein.

[0216] HSC for transplantation can be mixed with any isotonic buffered intravenous crystalloid solution having a physicochemical composition that closely reflects human plasma. Some examples include, but are not limited to, Plasmalyte, Normosol, and Lactated Ringers solution (LR). Thus, in some embodiments, the composition comprising CD34+ hematopoietic cells obtained by the methods described herein comprises an isotonic buffered intravenous crystalloid solution, such as those described above. The preparation and composition of such solutions contained in the composition are well known in the art.

[0217] In a sixth aspect, the present invention provides a kit of parts comprising CD34+ hematopoietic stem cells obtainable by and / or through the methods and / or uses described herein and / or the CD34+ hematopoietic stem cell population described herein.

[0218] In an alternative sixth aspect, the present invention provides a kit of parts for expanding CD34+ hematopoietic stem cells, comprising one or more estrogen receptor agonists, and / or a sample comprising one or more CD34+ hematopoietic stem cells and / or 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 present invention may include instructions explaining the steps described herein related to the method of the first aspect of the present invention and / or the use of the second aspect of the present invention. The instructions may include details of how to expand cells in a cord blood bank context using a tissue culture incubator (5% CO2, 98% humidity), a cell culture bag, and a centrifuge using good manufacturing practices (GMP).

[0219] In some embodiments, the kit further comprises growth factors and / or cytokines as described herein (provided as a mixture with one or more estrogen receptor agonists or provided as separate components). The kit may further comprise a serum-free medium suitable for expanding hematopoietic stem cells. The composition of such a medium is well known in the art.

[0220] In a seventh aspect, the present invention provides CD34+ hematopoietic stem cells obtainable by and / or by the methods and / or uses described herein and / or the CD34+ hematopoietic stem cell population described herein for use in medicine.

[0221] In an alternative seventh aspect, the present invention provides one or more cells derived from CD34+ hematopoietic stem cells obtainable by and / or by the methods and / or uses described herein, and / or derived from a CD34+ hematopoietic stem cell population described herein, for use in medicine.

[0222] In an eighth aspect, the present invention provides CD34+ hematopoietic stem cells obtainable by and / or by the methods and / or uses described herein and / or the CD34+ hematopoietic stem cell population described herein for use in treating and / or preventing a condition in a subject in need thereof.

[0223] In an alternative eighth aspect, the present invention provides one or more cells derived from CD34+ hematopoietic stem cells obtainable by and / or by the methods and / or uses described herein, and / or derived from the CD34+ hematopoietic stem cell population described herein, for use in treating and / or preventing a condition in a subject in need thereof.

[0224] In a ninth aspect, the present invention provides use of CD34+ hematopoietic stem cells obtainable by and / or through the methods and / or uses described herein and / or use of the CD34+ hematopoietic stem cell population described herein for the preparation of a medicament for treating and / or preventing a disorder in a subject in need thereof.

[0225] In an alternative ninth aspect, the present invention provides the use of one or more cells in the preparation of a medicament for treating and / or preventing a condition in a subject in need thereof, wherein the one or more cells are derived from CD34+ hematopoietic stem cells obtainable by and / or by the methods and / or preparations described herein, and / or derived from the CD34+ hematopoietic stem cell population described herein.

[0226] In a tenth aspect, the present invention provides a method for treating and / or preventing a condition in a subject in need thereof, comprising administering to a subject in need thereof CD34+ hematopoietic stem cells obtainable by and / or through the methods and / or uses described herein and / or the CD34+ hematopoietic stem cell population described herein.

[0227] In an alternative tenth aspect, the present invention provides a method for treating and / or preventing a condition in a subject in need thereof, comprising administering to a subject in need thereof one or more cells derived from CD34+ hematopoietic stem cells obtainable by and / or by the methods and / or subject described herein, and / or derived from a CD34+ hematopoietic stem cell population described herein.

[0228] In a further alternative tenth aspect, the present invention provides a method for treating and / or preventing a condition in a subject in need thereof, comprising the steps of:

[0229] a) providing a sample comprising one or more CD34+ hematopoietic stem cells;

[0230] b) culturing the cells of step a) in the presence of a nuclear estrogen receptor agonist to expand CD34+ hematopoietic stem cells; optionally differentiating the expanded CD34+ hematopoietic stem cells into one or more cells derived from CD34+ hematopoietic stem cells; and

[0231] c) administering the cells of step b) to a subject in need thereof.

[0232] The method of the further alternative tenth aspect of the invention may include the steps described herein in connection with the method of the first aspect of the invention and / or the use of the second aspect of the invention. As will be understood, 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.

[0233] In one embodiment, the condition is one or more conditions selected from the list consisting of: a condition requiring transplantation; a hematological condition and / or a hematological malignancy; a condition caused by a failure or dysfunction in normal blood cell production and maturation; immunosuppression in a subject with a malignancy and / or a solid tumor; an autoimmune disease and / or an immune condition; a genetic disorder; a cancer; a tumor; osteopetrosis; myelosclerosis; acquired hemolytic anemia; an infectious disorder causing primary or secondary immunodeficiency; acquired immunodeficiency; neutrophil actin deficiency; neutrophil membrane GP-180 deficiency; a bacterial infection (e.g., brucellosis infection, Listeria monocytogenes infection, Listerosis infection, tuberculosis infection and / or leprosy); parasitic infections (e.g. malaria and / or leishmaniasis); fungal infections; disorders involving an imbalance in the proportions of lymphoid cell groups and / or impaired immune function due to aging; phagocytic cell disorders; Kostmann agranulocytosis; chronic granulomatous disease; Chediak-Higachi syndrome; Wiskott-Aldrich syndrome; metabolic storage diseases; mucopolysaccharidoses; mucolipidoses; alpha 1-antitrypsin deficiency; neurological disorders; and / or various disorders involving immune mechanisms.

[0234] In one embodiment, the transplant is an allogeneic transplant or an autologous transplant.

[0235] In one embodiment, the disorder caused by the failure or dysfunction of normal blood cell production and maturation is one or more disorders selected from the list consisting of: pancytopenia (such as radiation- and / or chemotherapy-induced pancytopenia); aplastic anemia; thrombocytopenia; agranulocytosis; red blood cell aplasia; hyperproliferative stem cell disorder; Blackfan-Diamond syndrome; Blackfan-Diamond syndrome caused by drug use; Blackfan-Diamond syndrome caused by radiation; Blackfan-Diamond syndrome caused by infection; and / or idiopathic Blackfan-Diamond syndrome.

[0236] In one embodiment, the hematological condition and / or hematopoietic malignancy is one or more disorders selected from the list consisting of: Hodgkin's lymphoma; non-Hodgkin's lymphoma; acute lymphoblastic (lymphocytic) leukemia; acute myeloid leukemia; acute malignant myelosclerosis; chronic lymphocytic leukemia; chronic myeloid leukemia; multiple myeloma; myeloid metaplasia of unknown cause; Waldenstrom's macroglobulinemia; and / or polycythemia vera.

[0237] In one embodiment, immunosuppression in a subject with a malignancy and / or solid tumor is one or more conditions selected from the list consisting of: ovarian cancer; breast cancer; malignant melanoma; testicular cancer; gastric cancer; small cell lung cancer; rhabdomyosarcoma; Ewing's sarcoma; retinoblastoma; glioblastoma; neuroblastoma; and / or lymphoma.

[0238] In one embodiment, the autoimmune disease and / or immune condition is one or more disorders selected from the list consisting of: type I diabetes; rheumatoid arthritis; systemic lupus erythematosus; chronic hepatitis; and / or multiple sclerosis.

[0239] In one embodiment, the genetic disorder is one or more conditions selected from the list consisting of: anemia; familial aplasia; Bloom's syndrome; Fanconi's syndrome; pure red cell aplasia (PRCA); dyskeratosis congenita; Blackfan-Diamond syndrome; congenital dyserythropoiesis syndrome I-IV; Chwachmann-Diamond syndrome; dihydrofolate reductase deficiency; methylaminotransferase deficiency; pyruvate kinase deficiency; sickle cell disease; alpha thalassemia; beta thalassemia; gamma thalassemia; methemoglobinemia; 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; innate immune disorders; severe combined immunodeficiency disease (SCID) bare lymphocyte syndrome; ionophore-responsive combined immunodeficiency; combined immunodeficiency with capping disorder; nucleoside phosphorylase deficiency; granulocyte actin deficiency; infantile agranulocytosis; Gaucher disease; adenosine deaminase deficiency; Kostmann syndrome; reticular dysplasia; and / or congenital leukocyte dysfunction syndrome.

[0240] In one embodiment, the genetic disorder is one or more disorders selected from the list comprising or consisting of: cerebral palsy; autism spectrum disorder; Alzheimer's disease; multiple sclerosis, Parkinson's disease; Huntington's disease; stroke; motor neuron disease; traumatic brain injury; spinal cord injury; amyotrophic lateral sclerosis; hypoxic-ischemic brain injury; eye injury; and / or epilepsy.

[0241] The term "treatment" includes the following meanings: administering the CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells described herein in an amount, manner and / or pattern that is effective to improve the condition, symptoms or parameters associated with the condition or to prevent the progression or worsening of the condition (including secondary damage caused by the condition) to a statistically significant degree or to a degree detectable by medical technicians.

[0242] The term "prevention" includes the following meanings: administering the CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells described herein in an amount, manner and / or pattern effective to avoid the development of a condition, symptom or parameter associated with a condition to a statistically significant degree or a degree detectable by a medical technician.

[0243] The expanded CD34+ stem cell population produced as described herein can be administered by any convenient route known in the art, such as systemic or local administration by infusion or bolus, and can be administered together with other bioactive agents, such as any isotonic buffered intravenous crystalloid solution having a physicochemical 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 population is administered by infusion.

[0244] The subject can be administered alone (monotherapy) or in combination with other agents (combination therapy), or in an admixture or by separate, simultaneous or sequential administration. In the case of combination therapy, the amount and time of administration can be those that provide, for example, an additive or synergistic therapeutic effect. In addition, administration can be used as a primary treatment (e.g., first-line treatment) or as a secondary treatment (e.g., for subjects who have insufficient response to a previously administered therapy).

[0245] In one embodiment, one or more estrogen receptor agonists (and / or other growth factors or cytokines, if used) are removed or diluted (such as by washing or dilution) from the expanded CD34+ hematopoietic stem cells and / or one or more cells derived from CD34+ hematopoietic stem cells prior to administration to a subject.

[0246] Figure 1 : Fold expansion of UCB-HSPCs treated with 0, 1 and 10 nM E2 for 8 days (p<0.05).

[0247] Figure 2 : Flow cytometric analysis of expanded cells. No changes were observed in the surface phenotype of CD34+ and CD34+CD133+ cell populations.

[0248] Figure 3 : Fold expansion of CD34+ cells treated with 0, 1 and 10 nM E2 for 8 days (p<0.05).

[0249] Figure 4 : Flow cytometric analysis of expanded UCB-HSCs. Cells maintained their phenotype and stemness under control and all treatment conditions.

[0250] Figure 5: Expansion of CD34+CD38- HSCs in various growth factor and compound combinations tested. a) Number of cells (in millions), and b) fold expansion of cells after 14 days (left) and 21 days (right) of expansion (compared to control) (p<0.05).

[0251] Figure 6: Phenotype of cells expanded from CD34+CD38- HSCs in various growth factor and compound combinations tested. a) Fold expansion of CD34+ (HSPCs), and b) percentage of CD34+ cells after 14 days (left) and 21 days (right) of expansion (p<0.05).

[0252] Figure 7 : Fold expansion of CD34+CD133+ cells (LT-HSCs) expanded from CD34+CD38- HSCs for 14 days (left) and 21 days (right) in various growth factor and compound combinations tested (p<0.05).

[0253] Figure 8 : Evaluation of the hematopoietic multi-lineage potential of ex vivo expanded UCB-HSCs after 21 days of culture in various growth factor and compound combinations tested.

[0254] Figure 9: Expansion of CD34+CD38 selected HSCs in various serum (FBS) and serum-free media supplemented with combinations of growth factors and estrogen ligands. a) Number of cells (in millions) at day 14 (left) and day 21 (right) and, b) fold expansion of cells after 14 days (left) and 21 days (right) of culture (p<0.05).

[0255] Figure 10: Phenotype of cells expanded from CD34+CD38- selected HSCs in various serum (FBS) and serum-free media supplemented with combinations of growth factors and estrogen ligands. 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).

[0256] Fig.11 : Evaluation of the hematopoietic multi-lineage potential of ex vivo expanded CD34+ HSPCs after 21 days of culture in various serum-containing (FBS) and serum-free media supplemented with combinations of growth factors and estrogen ligands. Example

[0257] The following examples are included to demonstrate specific embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes of its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the present invention, and still similar or similar results can be obtained.

[0258] Overview

[0259] Transplantation of hematopoietic stem cells (HSC) to treat various hematological, oncological and immune disorders is very promising, but is often limited by the availability of matching donors. Umbilical cord blood (UCB) contains less mature HSCs, has greater telomere length, and therefore provides a better alternative. However, clinical use in adults is generally limited due to slow implantation caused by low stem cell counts. Ex vivo expansion of UCB-HSC to clinically relevant doses can overcome this obstacle and benefit patients. Research groups worldwide have adopted several approaches and methods, including the use of hematopoietic growth factors, cytokines and differentiation blockers such as Notch ligands, copper chelators, nicotinamide and aryl hydrocarbon receptor antagonists. Estrogen (E2) and estrogen receptors (ER) have been involved in the proliferation of specialized and directed progenitor cells, but have not yet been tested in the proliferation of UCB-HSC. Interestingly, E2, which is easily available in placenta and normal umbilical cord blood, has been involved in the growth and differentiation of lineage-directed progenitor cells in rodents. So far, relatively few studies have been known about the role of E2 in the expansion and self-renewal of HSCs with long-term repopulation potential. E2 has not been tested in the expansion of human UCB-derived hematopoietic stem cells in a completely xeno-free environment using defined serum-free growth media. Reliable and clinically useful UCB-HSC expansion not only facilitates allogeneic transplantation, but also provides a suitable platform to derive effector immune cells for ex vivo expansion and / or genetic modification to improve cellular immunotherapy, such as UCB-derived chimeric antigen receptor (CAR)-T and NK cells. Therefore, these enhanced anti-tumorigenic cells can be provided as an off-the-shelf product, which minimizes therapy time by avoiding the need to modify the patient's own cells.

[0260] The inventors have developed a new xeno-free method to effectively expand UCB-HSPC in vitro by using growth supplements containing estrogen ligands. In this way, we can uniquely achieve relevant therapeutic doses, and thereby promote the clinical use of UCB-HSC in autologous transplantation and allogeneic transplantation, and open the potential for using UCB CAR-T cells as effective adoptive cell immunotherapy. Compared with other reports, the present method is robust and unique in terms of reliably expanding UCB-HSC within a limited time span to provide a larger number of therapeutic cells with a sustained stem phenotype. Interestingly, the method effectively expands CD34+HSPC and long-term repopulation HSC (LT-HSC) to 1500 times and 90 times, respectively, in 21 days without affecting its multi-lineage differentiation potential, which has not been observed by others so far, and these cells will exemplify the advantages of expanded UCB-HSC in a clinical setting.

[0261] background

[0262] Hematological and non-hematological disorders can be treated with autologous or allogeneic hematopoietic stem cell (HSC) transplantation. However, this application is limited by the identification of suitable donors. Umbilical cord blood (UCB) contains less mature HSCs, has a greater telomere length, and therefore provides a better alternative source for HSC transplantation. However, compared with bone marrow or mobilized peripheral blood stem cell transplants, adult UCB transplants are usually clinically limited because of the low number of total nucleated cells (about 1 billion, TNC) and CD34+ progenitor cells (about 1.2 million), followed by slow / delayed implantation. Most healthy full-term births provide just over 1 million blood-forming stem cells (HSC) in their umbilical cord blood. Therefore, if the donation is below the threshold, a large number of UCB units are not considered optimal in most public cord blood banks. Higher nucleated cell counts per kg body weight (≥25 million-50 million cells / kg) and CD34 + (1-1.5x10 5 / kg) cell dose is associated with better outcomes of cord blood transplantation (1) and overcoming implantation delays. Therefore, the ex vivo expansion of umbilical cord blood hematopoietic stem cells (UCB-HSC) can help increase the cell dose available for transplantation. The ex vivo expansion of stem cells requires symmetrical division, and both daughter cells should maintain the characteristics of stem cells. The precise dose of cytokines, culture media, serum and other small molecules used for expansion is also extremely important. The ultimate goal of cord blood expansion should be to produce clinically safe, transplantable HSCs that can reliably repopulate recipients. Another acceptable alternative may be to generate more mature progenitor cells in vitro that can be rapidly implanted. The initial efforts to expand HSC began in the 1970s, and since then, many efforts have been made to expand cells to retain stemness and limit differentiation by using cytokines or stromal cells or a combination thereof, which has led to mixed success, and still continues to seek to stably expand reliable HSC populations from cord blood worldwide.

[0263] 1. Hematopoietic stem cell (HSC) phenotype and self-renewal pathway

[0264] HSCs are a rare subpopulation of the hematopoietic system that is capable of generating all mature blood cells throughout life (2). Thus, a unique feature of HSCs is a dynamic balance between quiescence, self-renewal, and differentiation that is governed by multiple mechanisms, such as genetic and epigenetic regulation, as well as microenvironmental (niche) factors.

[0265] Routine CD34 analysis by flow cytometry enumerates hematopoietic stem and progenitor cells, regardless of their subsets, although this may not predict true engraftment dynamics and immune reconstitution. Over the past two decades, the CD34 antigen has become widely used to identify human hematopoietic stem cells. Experiments using umbilical cord blood to identify human HSCs using CD34 as a sole marker for long-term repopulating HSCs (LT-HSCs) have yielded mixed results. Ishikawa et al. (3) strongly suggested that the phenotype of human CB stem cells is CD34. + CD38 - , while only CD34 + Positive cells represent not only LT-HSCs, but also short-term hematopoietic stem cells (ST-HSCs) and progenitor cell subsets (HSPCs) in the hematopoietic system hierarchy. Thus, primitive progenitor cells (LT-HSCs) lack expression of the cell surface antigen CD38 or express it only in small amounts, whereas, in contrast, CD38 is abundantly expressed on more mature progenitor cells. In addition, et al. have meticulously shown that all HSCs with long-term potential, including those with the ability to engraft into NOD / SCID or NSG mice, were isolated as CD133 + CD34 + CD38 -Therefore, long-term potential UCB-HSCs are phenotypically characterized by CD133 + CD34 + CD38 - .

[0266] In addition, monoclonal antibody identification has been used to identify the presence or absence of cell surface protein markers to identify and separate hematopoietic stem cells. Such markers include, but are not limited to, Lin, CD34, CD38, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, and HLADR and combinations thereof. For more information on this, you can find Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006 (http: / / stemcells.nih.gov / info / scireport / 2006report.htm).

[0267] Functional readouts of HSCs and HSPCs have been associated with cell survival, self-renewal, proliferation, differentiation, and migration (homing, mobilization, and chemotaxis) (10). These processes involve several intracellular signaling molecules, including but not limited to p21cip1 / waf1, p27kip1, Stat3, Stat5, Notch, Wnt, β-catenin, GSK-3, sonic hedgehog, bone morphogenetic proteins, members of the Hox family (such as Hoxb4), FoxO, Pu.1, GATA-1, Sirt1 (and other members of the sirtuin family of deacetylases), HIF-1α, Rheb2, and the rapamycin-sensitive m-TOR pathway (reviewed in (17)).

[0268] 2. Hematopoietic growth factors in self-renewal and proliferation

[0269] Hematopoietic growth factors are released under inflammation or cytopenia conditions and induce the mobilization and proliferation of HSC in vivo. These growth factors are also called colony stimulating factors (CSFs), including granulocyte colony stimulating factor (G-CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); macrophage colony stimulating factor (M-CSF); interleukin-3 (IL-3); IL-6; erythropoietin (EPO) and TPO (5). In the late 1990s, many of these cytokines were introduced into cell culture media to expand UCB-HSC in vitro to illustrate their ability to self-renew and reconstruct in mice. Most experimental evidence shows that a combination of multiple cytokines, such as stem cell factor (SCF), FLT-3 / FLK-2 ligand (Flt3-ligand), thrombopoietin (TPO), seems to be necessary for progenitor cell expansion. Among these growth factors, it is unanimously agreed that SCF is indispensable for stem cell and progenitor cell expansion, and even proves that SCF is a key factor for the survival of hematopoietic progenitor cells. Using this cytokine mixture, CD34+ cells can be expanded ex vivo by about several times relative to the value before expansion. The key to ex vivo HSC expansion is the increase of undifferentiated stem cells, rather than the expansion of differentiated and undifferentiated cells. Mohamed et al. (6) used SCF, GM-CSF, IL-3 and TPO for 7-day and 11-day ex vivo expansion of CB stem cells under defined conditions. They reported better expansion of CD34+ cells within 7 days, and adding more cytokines (IL-6 and FLT-3) did not affect the number of CD34+ cells, but led to the appearance of apoptotic cells. Yao et al. used a statistically formulated cytokine-restricted serum-free medium and reported a 20-fold expansion of CD34+CD133+ cells after 7 days of culture, a 723-fold expansion of CD34+CD38+ cells, and 8-fold long-term culture starting cells. Madkaikar et al. (7) combined different cytokines (SCF, IL-3, GM-CSF) with stromal cell support for HSC expansion and achieved a maximum of 21-fold expansion of CD34+ cells and nucleated cells. Another group (Wei et al.) identified 7 sets of culture conditions for expanding CD34+ cells containing a basic combination of SCF, IL-3 and IL-6 for 3, 7 and 14 days. The fold expansion rate of CD34+ cells ranged from 10 to 50 times compared to fresh UCB. Although a large number of recombinant cytokines are used to expand primitive hematopoietic progenitor cells, the optimal combination has not yet been approved for use in clinical practice. The most commonly used factors are stem cell factor (SCF), IL-3 and IL-6, G-CSF, thrombopoietin (TPO) and Flt-3 ligand (8,9). It should be noted that in addition to the group of factors, their concentrations and the order in which they are used are also important.

[0270] Fetal calf serum (FCS) contains a natural mixture of growth factors, adhesion mediators, minerals, lipids and hormones and is a standard component for culturing most human cell types, including HSCs. However, there is no consensus on the possibility of using FCS-supported expanded cells in a clinical setting. Disadvantages of serum include the difficulty in standardizing its composition, potential viral contamination, and a high risk of recipient immunity to foreign proteins (10, 11). Therefore, excluding FCS and using defined serum-free media for ex vivo expansion of HSPCs would greatly help optimize the cytokine combination and the acceptability of expanded cells for transplantation (12, 13).

[0271] 3. In vitro expansion strategy of cord blood HSC

[0272] A number of ex vivo expansion methods have been developed to increase the UCB cell dose, and results from traditional UCB expansion methods using cytokines alone have been disappointing due to variability in the concentration of each cytokine used.

[0273] Another approach is to use HSC differentiation blockers, such as nicotinamide analogs, copper chelators, induction of constitutive Notch signaling, or the use of aryl hydrocarbon receptor antagonists (StemReginin1). Many of these methods result in significant expansion of total nucleated cells and CD34+ cells compared to recipients of unoperated CBT, and the time for neutrophil or platelet engraftment in patients transplanted with expanded products is significantly shortened. These studies differ not only in the expansion methods, but also in terms of the cytokines used, patient populations, conditioning regimens, and transplant practices. Some of these methods start with unselected cell populations in umbilical cord blood, while other methods use selected subsets, such as CD34+ or CD133+. Another UCB cell expansion technology utilizes computer-automated "continuous perfusion" culture for 12 days and results in a 2.4-fold expansion of TNCs and a 0.5-fold expansion of CD34+ cells.

[0274] 3.1 Copper chelating agents

[0275] It has been reported that tetraethylenepentamine (TEPA), a polyamine copper chelator, together with the cytokines FLT-3, IL-6, TPO, and SCF for 3 weeks, expanded CD133+ selected HSCs by blocking cytokine-induced differentiation without affecting the subsequent differentiation and proliferation of mature committed cells (14). This resulted in an 89-fold expansion of CD34+ cells, a 30-fold expansion of CD34+CD38-, and a 172-fold expansion of colony forming units (CFU).

[0276] 3.2 Notch signaling

[0277] Another method to block EPC differentiation is to induce constitutive Notch signaling, which can establish immortalized cell lines that can be cultured in liquid culture medium with cytokines. Interestingly, the cell fate selection of both invertebrates and vertebrates is controlled by Notch signaling (15, 16), which inhibits certain differentiation pathways and allows cells to differentiate or self-renew along alternative pathways. Hematopoietic precursor cells express Notch receptors, and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursor cells (17-19), and also dominates T cells or B cell lineage specialization from common lymphoid precursor cells (20). Delaney et al. (21) used CB cells selected by CD34+, and transduced them with engineered Notch ligands (Delta1ext-IgG), and cultured for 16 days in the presence of IL-3, IL-6, TPO, SCF and flt-3 ligands, which resulted in an average expansion of 222 times of CD34+ cells.

[0278] 3.3 Niacinamide

[0279] Another technique to inhibit HSC differentiation involves the use of NAM, a member of the sirutin family of protein deacetylases (SIRT1), which plays an important role in determining the lifespan of many lower organisms (22) and in the self-renewal of mouse embryonic stem cells. It is known to inhibit the differentiation of HPCs and play an important role in the adhesion, migration and proliferation of stem cells (23). CD34+Lin-selected CB cells were expanded in medium containing SCF, FLT-3, IL-6 and TPO with NAM. After 3 weeks of culture, TNCs expanded an average of 486-fold and CD34+ cells expanded an average of 72-fold (24).

[0280] 3.4 Aryl hydrocarbon receptor antagonism

[0281] The University of Minnesota group presented results from a phase I / II study of CB expansion using StemReginin1 (SR1), an aryl hydrocarbon receptor antagonist, which facilitated proliferation of CD34+ cells in the absence of differentiation in the presence of SCF, flt-3 ligand, TPO, and IL-6. Culture for 21 days resulted in an average expansion of CD34+ cells of 328-fold (25).

[0282] 3.5 Estrogen

[0283] The female sex hormone estrogen (E2) plays a vital role in the sexual differentiation and reproduction of females and males. An important question is whether stem cells are targets of sex hormone regulation in tissues without sex-specific morphological differences. The terminal differentiation and function of some directed hematopoietic cells are regulated by sex hormones (26, 27), but the function of hematopoietic stem cells (HSC) is mostly similar in both sexes. In addition to the importance of E2 in reproduction, it has been shown that E2 regulates the proliferation and differentiation of multipotent and pluripotent stem cells (28, 29). In mice, E2 has been involved in promoting the cycle of bone marrow hematopoietic multipotent progenitor cells (MPP) and their differentiation into megakaryocyte-erythroid progenitor cells (MEP) (30). It has been reported that E2 enhances the proliferation and osteogenic differentiation of bone marrow mesenchymal stromal cells (BM-MSC) by activating Notch signaling and upregulating estrogen receptors (ER) (31). Interestingly, E2, which is readily available in the placenta and normal umbilical cord blood, has been implicated in the growth and differentiation of lineage-committed progenitors in rodents.

[0284] E2 acts on target cells by binding to nuclear receptors, of which two have been identified, estrogen receptor α (ERα) and β (ERβ) (32). The E2-ER complex binds to specific promoter regions on DNA to regulate the transcription of hundreds of target genes. This is called the genomic action of E2. E2 can also mediate rapid non-genomic actions by interacting with signaling cascades in the cytoplasm. ERα has been detected in non-hematopoietic cells in the bone marrow and in mouse B lymphocyte precursors (33) and human hematopoietic multipotent progenitor cells (MPP) (34). ERβ has also been found in non-hematopoietic cells in mouse bone marrow and human spleen (33), but there are no reports on the identity of ERβ in UCB-HSCs. We recently showed that loss of ERβ disrupts the proliferation of neural stem cells. These effects may be propagated through Notch-HES signaling, which is dysregulated by loss of ERβ (35). Interestingly, ERβ can mediate the expression of p21, p27, and Notch1 in both ligand-dependent and ligand-independent manners (36, 37), and ERβ has been shown to control NOTCH1 transcription in squamous cell carcinoma cells (38). Ligand-mediated effects of ERα are generally associated with cell proliferation, and ligand-mediated effects of ERβ are associated with a variety of cell cycle-regulated antiproliferative events and generally counteract excessive proliferation (anti-tumor effects).

[0285] Illing et al. (39) have reported that, independent of estrogen-induced bone growth, estradiol-treated mice increased the number of progenitor cells, but not long-term (LT) repopulating HSCs in the vascular compartment of the bone marrow. Interestingly, this study suggests that the effects of estradiol on stromal cells effectively favor HSPC cell frequency. Furthermore, these proliferative effects of E2 on BM HSCs are due to enhanced cycling (division), which leads to more short-term (ST) HSCs and depletion of LT-HSCs. This is further supported by the significant reduction in LT-HSCs derived from E2-treated donors when given to tertiary recipient mice. However, the effects of E2 on the proliferation of these cells may not be mediated through the classical ER.

[0286] During pregnancy, placental trophoblast cells increase maternal E2 levels by up to 1000 times (40), and this can be attributed to the maternal adaptation to the increased blood demand associated with fetal nutrition and physiological blood loss during childbirth. Based on these findings, Nakada et al. (41) have observed that the replication activity of HSC increases in pregnant female mice. They have reported that E2 promotes the enhanced cell cycle entry of HSC and multipotent progenitor cells (MPP), and increases the differentiation to megakaryocyte erythrocyte progenitor cells (MEP). Despite the increase in HSC proliferation and the production of erythrocytes and platelets, the number of HSC in pregnant mice is no different from that of control mice or male mice. These effects of estrogen are attributed to mediation by ERα, and estrogen promotes the asymmetric division of HSC to a large extent rather than the symmetric self-renewal in the bone marrow. However, it is worth noting that the inventors have widely attributed it to HSC self-renewal, although they have not observed the difference in the number of HSC in males or females and after ovariectomy. In their experiments, BrdU incorporation by HSCs did not reflect self-renewal (generating identical HSCs) but rather enhanced cell division, which could result in more committed progenitors.

[0287] Anguilera et al. (42) have attempted to determine the effects of E2 on specific HSPC subsets in mice and found that ERα and ERβ are differentially expressed in LT-HSCs, MPPs, and committed progenitors, which may indicate different roles for E2 signaling in different cell types. This study showed that in ST-HSCs and MPPs, ERα activation by selective ER regulators induced apoptosis, in part through Myc induction. In contrast, E2-mediated proliferation of quiescent LT-HSCs was associated with a loss of self-renewal activity and, thus, the ability to reconstitute hematopoiesis, which resulted in increased differentiation.

[0288] Kim et al. (43) have recently shown that supplementation of E2 in the growth medium improves the hematopoietic differentiation of human pluripotent stem cells (hPSCs) in vitro via an ERα-mediated pathway. The study reported that ERα was continuously expressed during the differentiation process and was exclusively hematopoietic phenotype. However, E2 signaling had no critical effect on the functional capacity of differentiated hematopoietic cells. In addition, the authors suggest that E2 signaling is involved in fate determination processes in early hematopoiesis, as E2 treatment produced a large number of angioblasts. Furthermore, E2 alone had no effect on the generation of hematopoietic cells from hPSCs, but increased cell generation by two-fold in the presence of hematopoietic growth factors. Paradoxically, the authors reported, in particular, an increase in erythropoietic differentiation from hPSCs after supplementation with E2. Interestingly, they also reported that the number of CD34+ blood cells in human umbilical cords could be increased by two-fold when cultured for 10 days in the presence of E2. The authors concluded that E2 enhances the proliferation of hematopoietic progenitor cells by maintaining their ER positivity and their functional capacity. However, the studies reported by Kim et al. focused primarily on the development of hematopoietic cells from human pluripotent stem cells and there are many pitfalls that do not clearly support their claim that E2 exerts a proliferative effect on HSCs rather than on later progenitor cells. Some of the problematic findings are as follows:

[0289] 1. The HSC markers used in the study, such as CD34+CD45+, do not truly reflect LT-HSCs with long-term repopulation capacity and therefore may not be able to reconstitute long-term hematopoiesis. (See 1. Hematopoietic stem cell (HSC) phenotype and self-renewal pathway)

[0290] 2. Even if the identification markers used in this study are acceptable, in their study, simple E2 supplementation only resulted in a maximum of 8% of cells expressing hematopoietic progenitor markers, which is not feasible for therapeutic applications due to the low cell yield.

[0291] 3. The authors used the terms hematopoietic stem cells and progenitors of CD34+CD45+ cells interchangeably in their experiments, whereas the true hematopoietic stem cell fraction is only CD133+CD34+CD38-, and the rest are multipotent or committed progenitors. CD133+CD34+CD38- has not been investigated in their study.

[0292] 4. This study mainly focused on the hematopoietic fate specification from pluripotent stem cells, and the study did not provide any experimental evidence to demonstrate the further expansion of differentiated HSCs in the presence of E2.

[0293] 5. This study is consistent with other studies conducted in mouse models, where E2 has been reported to enhance the proliferation of progenitor cells, but not HSCs, and subsequently increase differentiation into erythroid cells. The progenitor cell bias observed under the above experimental conditions is not conducive to the use of HSCs in expansion and transplantation, as these expanded fractions will not be able to properly reconstitute the recipient.

[0294] 6. The authors claim that UCB hematopoietic progenitor cells proliferate up to 2-fold in 10 days, which is minimal for clinical use. Furthermore, no information is provided about other growth factors used in combination with E2, and the assessment of the multi-lineage potential of E2-expanded progenitor cells is inaccurate, as the number of progenitor cells used for the assay is not mentioned, and therefore the number of CFUs shown is technically impossible to reproduce.

[0295] 7. The study extensively used fetal bovine serum as a medium supplement for culturing cord blood stem and progenitor cells, which may carry several unknown growth factors, hormones, matrix proteins, and other factors (which may contribute to the increased differentiation noted in hematopoietic progenitor cells despite the 2-fold expansion). In addition, the use of animal components for ex vivo expansion is not suitable for therapeutic purposes because they pose a risk of exposure to non-human molecules and pathogens that can elicit a strong immune response in humans.

[0296] Therefore, the above studies mainly focus on the effects of E2 on the proliferation of hematopoietic progenitor cells and their enhanced differentiation, but completely lack the demonstration that HSCs can be significantly expanded while retaining their self-renewal. In addition, this study took the opportunity to define hematopoietic development under the influence of E2, rather than the expansion of hematopoietic stem cells themselves.

[0297] Considering the in vivo rodent studies mentioned above, estrogen has been shown to increase, decrease, or have no effect on HSC numbers, depending on the methods / markers used to identify the cell population. Animal-based studies do not translate well to a human context because of differences in cell surface marker phenotype and growth factor requirements as well as telomere activity, which can lead to more frequent cell cycling and cell divisions in mice.

[0298] Although the proliferative capacity of E2 is well described in several mature tissues, little is known about the role of E2 in the expansion and self-renewal of human HSCs with preserved long-term repopulating potential. Furthermore, E2 has not been tested in the expansion of human UCB-derived hematopoietic stem cells in a completely xeno-free environment using therapeutically relevant defined serum-free growth media.

[0299] result

[0300] The few studies mentioned above suggesting that estrogen may mediate proliferative effects on cord blood-derived HSCs are of high clinical relevance, as large numbers of HSCs are required for therapeutic efficacy. However, these studies do not fully address this question, as they do not distinguish between HSCs and more differentiated progenitor cells, which leaves a large gap in interpretation. Furthermore, most of these studies used rodent models, which may not represent ideal conditions for human HSCs, at least with regard to differences in cell surface markers. To fully determine the ability of E2 to affect therapeutic HSC expansion, we explored the effects of E2 on a primitive (long-term) HSC population, namely, the CD34 + CD133 + Furthermore, we used more therapeutically relevant serum-free media with carefully selected chemically defined additives and a unique combination of growth factors, cytokines, and ER-selective ligands. Beyond our expectations, we observed that low doses of E2 resulted in significantly higher numbers of CD34 cells with preserved stemness. + Cells (CD34 + CD133 + or CD34 + CD38 - We have examined the dose response of E2 and / or ER subtype specific ligands in combination with specific concentrations of known cytokines and growth factors in defined serum-free medium.

[0301] Experiment 1

[0302] UCB HSCs were outsourced from a commercial supplier (Zen-Bio Inc., USA) and the cells were 80-95% CD34 + CD133 + HSC. HSC was cultured without feeder cells in a limited serum-free medium with an optimized mixture of growth factors and cytokines. 17β-estradiol (E2) and ER-specific estrogen ligands were added at different concentrations ranging from 0.5nM to 25nM. Cells were grown for 8 days and then counted with trypan blue to exclude dead cells. Compared with the control group, cell number was quantified and fold amplification was calculated. Compared with untreated cells 94.23 ± 6.97, the fold amplification of HSC cultured for 8 days in the presence of 1nM and 10nM doses of E2 was significantly higher, 129 ± 0.79 and 121.1 ± 3.98 ( Figure 1Interestingly, even in the absence of E2, the expansion of UCB-HSCs was still satisfactory compared to ex vivo expansion methods reported by others, indicating that our specific formulation of growth factors can robustly promote HSC expansion. However, the addition of E2 to this medium resulted in an even higher expansion of cells. Subsequently, flow cytometric analysis was used to assess the HSC stemness of the ex vivo expanded cells by gating the cells for CD45 expression, as recommended by ISHAGE, and confirmed that all treatments and control conditions contained 70-80% CD34 + cells and 24-26% CD34 + CD133 + cell( Figure 2 ). CD34 + CD133 + The numbers of CD34+ cells in the expanded cultures were surprisingly high, further complimenting our expansion method. Quantification of CD34+ cells in the expanded cultures showed that E2 treatment at 1 nM and 10 nM doses resulted in significantly higher expansion folds of 91.94 ± 3.11 and 95.07 ± 1.47 (95.07 ± 1.47, respectively) compared to 74.66 ± 6.68 (95.07 ± 1.47, respectively) in the control group. Figure 3 This is the highest CD34+ cell expansion ever achieved in the shortest time period (8 days), compared to expansions previously reported by others (186-fold / 14 days, 222-fold / 16 days, 118-fold / 18 days, and 425-fold / 21 days).

[0303] Importantly, the inventors did not find any significant differences in surface phenotype between untreated and E2-treated populations, suggesting that the expanded cells retained their stemness and that the treatment conditions did not affect their phenotype. This supports our hypothesis that estrogen ligands can be successfully used to promote robust ex vivo expansion of HSC populations from cord blood without affecting HSC phenotype ( Figure 4 ). (See 1. Hematopoietic stem cell (HSC) phenotype and self-renewal pathways). However, further studies on their ability to differentiate into myeloid and lymphoid lineages in vitro and their ability to repopulate recipients are warranted.

[0304] Experiment 2

[0305] In another experiment, to fully evaluate the effect of estrogen on UCB-HSC proliferation in the absence of Notch ligands, we expanded CD34+CD38- magnetically selected cells (80-90%) from umbilical cord blood (Zen Bio, USA) for 14 and 21 days in the presence of different growth factors, cytokines, estrogen and ER subtype-specific ligands. Cells were analyzed by flow cytometry as described in Experiment 1 above. The results of this experiment showed that the addition of GM-CSF and G-CSF to the culture medium significantly increased the number of expanded cells (in millions) compared to the control group (7.26±0.11 vs. 3.7±0.17) ( Figure 5a ). Addition of E2 to this cytokine cocktail also resulted in significant expansion of cells compared to the control (5.86 ± 0.11). Interestingly, addition of different concentrations of notch ligand (sDLL1) and IGF binding protein 2 (IGFBP2) to the standard culture medium formulation (FKT36, i.e., Flt-3, SCF, TPO, IL-3, and Il-6) had no significant effect on cell expansion ( Figure 5a However, the addition of both ligands to medium containing GM-CSF and G-CSF increased the cell number at day 14 (5.6 ± 0.72) but not at day 21 ( Figure 5a ). Surprisingly, the addition of 0.5 nm dose of E2 along with ERβ-selective LY3201 (0.5 nm) and ERα-selective PPT (1 nM) ligands significantly increased the number of expanded cells in both culture periods (day 14 and day 21). Similar effects were observed in terms of fold expansion of cells, where the addition of estrogen and its ligands in the absence of notch ligands significantly increased the fold expansion of both 14 and 21 day culture periods. In addition, the addition of E2 and its ligands in the presence of Notch ligands also resulted in an increased fold expansion of cells ( Figure 5b ).

[0306] The phenotypic characteristics of the expanded cells were evaluated by flow cytometry. As expected from the above observations, at day 14, the fold expansion of CD34+ HSPCs supplemented with GG was significantly higher compared to the control group ( Figure 6a However, at day 21, there was no significant difference in the fold expansion of CD34+ cells between all treatment groups, which may be due to the exhaustion of HSC self-renewal capacity at this time and thus reaching a stable phase. Interestingly, the percentage of CD34+ cells in total viable expanding cells did not change in both culture periods ( Figure 6b). These findings suggest that the expansion of UCB-HSCs under 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 referred to as LT-HSCs and also defined as CD38-, which were not much different from our starting cell population (CD34+CD38-HSCs). Surprisingly, the fold expansion of CD34+CD133+ cells was higher in GM-CSF and G-CSF supplemented media, while the addition of notch ligands had no significant effect at both 14 and 21 days of culture ( Figure 7 ). As expected from the above observations, the addition of estrogen significantly increased the fold expansion of CD34+CD133+ cells in both culture periods in the absence or presence of notch ligands. Furthermore, the addition of ER-selective ligands to the medium containing notch ligands also had significant expansion at day 21, but not at day 14, and this can be attributed in part to the highly selective nature of these ligands, which may take time to reach optimal effects in vitro.

[0307] Functional assessment and characterization of the repopulation capacity and long-term engraftment of ex vivo expanded HSPCs

[0308] The stem cell activity of ex vivo expanded HSC must be characterized and quantified. The ability of a single HSC to repopulate the entire hematopoietic system requires self-renewal and effective differentiation into each type of hematopoietic cell (37). However, direct evaluation of HSC function in humans is very limited, and alternative in vitro assays have therefore been developed for functional evaluation of immature HSC (38). Flow cytometry analysis is a widely accepted method that can prospectively identify and isolate HSC. Although it does not provide functional data, it can be used to assess repopulation capacity (39). Colony-forming cell assay (CFC) is a good in vitro method for identifying hematopoietic progenitor cells, which utilizes their ability to form multi-lineage colonies, which requires differentiation and some self-renewal capabilities. Functional evaluation of the most primitive HSC population can also be completed with in vitro cobblestone zone forming cells (CAFC) and long-term culture initiating cells (LTC-IC) assays (40), and both assays require a wider range of self-renewal capabilities than CFC assays. However, optimal assessment of HSCs requires in vivo engraftment into immunodeficient mice, such as NOD-SCID mice, and determination of long-term multilineage reconstitution and continuous reconstitution (41). We also expanded cord blood HSCs and assessed the cell surface phenotype and stem cell activity of the resulting cells during the culture process using an in vitro CFC assay.

[0309] CFC assays from the above experiment (#2) show that cells from all experimental groups (growth factor / ligand combinations) have the functional capacity of hematopoietic progenitor cells in addition to proliferation, and therefore retain their differentiation potential. In addition, all estrogen ligand-treated groups show the potential for HSPC differentiation into myeloid (CFU-GEMM, CFU-GM) and erythroid (CFU-E and BFU-E) lineages. Interestingly, in the culture medium supplemented with estrogen ligands, we did not observe any enhanced bias of the expanded HSC to the megakaryocyte-erythroid lineage.

[0310] Experiment 3

[0311] In another experiment, to fully evaluate the effect of estrogen on the proliferation of UCB-HSCs from cryopreserved cord blood units, we magnetically selected UCB-HSCs from cryopreserved cord blood units (Nationella Sahlgrenska Universitetssjukhuset Sweden) CD34+CD38- cells. As described in Experiments 1 and 2 above, cells were analyzed by flow cytometry. The results from this experiment show that serum-free medium (SFM / SPro34) and serum-containing medium (FBS medium) without standard growth factors known for HSC expansion (FKT) do not promote cell expansion at all. Surprisingly, in the absence of FKT factors, all viable cells die. However, with or without the addition of E2, the addition of these factors significantly promotes cell expansion in both serum-free and serum-containing medium. Compared with FKT medium alone, further addition of cytokines IL3 and IL6 (marked as 36) almost doubles cell expansion. Further addition of GM-CSF and G-CSF to 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 estrogen ligands significantly increased the number of expanded cells (in millions) compared to the control group (SFM / SPro34+FKT36, 3.7±0.1106) ( Figure 9aSurprisingly, the addition of E2 (at a dose of 10 nM) to a different serum-free medium with a mixture of FKT36GG resulted in a significant expansion of cells compared to the control group (SFM / SPro34+FKT36, 3.7 ± 0.1106). In addition, the addition of the ERβ selective ligand LY320 1 (1 nM) and the ERα selective ligand PPT (1 nM) also increased the number of expanded cells during the 14-day and 21-day culture periods ( Figure 9a Similar effects were observed in terms of fold expansion of cells, where the addition of estrogen and its ligands as well as our optimized growth factor / cytokine cocktail FKT36GG significantly increased fold expansion at both 14 and 21 days of culture ( Figure 9b ).

[0312] The phenotypic characteristics of the expanded cells were evaluated by flow cytometry. As expected from the above observations, at days 14 and 21, the expansion fold of CD34+ HSPCs was significantly higher in all serum-free media supplemented with FKT36GG compared to FBS media with standard FKT supplements or SFM / SPro34 media ( Fig.10a ). Different serum-free media performed relatively well using our optimized mixture of growth factors supplemented with estrogen ligands (FFKT36GG). We further analyzed the fold expansion of CD34+CD38- cells, which are often called long-term culture initiating cells (LTC-ICs) and are also considered primitive hematopoietic stem cells with long-term reconstitution capacity. Considering this fact, the starting population we used for this expansion experiment was also CD34+CD38-HSC. Surprisingly, at day 14, the fold expansion of CD34+CD38- cells in serum-free media (except StemMACS medium) supplemented with FKT36GG+E2 was almost two-fold higher or more (41.31-fold to 110.37-fold) compared to FKT36GG SFM / SPro34 medium alone (21.3-fold), whereas this effect was not evident at day 21, except for our in-house serum-free medium, which showed 223.79-fold (KISCO-H2) or 173.50-fold (KISCO-H1) expansion of CD34+CD38-LTC-IC HSCs compared to control medium (65.02-fold, SFM / SPro34+FKT36GG). Fig.10b ).

[0313] Functional assessment and characterization of the repopulation capacity and long-term engraftment of ex vivo expanded HSPCs

[0314] As described above in Experiment 2, we assessed the cell surface phenotype during culture, as well as the stem cell activity of the resulting cells, using an in vitro CFC assay.

[0315] The CFC assay from experiment (#3) showed that cells from all experimental groups (growth factor / ligand combinations) had the functional capacity of hematopoietic progenitor cells in addition to proliferation and thus retained their differentiation potential. In addition, compared with the thawed source cord blood unit (dashed line, Fig.11 ), all estrogen ligand-treated groups showed higher total colony forming units (CFU) of both myeloid (CFU-GEMM, CFU-GM) and erythroid (CFU-E and BFU-E) lineages compared to .

[0316] in conclusion

[0317] UCB-derived HSC is expanded in vitro to a therapeutically relevant number and is expected to treat malignant and non-malignant blood disorders. However, many methods for expanding cells have been reported, but reliable expansion in shorter and longer time periods has not been satisfactorily shown. In this article, we have demonstrated that UCB-HSC can be satisfactorily expanded with unique and optimal growth factors and ligand compositions without affecting its pluripotency under the conditions of shorter (8 days) and longer periods (14 days and 21 days). In addition, the culture medium supplement of the present application is significantly effective in the expansion of multipotent LT-HSCs with the addition of estrogen compounds, which enhances their circulation and helps to maintain self-renewal. The method was used to cause CD34+HSPC and CD34+CD133+LT-HSC to exceed 500 times and 30 times of expansion in 14 days. Interestingly, when the cells were cultured for 21 days, this expansion increased significantly and caused CD34+HSPC and CD34+CD133+LT-HSC to exceed 1500 times and 90 times of expansion. Thus, this study provides a reliable method to expand cord blood HSCs in both short and long ex vivo periods (1-3 weeks) without compromising their differentiation potential. This method far exceeds the number of expanded cells compared to any other method proposed elsewhere, which will exemplify the advantages of expanded UCB-HSCs in a clinical setting.

[0318] Table 1 - Selected abbreviations used in the examples

[0319]

[0320]

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[0365] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0366] Unless the context indicates otherwise, preferences and options for a given aspect, feature or parameter of the invention should be considered to have been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, any determined estrogen receptor agonist can be combined with any determined sample.

[0367] The present invention is also described as summarized in the following numbered paragraphs:

[0368] 1. A method for expanding CD34+ hematopoietic stem cells, comprising the following steps:

[0369] i) providing a sample comprising one or more CD34+ hematopoietic stem cells; and

[0370] ii) culturing the cells of step i) in the presence of one or more estrogen receptor agonists to expand CD34+ hematopoietic stem cells.

[0371] 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.

[0372] 3. The method of paragraph 2, wherein the CD34+ hematopoietic stem cells further comprise the marker CD133.

[0373] 4. The method of paragraph 2, wherein the CD34+ hematopoietic stem cells further contain a marker CD45 (低 / 中) .

[0374] 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.

[0375] 6. The method of any 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.

[0376] 7. The method of any 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 group consisting of: CD38 and / or Lin.

[0377] 8. The method of any one of paragraphs 1-7, wherein the CD34+ hematopoietic stem cell is a long-term repopulating hematopoietic stem cell (LT-HSC).

[0378] 9. The method of any of paragraphs 1-8, wherein the CD34+ hematopoietic stem cells comprise a persistent stemness phenotype.

[0379] 10. The method of any of paragraphs 1-9, wherein the culture medium is a serum-free culture medium.

[0380] 11. The method of any of paragraphs 1-10, wherein step ii) comprises culturing the cells of step i) in the absence of feeder cells.

[0381] 12. The method of any 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.

[0382] 13. The method of paragraph 12, wherein the one or more growth factors and / or cytokines are selected from the list consisting of: granulocyte-macrophage colony stimulating factor (GM-CSF); granulocyte colony stimulating factor (G-CSF); IGF binding protein 2 (IGFBP2); notch ligand; notch ligand sDLL1; interleukin-3 (IL-3); interleukin-6 (IL-6); Flt-3 ligand; stem cell factor or KIT ligand or steel factor (SCF); and / or thrombopoietin (TPO).

[0383] 14. The method of any 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, the isolated hematopoietic stem cells comprising CD34+ hematopoietic stem cells; and / or a blood sample; and / or a bone marrow sample.

[0384] 15. The method of paragraph 14, wherein the blood sample is one or more selected from the list consisting of: peripheral blood; mobilized peripheral blood; or umbilical cord blood.

[0385] 16. The method of any of paragraphs 1-15, wherein the estrogen receptor agonist is a nuclear estrogen receptor agonist and / or a membrane-bound estrogen receptor agonist.

[0386] 17. The method of paragraph 16, wherein the nuclear estrogen receptor agonist is an estrogen receptor alpha (ERα) agonist and / or an estrogen receptor beta (ERβ) agonist.

[0387] 18. The method of paragraph 17, wherein the ERa agonist is one or more molecules selected from the group consisting of: estrogen E2; estrogen El; estrogen E3; estrogen E4; and / or propylpyrazoletriol (PPT).

[0388] 19. The method of paragraph 17, wherein the ERβ agonist is one or more molecules selected from the group consisting of estrogen E2; estrogen E1; estrogen E3; estrogen E4; diarylpropionitrile (DPN); LY3201; and / or LY-500307.

[0389] 20. The method of paragraph 16, wherein the membrane-bound estrogen receptor agonist is one or more molecules selected from the group consisting of: a GPRC6A agonist; an ER-X agonist; and / or a Gq-mER agonist.

[0390] 21. The method of any 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 1 nM to about 10 nM.

[0391] 22. The method of any one of paragraphs 1-21, further comprising the step of isolating CD34+ hematopoietic stem cells.

[0392] 23. The method of paragraph 22, wherein the isolation of CD34+ hematopoietic stem cells comprises the use of flow cytometry and / or immunomagnetic cell separation and / or immunopanning.

[0393] 24. The method of any of paragraphs 1-23, wherein step ii) is performed for a period of about 8 days or more; for example, about 14 days or more; or about 21 days or more.

[0394] 25. The method of any one of paragraphs 1 to 24, wherein in the expanded CD34+ hematopoietic stem cells in step ii), there are one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ hematopoietic stem cells) of the CD34+ hematopoietic stem cell subset for every about two to about 20 CD34+ hematopoietic stem cells. (低 / 中) The ratio of CD133+CD38- hematopoietic stem cells).

[0395] 26. The method of any of paragraphs 1-25, wherein the number of expanded CD34+ hematopoietic stem cells of step ii) is increased by a fold change of about 88 or more.

[0396] 27. The method of any of paragraphs 1-26, wherein the number of expanded CD34+ hematopoietic stem cells of step ii) is increased by about 8,800% or more.

[0397] 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.

[0398] 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 CD34+ hematopoietic stem cells.

[0399] 30. The method of paragraph 29, further comprising the step of administering the one or more cells derived from CD34+ hematopoietic stem cells of step iii) to a subject in need thereof.

[0400] 31. The method of any of paragraphs 1-30, wherein the method is an ex vivo method and / or an in vitro method.

[0401] 32. Use of an estrogen receptor agonist for expanding one or more CD34+ hematopoietic stem cells.

[0402] 33. A population of CD34+ hematopoietic stem cells obtainable by and / or by a method as defined in any one of paragraphs 1 to 31 and / or by the use as defined in paragraph 32.

[0403] 34. One or more cells derived from CD34+ hematopoietic stem cells obtainable by and / or by the method defined in any one of paragraphs 1-31.

[0404] 35. The one or more cells of paragraph 34 or the method of any of paragraphs 1-31, wherein the one or more cells derived from CD34+ hematopoietic stem cells are cells selected from the list consisting of: myeloid progenitor cells; lymphoid progenitor cells; erythroid cells; erythrocytes; mast cells; megakaryocytes; platelet cells; mast cells; myeloblasts; basophils; neutrophils; eosinophils; monocytes; macrophages; dendritic cells; natural killer cells; T lymphocytes; and / or B lymphocytes.

[0405] 36. The one or more cells of paragraph 35 or the method of paragraph 35, wherein the T lymphocyte is a CAR-T cell.

[0406] 37. A composition comprising CD34+ hematopoietic stem cells obtainable by and / or by the method defined in any one of paragraphs 1 to 31, or a population of CD34+ hematopoietic stem cells as defined in paragraph 33.

[0407] 38. A kit comprising CD34+ hematopoietic stem cells obtainable by and / or by the method defined in any one of paragraphs 1 to 31, or a population of CD34+ hematopoietic stem cells as defined in paragraph 33.

[0408] 39. A kit of parts for expanding CD34+ hematopoietic stem cells, comprising one or more estrogen receptor agonists, and a sample comprising one or more CD34+ hematopoietic stem cells and / or means for obtaining a sample comprising one or more CD34+ hematopoietic stem cells.

[0409] 40. CD34+ hematopoietic stem cells obtainable by and / or by the method as defined in any one of paragraphs 1 to 31, or a population of CD34+ hematopoietic stem cells as defined in paragraph 33, for use in medicine.

[0410] 41. One or more cells derived from a CD34+ hematopoietic stem cell obtainable by and / or by a method as defined in any one of paragraphs 1 to 31, or derived from a population of CD34+ hematopoietic stem cells as defined in paragraph 33, for use in medicine.

[0411] 42. CD34+ hematopoietic stem cells obtainable by and / or by the method defined in any one of paragraphs 1 to 31, or a population of CD34+ hematopoietic stem cells as defined in paragraph 33, for use in treating and / or preventing a condition in a subject in need thereof.

[0412] 43. One or more cells derived from a CD34+ hematopoietic stem cell obtainable by and / or by the method defined in any one of paragraphs 1 to 31, or derived 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.

[0413] 44. Use of CD34+ hematopoietic stem cells obtainable by and / or by the method defined in any one of paragraphs 1 to 31 or a population of CD34+ hematopoietic stem cells as defined in paragraph 33 in the preparation of a medicament for treating and / or preventing a condition in a subject in need thereof.

[0414] 45. Use of one or more cells derived from a CD34+ hematopoietic stem cell obtainable by and / or by the method defined in any one of paragraphs 1 to 31 or derived from a CD34+ hematopoietic stem cell population as defined in paragraph 33 in the preparation of a medicament for treating and / or preventing a condition in a subject in need thereof.

[0415] 46. ​​A method for treating and / or preventing a disorder in a subject in need thereof, comprising administering one or more cells derived from a CD34+ hematopoietic stem cell obtainable by and / or by the method defined in any one of paragraphs 1-31 or derived from a CD34+ hematopoietic stem cell population as defined in paragraph 33.

[0416] 47. A method for treating and / or preventing a disorder in a subject in need thereof, comprising administering CD34+ hematopoietic stem cells obtainable by and / or by the method defined in any one of paragraphs 1-31, or a population of CD34+ hematopoietic stem cells as defined in paragraph 33.

[0417] 48. A method for treating and / or preventing a condition in a subject in need thereof, comprising the steps of:

[0418] a) providing a sample comprising one or more CD34+ hematopoietic stem cells;

[0419] b) culturing the cells of step a) in the presence of a nuclear estrogen receptor agonist to expand CD34+ hematopoietic stem cells; optionally differentiating the expanded CD34+ hematopoietic stem cells into one or more cells derived from CD34+ hematopoietic stem cells; and

[0420] c) administering the cells of step b) to a subject in need thereof.

[0421] 49. The CD34+ hematopoietic stem cells used in any of paragraphs 40-43, the use in any of paragraphs 44-45, or the method in any of paragraphs 46-48, wherein the condition is one or more conditions selected from the list consisting of: a condition requiring transplantation; a hematological condition and / or a hematological malignancy; a condition caused by failure or dysfunction of normal blood cell production and maturation; immunosuppression in subjects with malignancies and / or solid tumors; autoimmune diseases and / or immune conditions; genetic disorders; cancer; tumors; osteopetrosis; myelosclerosis; acquired hemolytic anemia; infectious conditions causing primary or secondary immunodeficiency; acquired immunodeficiency; neutrophil actin deficiency; neutrophil membrane GP-180 deficiency; bacterial infection Infections (e.g., brucellosis, listerosis, tuberculosis, and / or leprosy); parasitic infections (e.g., malaria and / or leishmaniasis); fungal infections; disorders involving an imbalance in the proportions of lymphoid cell groups and / or impaired immune function due to aging; phagocytic cell disorders; Kostmann agranulocytosis; chronic granulomatous disease; Chediak-Higachi syndrome; Wiskott-Aldrich syndrome; metabolic storage diseases; mucopolysaccharidoses; mucolipidoses; alpha 1-antitrypsin deficiency; and / or various disorders involving immune mechanisms.

[0422] 50. A method, use, population of CD34+ hematopoietic stem cells, one or more cells derived from CD34+ hematopoietic stem cells, composition, kit or method for treating and / or preventing a condition substantially as described herein with reference to the description and / or accompanying drawings.

Claims

1. A method for expanding CD34+ hematopoietic stem cells, comprising the following steps: 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 agonists 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 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); 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, wherein the isolated hematopoietic stem cells comprise 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; mobilized 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 estrogen receptor alpha (ERα) agonist or an estrogen receptor beta (ERβ) agonist.

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

13. The method of claim 11, wherein the ERβ agonist is one or more molecules selected from the group consisting of estrogen E2; estrogen El; 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 1 nM to about 10 nM.

15. The method of any one of claims 1-14, wherein step ii) is performed for a period of about 8 days or more; e.g., about 14 days or more; or about 21 days or more.

16. The method of any one of claims 1 to 15, wherein in the expanded CD34+ hematopoietic stem cells in step ii), there are one or more CD34+ hematopoietic stem cells (preferably CD34+CD133+ hematopoietic stem cells and / or CD34+CD133+CD38- hematopoietic stem cells and / or CD34+CD45+ hematopoietic stem cells) of the CD34+ hematopoietic stem cell subset for every about two to about 20 CD34+ hematopoietic stem cells. (低 / 中) The ratio of CD133+CD38- 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 by and / or by the method as defined in any one of claims 1 to 18 and / or the use as defined in claim 19.

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

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

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

24. CD34+ hematopoietic stem cells obtainable by and / or by a method as defined in any one of claims 1 to 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 use according to claim 23 or 24, wherein the condition is one or more conditions selected from the list consisting of: a condition requiring transplantation; a hematological condition and / or a hematological malignancy; a condition caused by failure or dysfunction of normal blood cell production and maturation; immunosuppression in subjects with malignancies and / or solid tumors; autoimmune diseases and / or immune conditions; genetic disorders; cancer; tumors; osteopetrosis; myelosclerosis; acquired hemolytic anemia; infectious disorders causing primary or secondary immunodeficiency; acquired immunodeficiency; neutrophil actin deficiency; neutrophil membrane GP-180 deficiency; bacterial infections (e.g., brucellosis, listeriosis, tuberculosis, and / or leprosy); parasitic infections (e.g., malaria and / or leishmaniasis); fungal infections; disorders involving an imbalance in the proportion of lymphoid cell groups and / or impaired immune function due to aging; phagocytic cell disorders; Kostmann agranulocytosis; chronic granulomatous disease; Chediak-Higachi syndrome; Wiskott-Aldrich syndrome; metabolic storage diseases; mucopolysaccharidosis; mucolipidosis; alpha 1-antitrypsin deficiency; and / or various disorders involving immune mechanisms.

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