WO2017193009A1

WO2017193009A1 - Maintenance, enrichment, enhancement and expansion of human hematopoietic stem cells

Info

Publication number: WO2017193009A1
Application number: PCT/US2017/031325
Authority: WO
Inventors: Timothy Webb
Original assignee: Transfusion Health, Llc
Priority date: 2016-05-05
Filing date: 2017-05-05
Publication date: 2017-11-09

Abstract

This invention is directed to, inter alia, methods, systems, and compositions for the enrichment, maintenance, enhancement, and expansion of hematopoietic stem cells derived from sources of non-mobilized peripheral blood.

Description

MAINTENANCE, ENRICHMENT, ENHANCEMENT AND EXPANSION OF HUMAN

HEMATOPOIETIC STEM CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62/322,438, filed May 5, 2016, the contents of which are incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0002] This invention is directed to, inter alia, methods and systems for maintaining and/or enhancing the expansion of hematopoietic stem cells in culture, media for culturing hematopoietic stem cells, and therapeutic compositions comprising the same for treatment of hematologic disorders.

BACKGROUND

[0003] The maintenance of the hematopoietic system relies on primitive pluripotent hematopoietic stem cells (HSCs) that have the capacity to self-renew and repopulate all the blood cell lineages with relevant progenitor cells. Due to their capacity for self-renewal and their pluripotent, long term reconstituting potential, HSCs have long been considered ideal for transplantation to reconstitute the hematopoietic system after treatment for various hematologic disorders or as a target for the delivery of therapeutic genes. Additionally, human HSCs have potential applications for restoring the immune system in autoimmune diseases and in the induction of tolerance for allogenic solid organ transplantation.

[0004] The classical hematopoietic expansion cytokines thrombopoietin (TPO), stem cell factor (SCF), interleukin-3 (IL-3) and fins-related tyrosine kinase 3 ligand (Flt31) are insufficient for the true maintenance and expansion of HSCs. In these cultures, HSCs generally lose their potency within a week. Cord blood may be one of the best sources for HSCs available due to the relative potency of the cells and ease of access. Cord blood banks have extensive, preserved stocks of cells that can be rapidly employed for therapeutic use. However, without extensive expansion of a single cord unit, each cord is unlikely to be used for more than one therapeutic dose or application.

[0005] Considering the therapeutic benefits that maintenance and expansion, or enhancement of HSCs and/or early hematopoietic progenitor cells would enable, it is critical that new, aggressive, efficient, yet safe protocols and reagents be developed to meet this goal. To date, no method has been developed that convincingly and reliably fills this unmet need.

[0006] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

SUMMARY

[0007] Provided herein, inter alia, are methods and compositions for the rapid expansion, enrichment, maintenance, enhancement, and expansion of hematopoietic stem cells derived from one or more sources of CD34+ cells.

[0008] Accordingly, in some aspects, provided herein are methods for expanding hematopoietic stem cells in culture, the method including contacting a source of CD34+ cells in culture with an effective amount of one or more agents that bind a tetraspanin, thereby expanding hematopoietic stem cells in the culture. In some aspects, source of CD34+ cells is bone marrow, cord blood, mobilized peripheral blood, or non-mobilized peripheral blood. In some aspects, the source of CD34+ cells is non-mobilized peripheral blood. In some aspects, the source of CD34+ cells includes: (a) CD34+ hematopoietic progenitors; (b) CD34+ early hematopoietic progenitors and/or stem cells; and/or (c) CD133+ early hematopoietic progenitors and/or stem cells.

[0009] In some aspects, the one or more agents that bind a tetraspanin are one or more agents selected from: an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and/or a small molecule chemical compound. In some aspects, the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof. In some aspects, the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof. [0010] In some aspects, the tetraspanin is tetraspanin 29 (TSPAN29). In some aspects, the tetraspanin is tetraspanin 28 (TSPAN28). In some aspects, the method further includes culturing the cells under low oxygen conditions. In some aspects, low oxygen conditions include an atmosphere containing about 5% oxygen or less. In some aspects, low oxygen conditions include an atmosphere containing about 10% oxygen or less. In some aspects, the method further includes contacting the cells with one or more agents selected from: thrombopoietin (TPO), stem cell factor (SCF), hepatocyte growth factor (HGF), P38 MAPK inhibitor, retinoic acid receptor (RAR) inhibitors or modulators, epidermal growth factor (EGF), JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors. In some aspects, the method further includes contacting the cells with thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L). In some aspects, the method further includes contacting the cells with a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor. In some aspects, the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670.

[0011] In some aspects, the method stabilizes the hematopoietic stem cell phenotype. In some aspects, the hematopoietic stem cell phenotype includes: CD45+, CD34+, CD133+, CD90+, CD45RA-, CD38 low/-, and negative for major hematopoietic lineage markers including CD2, CD3, CD4, CD5, CD8, CD14, CD16, CD19, CD20, CD56. In some aspects, CD133+ and/or CD90+ positive cells are increased compared to cells in culture that are not contacted with one or more agents that bind a tetraspanin. In some aspects, the cells exhibit at least about two times the number of CD 133+ and/or CD90+ positive cells compared to cells in culture that are not contacted with one or more agents that bind a tetraspanin. In some aspects, the source of the CD34+ cells is a human being.

[0012] In some aspects, provided herein are methods for expanding hematopoietic stem cells in culture including: (a) (i) a base medium or (ii) a feed medium; and (b) one or more agents that bind a tetraspanin. In some aspects, the one or more agents that bind a tetraspanin are one or more agents selected from: an antibody or functional binding fragment thereof, a non- antibody binding polypeptide, an aptamer, and a small molecule chemical compound. In some aspects, the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof. In some aspects, the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof. In some aspects, the tetraspanin is tetraspanin 29 (TSPAN29). In some aspects, the tetraspanin is tetraspanin 28 (TSPAN28).

[0013] In some aspects, the medium further includes: (c) one or more agents selected from: thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANGl), VEGF165, IL-10, laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OAC1), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C, and ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS). In some aspects, the medium further includes: (c) thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS). In some aspects, the medium further includes a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor. In some aspects, the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670. In some aspects, the FBS is heat inactivated. In some aspects, the base medium is a base salt medium. In some aspects, the base salt medium is alpha MEM. In some aspects, the base salt medium includes 320-380mOsm CaCl₂.

[0014] In some aspects, provided herein are methods for expanding hematopoietic stem cells in culture, the method including: contacting a source of CD34+ cells in culture with a medium as described above, thereby expanding hematopoietic stem cells in the culture.

[0015] In some aspects, provided herein are systems for expanding hematopoietic stem cells in culture, the system including: (a) a source of CD34+ cells in culture; and (b) a medium as described above.

[0016] In some aspects, the source of CD34+ cells includes: bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood. In some aspects, the source of CD34+ cells is non-mobilized peripheral blood. In some aspects, the source of CD34+ cells includes one or more of (a) CD34+ hematopoietic progenitors; (b) CD34+ early hematopoietic progenitors and/or stem cells; and/or (c) CD 133+ early hematopoietic progenitors and/or stem cells.

[0017] In some aspects, the system further includes (c) an atmosphere containing low oxygen. In some aspects, the atmosphere contains about 5% oxygen or less. In some aspects, the atmosphere contains about 10% oxygen or less. In some aspects, the source of CD34+ cells is a human being.

[0018] In some aspects, provided herein is a kit including: (a) (i) a base medium or (ii) a feed medium; and (b) one or more agents that bind a tetraspanin. In some aspects, the kit further includes: (c) written instructions for maintaining and/or expanding hematopoietic stem cells in culture.

[0019] In some aspects, the one or more agents that bind a tetraspanin are one or more agents selected from: an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and a small molecule chemical compound. In some aspects, the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof In some aspects, the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof. In some aspects, the tetraspanin is tetraspanin 29 (TSPAN29). In some aspects, the tetraspanin is tetraspanin 28 (TSPAN28).

[0020] In some aspects, the kit further includes: (d) one or more agents selected from: thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10, laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OAC1), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS). In some aspects the kit further includes: (d) thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms- related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS). In some aspects, the kit further includes a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor. In some aspects, the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670. In some aspects, the FBS is heat inactivated. In some aspects, the base medium is a base salt medium. In some aspects, the base salt medium is alpha MEM. In some aspects, the base salt medium includes 320-380mOsm CaCl₂.

[0021] In some aspects, provided herein is a population of hematopoietic stem cells produced by the methods described above.

[0022] In some aspects, provided herein is a therapeutic agent comprising the population of hematopoietic stem cells described above.

[0023] In some aspects, provided herein is a method of treating an individual in need of hematopoietic reconstitution, comprising administering to said individual the therapeutic agent described above. In some aspects, the individual is a bone marrow donor or recipient. In some aspects, the individual is diagnosed with cancer. In some aspects, the method is used as a supplemental treatment in addition to chemotherapy. In some aspects, the method is used to shorten the time between chemotherapy treatments. In some aspects, the individual is diagnosed with an autoimmune disease.

[0024] In some aspects, provided herein is a method for producing a cell culture media for culturing hematopoietic stem cells (HSC), the method including: combining (a) a base or a feed medium; and (b) one or more agents that bind a tetraspanin. In some aspects, the method further includes: combining (c) thrombopoietin (TPO) and/or stem cell factor (SCF). In some aspects, the method further includes: (d) combining one or more of insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10, laminin, caspase inhibitor(s),

epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OAC1), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fins-related tyrosine kinase 3 ligand (FLT3L), VEGF-C, and

ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS). In some aspects, the method further includes (d) adding insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS) to cell culture media for culturing hematopoietic stem cells (HSC). In some aspects, the method further includes adding a retinoic acid receptor (RA ) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor to cell culture media for culturing hematopoietic stem cells (HSC). In some aspects, the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670. In some aspects, the FBS is heat-inactivated FBS. In some aspects, the one or more agents that bind a tetraspanin are one or more agents selected from: an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and a small molecule chemical compound. In some aspects, the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof. In some aspects, the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof. In some aspects, the tetraspanin is tetraspanin 29 (TSPAN29). In some aspects, the tetraspanin is tetraspanin 28 (TSPAN28).In some aspects, the base or feed medium is Alpha MEM.

[0025] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 depicts a light micrograph of hematopoietic stem cells cultured in C5- 3H1B media with addition of anti-CD9 antibody.

[0027] FIG. 2 depicts CD34+ cells cultured over time both with and without the addition of anti-CD9 antibody to C5-3H1B media. The bar on the left for each condition represents the number of cells after 3 days in culture; the bar on the right for each condition represents the number of cells after 7 days in culture.

[0028] FIG. 3 depicts CD133+ cells measured as a percentage of CD34+ cells over time both with and without the addition of anti-CD9 antibody to C5-3H1B media. The bar on the left for each condition represents the number of cells after 3 days in culture; the bar on the right for each condition represents the number of cells after 7 days in culture. [0029] FIG. 4A, FIG. 4B, and FIG. 4C depict addition of anti-human CD9 directly to cultures augments the maintenance and enhancement of the hematopoietic stem cell phenotype. FIG. 4 A depicts addition of anti-CD9 to the "base" condition resulted in twice as many CD133+, CD90+ cells on day 3, with an additional doubling of cell numbers between days 3 and 7. The bar on the left for each condition represents the number of cells after 3 days in culture; the bar on the right for each condition represents the number of cells after 7 days in culture. FIG. 4B depicts that fluorescence intensity - and therefore concentration of the stem cell marker CD133 on the cell surface - increases rapidly with the addition of anti-CD9 to culture. The bar on the left for each condition represents the number of cells after 0 days in culture; the middle bar for each condition represents the number of cells after 3 days in culture; and the bar on the right for each condition represents the number of cells after 7 days in culture. FIG. 4C depicts

fluorescence intensity - and therefore concentration of the stem cell marker CD90 on the cell surface - increases rapidly with the addition of anti-CD9 to culture. The bar on the left for each condition represents the number of cells after 0 days in culture; the middle bar for each condition represents the number of cells after 3 days in culture; and the bar on the right for each condition represents the number of cells after 7 days in culture. Cultures were initiated with the same number of CD34+ and CD133+ cells. Fluorescence intensities shown below were calculated using the same cell populations shown in FIG. 4A.

[0030] FIG. 5 depicts addition of high osmolality CaCl₂ directly to cultures augments the maintenance and enhancement of the hematopoietic stem cell phenotype. CaCl₂ at a final osmolarity of 340mOsm was added to alpha MEM culture conditions. After 3 days, cells grown in high osmolarity CaCl₂ showed increased cell number when compared to those cells with and without anti-human CD9 in a culture media comprising a 50:50 mix of alpha MEM (constituents shown below in Table 2, available from ThermoFisher Scientific, Carlsbad, CA, Cat. No.

41061029) with 10% FBS and Peprotech's hESC media (Cat. No. BM-hESC, Peprotech, Rocky Hills, NJ) (shown in FIG. 5 as ESC/MEM or EM).

[0031] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG. 6E depict addition of SF 1670 to cultures including a tetraspanin improves the maintenance and enhancement of the hematopoietic stem cell phenotype. FIG. 6 A depicts addition of SF1670 and a tetraspanin (+CD9+SF) resulted in reduction in the number of CD45+ cells on days 7 and 14 as compared to addition of CD9 to base conditions (+CD9) and base conditions. FIG. 6B, 6C, and 6D depict the increase in CD34+ cells (6B), CD 133+ cells (6C), and CD90+ (6D) at days 7 and 14 when culture medium includes SF1670 and a tetraspanin (+CD9+SF) as compared to +CD9 or base conditions. FIG. 6E depicts the increase in CD90+/CD38 low/- cells at days 7 and 14 when culture medium includes SF1670 and a tetraspanin (+CD9+SF) as compared to +CD9 or base conditions. CD90+/CD38 low/- is a measure for the most primitive HSC cells. For each referenced Figure, the bar on the left for each condition represents the number of cells after 0 days in culture; the middle bar for each condition represents the number of cells after 7 days in culture; and the bar on the right for each condition represents the number of cells after 14 days in culture.

[0032] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E depict addition of SF1670 and ER50891 to cultures including a tetraspanin improves the maintenance and enhancement of the hematopoietic stem cell phenotype. FIG. 7A depicts the addition of SF1670 and anti-human CD9 antibodies (+SF conditions), or addition of ER50891 to +SF conditions (+SF+ER) resulted in reduction in the number of CD45+ cells on days 4 and 7 as compared to base conditions. FIG. 7B, 7C, and 7D depict the increase in CD34+ cells (7B), CD 133+ cells (7C), and CD90+ (7D) at days 4 and 7 when culture medium includes +SF and +SF+ER conditions as compared to base conditions. FIG. 7E depicts the increase in CD90+/CD38 low/- cells at days 4 and 7 when culture medium includes SF1670 and ER50891 (+SF+ER) as compared to +SF and base conditions. CD90+/CD38 low/- is a measure for the most primitive HSC cells. For each referenced Figure, the bar on the left for each condition represents the number of cells after 0 days in culture; the middle bar for each condition represents the number of cells after 4 days in culture; and the bar on the right for each condition represents the number of cells after 7 days in culture.

[0033] FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, and FIG. 8E depict addition of SF 1670 and ER50891 to cultures including a tetraspanin improves the maintenance and enhancement of the hematopoietic stem cell phenotype. FIG. 8A shows that addition of SF1670, anti-human CD9 antibodies, and growth factors IGF-1 and HGH (+SF conditions), or +SF conditions with ER50891 (+SF+ER conditions) resulted in reduction in the number of CD45+ cells on days 7 and 13 as compared to base conditions. FIG. 8B, 8C, and 8D depict the increase in CD34+ cells (8B), CD 133+ cells (8C), and CD90+ (8D) at day 13 when culture medium includes +SF+ER conditions as compared to +SF and base conditions. FIG. 8E depicts the increase in CD90+/CD38 low/- cells at days 7 and 13 when culture medium includes +SF+ER conditions as compared to Conditions +SF and base. CD90+/CD38 low/- is a measure for the most primitive HSC cells. For each referenced Figure, the bar on the left for each condition represents the number of cells after 0 days in culture; the middle bar for each condition represents the number of cells after 7 days in culture; and the bar on the right for each condition represents the number of cells after 13 days in culture.

[0034] FIG. 9A and FIG. 9B depict the stem cell preserving effects using anti-CD9 antibodies. FIG. 9A depicts the relative loss of CD 133+ cells when samples are cultured without an anti-CD9 antibody (-CD9 conditions) as compared to +CD9 conditions. The results are shown as a percentage of cells as compared to the +anti-CD9 condition. Over the course of 14 days in culture, the relative amount of CD 133+ cells decrease when cultured without anti- CD9 antibodies. FIG. 9B depicts the relative loss of CD90+ cells when samples are cultured without an anti-CD9 antibody (-CD9 conditions) as compared to +CD9 conditions. The results are shown as a percentage of cells relative to the +anti-CD9 condition.

[0035] FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, and FIG. 10E show that particular cytokines and growth factors such as pleotroiphin (PTN) do not substantially improve maintenance or enhancement of hematopoietic stem cells under the tested conditions. FIG. 10A, 10B, IOC, and 10D show that addition of PTN does not increase the relative amount of CD45+ cells on days 7 and 10 as compared to SF or SF+H+I conditions. FIG. 10E shows that addition of PTN does not significantly alter the amount of CD90+/CD38 low/- cells at days 7 and 10 as compared to SF and SF+H+I conditions. CD90+/CD38 low/- is a measure for the most primitive HSC cells. For each referenced Figure, the bar on the left for each condition represents the number of cells after 0 days in culture; the middle bar for each condition represents the number of cells after 7 days in culture; and the bar on the right for each condition represents the number of cells after 10 days in culture.

[0036] FIG. 11A and FIG. 11B depict the stem cell preserving effects using anti-CD9 antibodies. FIG. 11 A depicts the relative amount of CD34+ cells in -CD9 antibody and +CD9 antibody conditions. The results are shown as a percentage of cells as compared to the +CD9 condition. Over the course of 9 days in culture, the relative amount of CD34+ cells decrease when cultured without anti-CD9 antibodies. FIG. 11B depicts the relative amount of CD133+ in -CD9 and +CD9 conditions. The results are shown as a percentage of cells relative to the +CD9 condition. Over the course of 9 days in culture, the relative amount of CD 133+ cells decrease when cultured without anti-CD9 antibodies.

[0037] FIG. 12A and FIG. 12B depict the stem cell preserving effects using anti-CD81 antibodies. FIG. 12 A depicts the relative amount of CD34+ cells in -CD81 antibody and +CD81 antibody conditions. The results are shown as a percentage of cells as compared to the +CD81 condition. Over the course of 9 days in culture, the relative amount of CD34+ cells decrease when cultured without anti-CD81 antibodies. FIG. 12B depicts the relative amount of CD133+ in -CD81 and +CD81 conditions. The results are shown as a percentage of cells relative to the +CD81 condition. Over the course of 9 days in culture, the relative amount of CD 133+ cells decrease when cultured without anti-CD81 antibodies.

DETAILED DESCRIPTION

[0038] The invention described herein provides, inter alia, methods and compositions for the enrichment, maintenance, enhancement, and expansion of hematopoietic stem cells (HSCs) derived from one or more sources of CD34+ cells (such as, non-mobilized peripheral blood). Peripheral blood is known to reliably carry a small number of CD34+ hematopoietic progenitors and an even smaller number of CD34+ and CD 133+ early hematopoietic progenitors and stem cells. Being the source with the least potent, least enriched, most dilute and unpractically small numbers of apparent stem cells by nature, stem cell scientists have generally concluded that this source is unlikely to be therapeutically relevant compared to other potential sources of HSCs, such as bone marrow cells, mobilized peripheral blood, cord blood, and even embryonic or induced pluripotent stem cell (also known as iPS)-sourced CD34+ cells. Despite failed efforts to expand blood stem cells using more potent sources of cells, such as bone marrow and cord blood, there is some evidence that mitogenic, survival promoting, and quiescence inducing factors can impact the phenotype of these cells in positive ways and even help maintain them for some time in vitro.

[0039] The inventors of the present invention have surprisingly discovered that multipotent blood stem cells and progenitors can be successfully maintained, expanded, and enhanced by culturing these cells in a medium containing one or more agents that bind to a tetraspanin. In particular, the methods and compositions of the present invention are not only able to successfully derive HSCs from conventional sources, such as bone marrow, cord blood, and mobilized peripheral blood, but also from non-conventional sources such as non-mobilized peripheral blood. As such, the methods and compositions described herein provide for the generation of a therapeutically relevant stem cell transplant product derived from an easy to access and permanently available tissue source, without the need to expose the donor to significant risk or pain and which is more readily available than cord blood.

I. General Techniques

[0040] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, fourth edition (Sambrook et al., 2012) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as "Sambrook"); Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987, including supplements through 2014); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Antibodies: A

Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (Greenfield, ed., 2014), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000, (including supplements through 2014), Gene

Transfer and Expression in Mammalian Cells (Makrides, ed., Elsevier Sciences B.V.,

Amsterdam, 2003), and Current Protocols in Immunology (Horgan K and S. Shaw (1994) (including supplements through 2014).

Π. Definitions

[0041] Hematopoietic cells encompass not only HSCs, but also erythrocytes, neutrophils, monocytes, platelets, megakaryocytes, mast cells, eosinophils and basophils, B and T

lymphocytes and NK cells as well as the respective lineage progenitor cells.

[0042] As used herein, "maintaining the expansion" of HSCs refers to the culturing of these cells such that they continue to divide rather than adopting a quiescent state and/or losing their multipotent characteristics. Multipotency of cells can be assessed using methods known in the art using known multipotentcy markers. Example multipotency markers includes CD133+, CD90+, CD38 low/-, CD45RA negativity but overall CD45 positivity, and CD34. In some examples, CD34 low/- cells may be hematopoietic stem cells. In examples, where CD34 low/- cells are hematopoietic stem cells, these cells express CD133.

[0043] As used herein the term "cytokine" refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. The cytokines may be human in origin, or may be derived from other species when active on the cells of interest. Included within the scope of the definition are molecules having similar biological activity to wild type or purified cytokines, for example produced by recombinant means; and molecules which bind to a cytokine factor receptor and which elicit a similar cellular response as the native cytokine factor.

[0044] The term "culturing" refers to the propagation of cells on or in media (such as any of the media disclosed herein) of various kinds.

[0045] As used herein, the term "mobilized blood" refers to cells which have been exposed to an agent that promotes movement of the cells from the bone marrow into the peripheral blood and/or other reservoirs of the body (e.g., synovial fluid) or tissue. Conversely, the phrase "non-mobilized peripheral blood" refers to a blood sample obtained from an individual who has not been exposed to an agent that promotes movement of the cells from the bone marrow into the peripheral blood and/or other reservoirs of the body.

[0046] "Tetraspanins," (also called "tetraspans" or "the transmembrane 4 superfamily" (TM4SF)) as used herein, refer to a family of membrane proteins found in all multicellular eukaryotes that have four transmembrane domains, intracellular N- and C-termini and two extracellular domains: one called the small extracellular domain or loop (SED/SEL or ECl) and the other, longer (typically 100 amino acid residue), domain called the large extracellular domain/loop (LED/LEL or EC2). There are 34 tetraspanins in mammals, 33 of which have also been identified in humans. Tetraspanins display numerous properties that indicate their physiological importance in cell adhesion, motility, activation and proliferation, as well as their contribution to pathological conditions such as metastasis or viral infection.

[0047] An "individual" can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. In one aspect, an individual is a human.

[0048] "Treatment," "treat," or "treating," as used herein covers any treatment of a disease or condition of a mammal, for example, a human, and includes, without limitation: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, i.e., arresting its development; (c) relieving and or ameliorating the disease or condition, i.e., causing regression of the disease or condition; or (d) curing the disease or condition, i.e., stopping its development or progression. The population of individuals treated by the methods of the invention includes individuals suffering from the undesirable condition or disease, as well as individuals at risk for development of the condition or disease.

[0049] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention

[0050] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

Ill, Compositions of the Ifiventioa

[0052] Provided herein are cell cultures of expanded hematopoietic stem cells (HSC), cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells in culture, and populations of cells containing HSCs made from the methodology described herein. Hematopoietic stem cell can include mammalian and avian hematopoietic stem cells. A population of hematopoietic cells can have the potential for in vivo therapeutic application. The medium includes a base medium or a feed medium as well as one or more agents that bind a tetraspanin. Any suitable base or feed medium for culturing mammalian cells can be used in the context of the present invention and can include, without limitation, such commercially available media as DMEM medium, DM medium, 199/109 medium, HamF10/F12 medium, McCoy's 5 A medium, Alpha MEM medium (without and with phenol red), and RPMI 1640 medium. In some embodiments, the base or feed medium is Alpha MEM medium (without phenol red).

Tetraspanin hindiri a2ent$

[0053] The cell culture media for use in the methods disclosed herein contains one or more tetraspanin binding agents. Any agent capable of binding to a tetraspanin family member can be used as a component of the media compositions disclosed herein. Tetraspanin binding agents can include, without limitation, one or more of an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, a small molecule chemical compound or any combination thereof.

[0054] Tetraspanin binding agents are capable of binding any tetraspanin family member including, without limitation, one or more of TSPAN1 , TSPAN2, TSPAN3, TSPAN4 (NAG- 2), TSPAN5, TSPAN6, TSPAN7 (CD231/TALLA-1/A15), TSPAN8 (CO-029), TSPAN9 (NET-5), TSPAN10 (OCULOSPANIN), TSPAN11 (CD151-like), TSPAN12 (NET-2),

TSPAN13 (NET-6), TSPAN14, TSPAN15 (NET-7), TSPAN16 (TM4-B), TSPAN17,

TSPAN18, TSPAN19, TSPAN20 (UPlb, UPK1B), TSPAN21 (UPla, UPK1A), TSPAN22 (RDS, PRPH2), TSPAN23 (ROM1), TSPAN24 (CD151), TSPAN25 (CD53), TSPAN26

(CD37), TSPAN27 (CD82), TSPAN28 (CD81), TSPAN29 (CD9), TSPAN30 (CD63),

TSPAN31 (SAS), TSPAN32 (TSSC6), TSPAN33, and/or TSPAN34. In some embodiments, the tetraspanin binding agent specifically binds to TSPAN29 (CD9). In some embodiments, the tetraspannin binding agent specifically bindts to TSPAN28 (CD81).

[0055] The tetraspanin binding agent can be an antibody, such as a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antibody fragment thereof. An "antibody fragment," as used herein, refers to a fragment of a complete immunoglobulin, or a part of a polypeptide including a site where an antigen may bind. The antibody fragment may be, for example, F(ab')2, Fab', Fab, Fv, or scFv. A Fab has a structure that includes a light chain variable region, a heavy chain variable region, and a first constant region (CHI) of the heavy chain. Also, a Fab has one antigen binding site. Fab' is different from a Fab in that a Fab' has a hinge region including at least one cysteine residue at a C-terminal of the CHI domain of the heavy chain. A F(ab')2 is produced by an S-S binding between the cysteine residues of the hinge region of the Fab'. Fv is a minimal antibody fragment having only the heavy chain variable region and the light chain variable region. A two-chain Fv has a non-covalent bond between the heavy chain variable region and the light chain variable region, and a single -chain Fv (scFv) generally has a covalent bond by a peptide linker between the heavy chain variable region and the light chain variable region. A scFv may also form a dimer structure as in a two-chain Fv by directly binding the heavy chain variable region and the light chain variable region at the C- terminals thereof. The antibody fragment may be prepared using a protein hydrolase or by recombinant DNA technology. For example, a Fab may be obtained by restriction fragmentation of the complete antibody using papain and a F(ab')2 fragment may be obtained by fragmenting the complete antibody using a protease such as pepsin.

[0056] The concentration of tetraspanin binding agents in any of the media disclosed herein can range from about 1-12 μg/mL, about 2-10 μg/mL, about 4-8 μg/mL, about 3-6 μg/mL, about 1-8 μg/mL, or about 1-5 μg/mL, such as at least about 0.5 μg/mL, 1 μg/mL, 1.5 μg/mL, 2 μg/mL, 2.5 μg/mL, 3 μg/mL, 3.5 μg/mL, 4 μg/mL, 4.5 μg/mL, 5 μg/mL, 5.5 μg/mL, 6 μg/mL, 6.5 μg/mL, 7 μg/mL, 7.5 μg/mL, 8 μg/mL, 8.5 μg/mL, 9 μg/mL, 9.5 μg/mL, 10 μg/mL, 10.5 μg/mL, 11 μg/mL, 11.5 μg/mL, 12 μg/mL, or more, inclusive of all values falling in between. In some embodiments, the concentration of tetraspanin binding agents in the media is 5 μg/mL. In some embodiments, the concentration of tetraspanin binding agents can be at or below

0^g/mL. For example, in some embodiments, the concentration of tetraspanin binding agents includes 0.05μg/mL, 0.10μg/mL, 0.15μg/mL, 0.20μg/mL, 0.25μg/mL, 0.30μg/mL, 0.35μg/mL, 0.40μg/mL, 0.45μg/mL, or O^g/mL, including all values falling in between. In some embodiments, the concentration of tetraspanin binding agents in the media is about 0. 5μg/mL.

[0057] In some embodiments, the tetraspanin binding agent is one or more anti- tetraspanin 29 (CD9) antibodies or fragments thereof. CD9 is reported to be involved in cell fusion, adhesion, motility, proliferation, and signaling, has been implicated in the metastatic process, as inhibitor of cell invasion and metastasis, or as pro-metastatic, depending on the context (Rappa et al., Oncotarget. 2015 Apr 10;6(10):7970-91). Antibodies to CD9 are commercially available and include HI9a (BioLegend, San Diego, CA) and anti-CD9 clone M- L13 (BD Biosciences, San Jose, CA). Additional antibodies to CD9 also commercially available include PA5-11556 (Invitrogen, Carlsbad, CA) and C-4 (Santa Cruz Biotechnology, Dallas, TX). In one embodiment, the HSC cell culture media for use in any of the methods disclosed herein includes more than one tetraspanin binding agent (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more). In another embodiment, the HSC cell culture media contains two CD9 binding agents, such as, without limitation, HI9a and M-L13.

[0058] In some embodiments, the tetraspanin binding agent is one or more anti- tetraspanin 28 (CD81) antibodies or fragments thereof. CD81 is reported to be involved in signal transduction including the regulation of cell activation and motility. Antibodies to CD81 are commercially available and include 1D6 and NBP2-20564 (both from Novus Biologicals, Littleton, CO). In one embodiment, the HSC cell culture media for use in any of the methods disclosed herein includes more than one tetraspanin binding agent (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more). In another embodiment, the HSC cell culture media contains two CD81 binding agents, such as, without limitation, 1D6 and NBP2-20564.

[0059] In some embodiments, binding agents selective for two or more tetraspanins may be used concurrently. In some embodiments, the multiple tetraspanin binding agents may have an additive or positive effect (e.g. the expansion and enhancement effect of binding agents for two or more tetraspanins is enhanced in comparison to binding agents selective for only one tetraspanin.) In some embodiments, concurrent use of binding agents selective for two or more tetraspanins may have a deleterious effect (e.g. the expansion and enhancement effect of binding agents for two or more tetraspanins is dampened in comparison to binding agents selective for only one tetraspanin.)

[0060] In some embodiments, binding agents selective for tetraspanins may be used concurrently with binding agents selective for megakaryocyte surface markers (e.g. CXCR4, Integrin alpha 2b/CD41, glycoprotein V/CD42d, LIF R alpha, CXCRl/IL-8 RA, SLAM/CD 150, CXCR2/IL-8 RB, and Thrombopoietin R/Tpo R.). In some embodiments, the one or more tetraspanin binding agents used in conjunction with one or more megakaryocyte surface marker binding agents may have an additive or positive effect (e.g. the expansion and enhancement effect of the combination of binding agents is enhanced in comparison to binding agents selective for tetraspanins alone.) In some embodiments, the one or more tetraspanin binding agents used in conjunction with one or more megakaryocyte surface marker binding agents may have a deleterious effect (e.g. the expansion and enhancement effect of the combination of binding agents is dampened in comparison to binding agents selective for tetraspanins alone.) For example, the enhancement and expansion effects of culture conditions described herein including an anti-CD9 antibody may be hindered by concurrent culture with an anti-CD41 antibody.In some embodiments, tetraspanin binding agents may be used in conjunction with binding agents selective for other cytokines. In some embodiments these cytokines are tetrapsansins themselves, or may be megakaryote surface markets. In some embodiments, IL-3 binding agents may be used in conjunction with tetraspanin binding agents. In some

embodiments, an IL-3 binding agent maybe an antibody against IL-3. In some embodiments, co- culture with one or more tetraspanin binding agents and one or more cytokine binding agents may have an additive or positive effect on enhancement and expansion of multipotent cells.

B. Cytokines and Growth Factors

[0061] The cell culture media (e.g. base media or feed media) for use in the methods disclosed herein can contain one or more cytokines or growth factors. These agents promote the survival, maintenance, expansion, enhancement or enrichment of HSCs and can be procured via commercially available sources.

[0062] Cell culture media for culturing HSCs can include thrombopoietin (TPO).

Thrombopoietin is a glycoprotein hormone produced by the liver and kidney which regulates the production of platelets. It stimulates the production and differentiation of megakaryocytes, the bone marrow cells that bud off large numbers of platelets. The cell culture media compositions for use in the methods of the present invention can include about 50-250 ng/mL of TPO such as about 75-225 ng/mL, about 100-200 ng/mL, or about 125-175 ng/mL, or such as any of about 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 141 ng/mL, 142 ng/mL, 143 ng/mL, 144 ng/mL, 145 ng/mL, 146 ng/mL, 147 ng/mL, 148 ng/mL, 149 ng/mL, 150 ng/mL, 151 ng/mL, 152 ng/mL, 153 ng/mL, 154 ng/mL, 155 ng/mL, 156 ng/mL, 157 ng/mL, 158 ng/mL, 159 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 245 ng/mL, or 250 ng/mL or more TPO, including values falling in between these concentrations. In some embodiments, the

concentration of TPO in the media is about 150ng/mL.

[0063] Any of the cell culture media disclosed herein can also include stem cell factor (also known as SCF, KIT-ligand, KL, or steel factor). SCF is a cytokine that binds to the c-KIT receptor (CD117) and which plays a role in the regulation of HSCs in bone marrow. SCF has been shown to increase the survival of HSCs in vitro and contributes to the self-renewal and maintenance of HSCs in-vivo. The cell culture media compositions for use in the methods of the present invention can include about 5-100 ng/mL of SCF, such as about 10-90 ng/mL, about 20- 80, ng/mL about 30-70 ng/mL, about 40-60 ng/mL, or about 45-55 ng/mL, or such as any of about 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46 ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53 ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL, 59 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL or more SCF, including values falling in between these concentrations. In some embodiments, the cell culture media compositions for use in the methods of the present invention can include concentrations at lOOng/mL or above. Accordingly, concentrations of SCF also include l lOng/mL, 115ng/mL, 120ng/mL, 125ng/mL, 130ng/mL, 135ng/mL, 140ng/mL, 145ng/mL, 150ng/mL, 155ng/mL 160ng/mL, 165ng/mL, 170ng/mL, 175ng/mL, 180ng/mL 185ng/mL, 190ng/mL, 200ng/mL, or more SCF, including values falling in between these concentrations. In some embodiments, the concentration of SCF in the media is about lOOng/mL.

[0064] The cell culture media disclosed herein can also contain insulin-like growth factor 1 (IGF-1; also called somatomedin C). IGF-1 is a hormone similar in molecular structure to insulin. It plays an important role in childhood growth and has anabolic effects in adults. The cell culture media compositions for use in the methods of the present invention can include about 100-400 ng/mL IGF-1, such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL, about 225-275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL or more IGF-1, including values falling in between these concentrations. In some embodiments, the concentration of IGF-1 is the media is about 250ng/mL

[0065] The cell culture media for culturing HSCs provided herein can further include fms-related tyrosine kinase 3 ligand (FLT3L). FLT3L is a cytokine that stimulates cell growth, proliferation, and differentiation. The cell culture media compositions for use in the methods of the present invention can include about 20-400 ng/mL FLT3L, such as about 40-375 ng/mL, about 60-350 ng/mL, about 80-325 ng/mL, about 100-300 ng/mL, about 140-275 ng/mL, about 160-260 ng/mL, or about 180-255 ng/mL, or such as any of about 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL,100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL or more FLT3L, including values falling in between these concentrations. In some embodiments, the concentration of FLT3L in the media is about lOOng/mL.

[0066] The cell culture media for culturing HSCs provided herein can further include human growth hormone (HGH). HGH is a protein hormone that stimulates cell growth, proliferation, and differentiation. The cell culture media compositions for use in the methods of the present invention can include about 100-400 ng/mL EGF, such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL, about 225-275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL or more EGF, including values falling in between these concentrations. In some embodiments, the concentration of HGH in the media is about 250ng/mL.

[0067] The cell culture media for culturing HSCs provided herein can further include epidermal growth factor (EGF). EGF is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. The cell culture media compositions for use in the methods of the present invention can include about 100-400 ng/mL EGF, such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL, about 225- 275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL or more EGF, including values falling in between these concentrations.

[0068] Any of the cell culture media disclosed herein can also include hepatocyte growth factor (HGF). HGF is a paracrine cellular growth, motility and morphogenic factor. It is secreted by mesenchymal cells and acts primarily upon epithelial cells and endothelial cells, but also acts on hematopoietic progenitor cells and T cells. HGF has been shown to have a major role in embryonic organ development, specifically in myogenesis, in adult organ regeneration and in wound healing. The cell culture media compositions for use in the methods of the present invention can include about 100-400 ng/mL HGF, such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL, about 225-275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL or more HGF, including values falling in between these concentrations.

[0069] The cell culture media disclosed herein can also contain pleiotrophin (PTN). PTN is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. The cell culture media compositions for use in the methods of the present invention can include about 100-400 ng/mL PTN, such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL, about 225-275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL or more PTN, including values falling in between these

concentrations. In some embodiments, PTN does not improve the maintenance or enhancement of hematopoietic stem cells.

[0070] In further embodiments, the cell culture media compositions disclosed herein can additionally contain basic fibroblast growth factor (bFGF, FGF2 or FGF-β). bFGF is a critical component of human embryonic stem cell culture medium. However, while the growth factor is necessary for the cells to remain in an undifferentiated state, the mechanisms by which it does this are poorly defined. The cell culture media compositions for use in the methods of the present invention can include about 25-225 ng/mL of bFGF such as about 50-200 ng/mL, about 100-200 ng/mL, about 100-150 ng/mL, or about 115-135 ng/mL, or such as any of about 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 116 ng/mL, 117 ng/mL, 118 ng/mL, 119 ng/mL, 120 ng/mL, 121 ng/mL, 122 ng/mL, 123 ng/mL, 124 ng/mL, 125 ng/mL, 126 ng/mL, 127 ng/mL, 128 ng/mL, 129 ng/mL, 130 ng/mL, 131 ng/mL, 132 ng/mL, 133 ng/mL, 134 ng/mL, 135 ng/mL, 140 ng/mL, 141 ng/mL, 142 ng/mL, 143 ng/mL, 144 ng/mL, 145 ng/mL, 146 ng/mL, 147 ng/mL, 148 ng/mL, 149 ng/mL, 150 ng/mL, 151 ng/mL, 152 ng/mL, 153 ng/mL, 154 ng/mL, 155 ng/mL, 156 ng/mL, 157 ng/mL, 158 ng/mL, 159 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 245 ng/mL, or 250 ng/mL or more bFGF, including values falling in between these concentrations.

[0071] Any of the cell culture media disclosed herein can also include angiopoietin 1 (ANG1). ANG1 is a member of the angiopoietin family of vascular growth factors that play a role in embryonic and postnatal angiogenesis. The cell culture media compositions for use in the methods of the present invention can include about 25-225 ng/mL of ANG1 such as about 50- 200 ng/mL, about 100-200 ng/mL, about 100-150 ng/mL, or about 115-135 ng/mL, or such as any of about 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 116 ng/mL, 117 ng/mL, 118 ng/mL, 119 ng/mL, 120 ng/mL, 121 ng/mL, 122 ng/mL, 123 ng/mL, 124 ng/mL, 125 ng/mL, 126 ng/mL, 127 ng/mL, 128 ng/mL, 129 ng/mL, 130 ng/mL, 131 ng/mL, 132 ng/mL, 133 ng/mL, 134 ng/mL, 135 ng/mL, 140 ng/mL, 141 ng/mL, 142 ng/mL, 143 ng/mL, 144 ng/mL, 145 ng/mL, 146 ng/mL, 147 ng/mL, 148 ng/mL, 149 ng/mL, 150 ng/mL, 151 ng/mL, 152 ng/mL, 153 ng/mL, 154 ng/mL, 155 ng/mL, 156 ng/mL, 157 ng/mL, 158 ng/mL, 159 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 245 ng/mL, or 250 ng/mL or more ANG1, including values falling in between these concentrations.

[0072] Interleukin 10 (IL-10) can also be a component of any of the cell culture media compositions disclosed herein. IL-10 is a cytokine with multiple, pleiotropic, effects in immunoregulation and inflammation. It downregulates the expression of Thl cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κΒ activity, and is involved in the regulation of the JAK-STAT signaling pathway. The cell culture media compositions for use in the methods of the present invention can include about 1-25 ng/mL of IL-10 such as about 5-20 ng/mL, 10-20 ng/mL, or 12-18 ng/mL, such as any of about 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, or 25 ng/mL of IL-10. [0073] Interleukin 3 (IL-3) can also be a component of any of the cell culture media compositions disclosed herein. IL-3 is a cytokine with multiple, pleiotropic, effects in immunoregulation and inflammation. The cell culture media compositions for use in the methods of the present invention can include about 1-25 ng/mL of IL-3 such as about 5-20 ng/mL, 10-20 ng/mL, or 12-18 ng/mL, such as any of about 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, or 25 ng/mL of IL-3.

[0074] The cell culture media disclosed herein can also contain vascular endothelial growth factor 165 (VEGF165), which belongs to the PDGF/VEGF growth factor family. Many cell types secrete VEGF165, which it is a potent angiogenic factor and mitogen that stimulates proliferation, migration, and formation of endothelial cells. The cell culture media compositions for use in the methods of the present invention can include about 5-100 ng/mL of VEGF165, such as about 10-90 ng/mL, about 20-80, ng/mL about 30-70 ng/mL, about 40-60 ng/mL, or about 45-55 ng/mL, or such as any of about 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46 ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53 ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL, 59 ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL or more VEGF165, including values falling in between these concentrations.

[0075] The cell culture media disclosed herein can also contain vascular endothelial growth factor C (VEGF-C), which belongs to the PDGF/VEGF growth factor family. Many cell types secrete VEGF-C, which functions in angiogenesis, and endothelial cell growth, stimulating proliferation and migration and also has effects on the permeability of blood vessels. The cell culture media compositions for use in the methods of the present invention can include about 50- 1000 ng/mL of VEGF-C, such as about 100-900 ng/mL, about 200-800, ng/mL about 300-700 ng/mL, about 400-600 ng/mL, or about 450-550 ng/mL, or such as any of about 50 ng/mL, 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL, 410 ng/mL, 420 ng/mL, 430 ng/mL, 440 ng/mL, 450 ng/mL, 460 ng/mL, 470 ng/mL, 480 ng/mL, 490 ng/mL, 500 ng/mL, 510 ng/mL, 520 ng/mL, 530 ng/mL, 540 ng/mL, 550 ng/mL, 560 ng/mL, 570 ng/mL, 580 ng/mL, 590 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, 1000 ng/mL or more VEGF-C, including values falling in between these concentrations.

[0076] In yet additional embodiments, the cell culture media compositions disclosed herein can contain laminins, which are high-molecular weight (~400kDa) proteins of the extracellular matrix. They are a major component of the basal lamina (one of the layers of the basement membrane), a protein network foundation for most cells and organs. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, and adhesion. The cell culture media compositions for use in the methods of the present invention can include about 500-1000 ng/mL laminin, such as about 600-900 ng/mL, about 700-800 ng/mL, about 725-775 ng/mL, or about 745-755 ng/mL, or such as any of about 500 ng/mL, 525 ng/mL, 550 ng/mL, 575 ng/mL, 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 705 ng/mL, 710 ng/mL, 715 ng/mL, 720 ng/mL, 725 ng/mL, 730 ng/mL, 735 ng/mL, 740 ng/mL, 741 ng/mL, 742 ng/mL, 743 ng/mL, 744 ng/mL, 745 ng/mL, 746 ng/mL, 747 ng/mL, 748 ng/mL, 749 ng/mL, 750 ng/mL, 751 ng/mL, 752 ng/mL, 753 ng/mL, 754 ng/mL, 755 ng/mL, 756 ng/mL, 757 ng/mL, 758 ng/mL, 759 ng/mL, 760 ng/mL, 765 ng/mL, 770 ng/mL, 775 ng/mL, 780 ng/mL, 785 ng/mL, 790 ng/mL, 795 ng/mL, 800 ng/mL, 825 ng/mL, 850 ng/mL, 875 ng/mL, 900 ng/mL, 925 ng/mL, 950 ng/mL, 975 ng/mL, 1000 ng/mL or more laminin, including values falling in between these concentrations.

C. Other small molecules

[0077] The cell culture media for use in the methods disclosed herein can additionally contain various small molecule inhibitors, such as a caspase inhibitors, DNA methylation inhibitors, P38 MAPK inhibitors, glycogen synthase kinase 3 (GSK3) inhibitors, phosphatase and tensin homolog (PTEN) inhibitors, and/or JAK/STAT inhibitors. In one embodiment, the DMSO concentration of the cell culture media does not exceed 0.025% v/v.

[0078] In some embodiments, the cell culture media for use in the methods disclosed herein includes one or more a caspase inhibitors. Caspases are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation. As of November 2009, twelve caspases have been identified in humans. There are two types of apoptotic caspases: initiator (apical) caspases and effector (executioner) caspases. Initiator caspases (e.g., CASP2, CASP8, CASP9, and CASP10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (e.g., CASP3, CASP6, CASP7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process. The cell culture media compositions for use in the methods of the present invention can include about 1 -10 μg/mL caspase inhibitor, such as any of about 2-8 μg/mL, about 3-7 μg/mL, or about 4-6 μg/mL, or such as any of about 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL or more caspase inhibitor. In one embodiment, the caspase inhibitor is Z-VAD-FMK.

[0079] The cell culture media for use in the methods disclosed herein can include one or more DNA methylation inhibitors. DNA methylation is a process by which methyl groups are added to DNA which modifies its function. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. The cell culture media compositions for use in the methods of the present invention can include about 300-700 nM DNA methylation inhibitors, such as about 350-650 nM, about 400-600 nM, about 450-550 nM, about 475-525 nM, or about 490-510 nM or such as any of about 300 nM, 325 nM, 350 nM, 400 nM, 425 nM, 430 nM, 435 nM, 440 nM, 445 nM, 450 nM, 455 nM, 460 nM, 465 nM, 470 nM, 475 nM, 480 nM, 485 nM, 490 nM, 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497 nM, 498 nM, 499 nM, 500 nM, 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506 nM, 507 nM, 508 nM, 509 nM, 510 nM, 515 nM, 520 nM, 525 nM, 530 nM, 535 nM, 540 nM, 545 nM, 550 nM, 555 nM, 560 nM, 565 nM, 570 nM, 575 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, or more DNA methylation inhibitors, including values falling in between these concentrations. In some embodiments, the DNA methylation inhibitor is epigallocatechin gallate (EGCG). In other embodiments, the cell culture media compositions for use in the methods of the present invention can include about 0.25-3 uM DNA methylation inhibitors, such as about 0.5-2.5 uM, about 1-2 uM, or about 1.25- 1.75 uM, such as any of about 0.5 uM, 1 μΜ, 1.5 μΜ, 2 μΜ, 2.5 μΜ, or 3 μΜ or more DNA methylation inhibitors, including values falling in between these concentrations. In some embodiments, the DNA methylation inhibitor is Oct4-activating compound 1 (OAC1).

[0080] Any of the cell culture media disclosed herein can also include a P38 MAPK inhibitor. P38 mitogen-activated protein kinases are a class of mitogen-activated protein kinases that are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell differentiation, apoptosis and autophagy. The cell culture media compositions for use in the methods of the present invention can include about 400-800 nM P38 MAPK inhibitor, such as about 500-700 nM, about 550-650 nM, about 600-650 nM, or about 615-635 nM, or such as any of about 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 525 nM, 550 nM, 575 nM, 600 nM, 605 nM, 610 nM, 615 nM, 616 nM, 617 nM, 618 nM, 619 nM, 620 nM, 621 nM, 622 nM, 623 nM, 624 nM, 625 nM, 626 nM, 627 nM, 628 nM, 629 nM, 630 nM, 631 nM, 632 nM, 633 nM, 634 nM, 635 nM, 640 nM, 645 nM, 650 nM, 655 nM, 660 nM, 665 nM, 670 nM, 675 nM, 680 nM, 685 nM, 690 nM, 695 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, or more P38 MAPK inhibitor, including values falling in between these concentrations. In some embodiments, the P38 MAPK inhibitor is BIRB796.

[0081] In yet additional embodiments, the cell culture media compositions disclosed herein can contain a glycogen synthase kinase 3 (GSK3) inhibitor. GSK3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. Phosphorylation of a protein by GSK-3 usually inhibits the activity of its downstream target. GSK-3 is active in a number of central intracellular signaling pathways, including cellular proliferation, migration, glucose regulation, and apoptosis. The cell culture media compositions for use in the methods of the present invention can include about 0.25-2 uM GSK3 inhibitor, such as about 0.5-1.5 uM, or 1.75-1.25 uM, such as about 0.25 uM, 0.3 uM, 0.4 uM, 0.5 uM, 0.6 uM, 0.7 uM, 0.8 uM, 0.9 uM, 1 uM, 1.1 uM, 1.2 uM, 1.3 uM, 1.4 uM, 1.5 uM, 1.6 uM, 1.7 uM, 1.8 uM, 1.9 uM, or 2 uM or more GSK3 inhibitor, including values falling in between these concentrations. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0082] In further embodiments, the cell culture media compositions disclosed herein can additionally contain a retinoic acid receptor (RAR) antagonist. The RAR is a nuclear receptor as well as a transcription factor that is activated by both all-trans retinoic acid and 9-cis retinoic acid. The cell culture media compositions for use in the methods of the present invention can include about 100-300 nM RAR antagonist, such as about 125-275 nM, about 150-250, or about 175-225, or such as any of about 100 nM, 105 nM, 110 nM, 115 nM, 120 nM, 125 nM, 130 nM, 135 nM, 140 nM, 145 nM, 150 nM, 155 nM, 160 nM, 165 nM, 170 nM, 175 nM, 180 nM, 185 nM, 190 nM, 191 nM, 192 nM, 193 nM, 194 nM, 195 nM, 196 nM, 197 nM, 198 nM, 199 nM, 200 nM, 201 nM, 202 nM, 203 nM, 204 nM, 205 nM, 206 nM, 207 nM, 208 nM, 209 nM, 210 nM, 215 nM, 220 nM, 225 nM, 230 nM, 235 nM, 240 nM, 241 nM, 242 nM, 243 nM, 244 nM, 245 nM, 246 nM, 247 nM, 248 nM, 249 nM, 250 nM, 251 nM, 252 nM, 253 nM, 254 nM, 255 nM, 256 nM, 257 nM, 258 nM, 259 nM, 260 nM, 265 nM, 270 nM, 275 nM, 280 nM, 285 nM, 290 nM, 295 nM, 300 nM or more RAR antagonist, including values falling in between these concentrations. The cell culture media compositions for use in the methods of the present invention also include RAR antagonist at concentrations at or below ΙΟΟηΜ. Accordingly, concentrations of RAR antagonist also include 50nM, 75nM, ΙΟΟηΜ, 125nM, 150nM, 175nM, 200nM, 225nM, 250nM, 275nM and 300nM, including values falling in between these concentrations. In some embodiments, the RAR antagonist is ER50891. In some embodiments, the concentration of ER50891 is about lOOnM.

[0083] In other embodiments, the cell culture media for use in the methods disclosed herein includes a phosphatase and tensin homolog (PTEN) inhibitor. PTEN is a tumor suppressor that is mutated in a large number of cancers at high frequency. This protein negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating Akt/PKB signaling pathway. The cell culture media compositions for use in the methods of the present invention can include about 300-700 nM PTEN inhibitor, such as about 350-650 nM, about 400-600 nM, about 450-550 nM, about 475-525 nM, or about 490-510 nM or such as any of about 300 nM, 325 nM, 350 nM, 400 nM, 425 nM, 430 nM, 435 nM, 440 nM, 445 nM, 450 nM, 455 nM, 460 nM, 465 nM, 470 nM, 475 nM, 480 nM, 485 nM, 490 nM, 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497 nM, 498 nM, 499 nM, 500 nM, 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506 nM, 507 nM, 508 nM, 509 nM, 510 nM, 515 nM, 520 nM, 525 nM, 530 nM, 535 nM, 540 nM, 545 nM, 550 nM, 555 nM, 560 nM, 565 nM, 570 nM, 575 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, or more PTEN inhibitor, including values falling in between these concentrations. The cell culture media compositions for use in the methods of the present invention also include PTEN inhibitors at concentrations at or below 300nM. Accordingly, concentrations of PTEN inhibitor also include 50nM, 75nM, lOOnM, 125nM, 150nM, 175nM, 200nM, 225nM, 250nM, 275nM and 300nM, including values falling in between these concentrations. In some embodiments, the PTEN inhibitor is SF1670. In some embodiments, the concentration of SF1670 is about 250nM. [0084] The cell culture media disclosed herein can also include a JAK/STAT inhibitor. The JAK-STAT signaling pathway transmits information from extracellular chemical signals to the nucleus resulting in DNA transcription and expression of genes involved in immunity, proliferation, differentiation, apoptosis and oncogenesis. The cell culture media compositions for use in the methods of the present invention can include about 300-700 nM JAK/STAT inhibitor, such as about 350-650 nM, about 400-600 nM, about 450-550 nM, about 475-525 nM, or about 490-510 nM or such as any of about 300 nM, 325 nM, 350 nM, 400 nM, 425 nM, 430 nM, 435 nM, 440 nM, 445 nM, 450 nM, 455 nM, 460 nM, 465 nM, 470 nM, 475 nM, 480 nM, 485 nM, 490 nM, 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497 nM, 498 nM, 499 nM, 500 nM, 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506 nM, 507 nM, 508 nM, 509 nM, 510 nM, 515 nM, 520 nM, 525 nM, 530 nM, 535 nM, 540 nM, 545 nM, 550 nM, 555 nM, 560 nM, 565 nM, 570 nM, 575 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, or more

JAK/STAT inhibitor, including values falling in between these concentrations. In some embodiments, the JAK/STAT inhibitor is Tofacitinib.

[0085] In addition to the inhibitor molecules described above, any of the cell culture media compositions disclosed herein can also contain fetal bovine serum (FBS) in concentrations ranging from 1-20% v/v, such as about 2-18% v/v, about 5-15% v/v, about 7.5-12.5% v/v or such as any of about 1% v/v, 2% v/v, 3% v/v, 4% v/v, 5% v/v, 6% v/v, 7% v/v, 8% v/v, 9% v/v, 10% v/v, 11% v/v, 12% v/v, 13% v/v, 14% v/v, 15% v/v, 16% v/v, 17% v/v, 18% v/v, 19% v/v, or 20% v/v or more FBS, including values falling in between these percentages. In some embodiments, the FBS is heat inactivated FBS. In some embodiments, the concentration of FBS in the medium is about 10% v/v.

[0086] In addition to the inhibitor molecules described above, any of the cell culture media compositions disclosed herein can also contain added salts, for example KC1, NaCl, MgCl, or CaCl₂. In one example, CaCl₂ may be added to achieve in concentrations ranging from 300-380mOsm, such as about 300mOsm, about 310mOsm, about 320mOsm, about 330mOsm, about 340mOsm, about 350mOsm, about 360mOsm, about 370 mOsm, about 380mOsm, or more CaCl₂, including values falling in between these numbers. High osmolarity CaCl₂ may also be used to select against non-multipotent cells, selecting for an HSC phenotype. [0087] In addition to the inhibitor molecules described above, any of the cell culture media compositions disclosed herein may be adjusted to comprise an overall higher osmolarity. Multipotent stem cells may be better adapted to withstand atypical osmolarity (e.g., a high osmolarity media may select against non-stem cell phenotypes.) Osmolarity may be adjusted, for example, by the addition of salts as above, or by glucose.

[0088] In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes one or more agents that bind a tetraspanin, TPO, SCF, FLT3L, HGH, and IGF-1. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells further includes a retinoic acid receptor (RA ) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor. In some embodiments, the PTEN inhibitor is SF1670. In some embodiments, the RAR inhibitor is ER50891.

[0089] In some embodiments, the base or feed medium in the above described cell culture media is Alpha MEM medium (without phenol red). In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells further includes head inactivated FBS.

[0090] In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to

TSPAN29 (CD9). In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN29 (CD9) and SF1670. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN29 (CD9) and ER50891. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN29 (CD9), SF1670, and ER50891.

[0091] In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to

TSPAN28 (CD81). In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN28 (CD81) and SF1670. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN28 (CD81) and ER50891. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN28 (CD81), SF1670, and ER50891.

[0092] In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to

TSPAN29 (CD9) and TSPAN28 (CD81). In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN29 (CD9), an antibody that specifically binds to TSPAN28 (CD81), and SF1670. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN29 (CD9), an antibody that specifically binds to TSPAN28 (CD81), and ER50891. In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes an antibody that specifically binds to TSPAN29 (CD9), an antibody that specifically binds to TSPAN28 (CD81), SF1670, and ER50891.

[0093] In some embodiments, the cell culture media for maintaining and/or enhancing the expansion of hematopoietic stem cells includes a based or feed medium of Alpha MEM medium (without phenol red); and further includes an antibody that binds to TSPAN29, TPO, SCF, FLT3L, IGF-1, SF1670, ER50891, and heat inactivated FBS.

[0094] Populations of cells containing HSCs provided herein confer the advantages found in cord blood. A person of skill in the art would readily recognize the characteristics of stem cells from cord blood and the advantageous properties therein. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the populations of cells containing HSCs provided herein are expanded HSC cells. In some embodiments, the expanded HSC cells in the populations of cells have retained their stem cell phenotype for an extended period of time. For example, in some embodiments, populations of cells containing HSCs include expanded HSC cells with cell surface phenotypes that include CD45+, CD34+, CD133+, CD90+, CD45RA-, and/or CD38 low/- and have been cultured in vitro for at least 3, 7, 10, 13, 14, 20, 25, 30, 40, or 50 or more days. In some embodiments, populations of cells containing HSCs include expanded HSC cells with cell surface phenotypes that includes CD 133+ and/or CD90+ and have been cultured in vitro for at least 3, 7, 10, 13, 14 or more days.

IV, Methods of the Invention

A. Maintaining and/or enhancing the expansion of hematopoietic stem cells in culture

[0095] Provided herein are methods for maintaining and/or enhancing the expansion of hematopoietic stem cells (HSCs) in culture. The method involves contacting a source of CD34+ cells in culture with one or more agents that bind a tetraspanin.

1. Sources of CD34+ cells

[0096] The methods of the present invention require a source of CD34+ blood cells, or in some examples CD341ow/-, CD133+ cells. These cells can be obtained from tissue sources such as, e.g., bone marrow, cord blood, placental blood, mobilized peripheral blood, non-mobilized peripheral blood, or the like, or combinations thereof.

[0097] CD34+ cells can, in certain embodiments, express or lack the cellular marker CD133. Thus, in specific embodiments, the hematopoietic cells useful in the methods disclosed herein are CD34+CD133+ or CD34+CD133-. In other embodiments, CD34+ cells can express or lack the cellular marker CD90. As such, in these embodiments, the hematopoietic cells useful in the methods disclosed herein are CD34+CD90+ or CD34+CD90-. Thus, populations of CD34+cells, or in some examples CD341ow/-, CD 133+ cells, can be selected for use in the methods disclosed herein on the basis of the presence of markers that indicate an undifferentiated state, or on the basis of the absence of lineage markers indicating that at least some lineage differentiation has taken place.

[0098] CD34+ cells used in the methods provided herein can be obtained from a single individual, e.g., from a source of non-mobilized peripheral blood, or from a plurality of individuals, e.g., can be pooled. Where the CD34+ cells are obtained from a plurality of individuals and pooled, it is preferred that the hematopoietic cells be obtained from the same tissue source. Thus, in various embodiments, the pooled hematopoietic cells are all from, for example, placenta, umbilical cord blood, peripheral blood (mobilized or non-mobilized), and the like.

[0099] Interestingly, cells enhanced and expanded by methods of the present invention are phenotypically similar to cord blood. Accordingly, it may be possible to use cells expanded and enhanced by methods described herein as a source for further expansion and enhancement. For example, it may be possible, following an initial expansion and enhancement to allow, or gently encourage, cells toward differentiation. These cells may be allowed to expand and can then be brought back from a differentiated, or near differentiated state, by following the methods of the invention utilized in the initial expansion/enhancement step. This expansion of differentiated, or nearly differentiated cells which can then be returned to a multipotent state may occur over multiple cycles.

[0100] CD34+ cells, or in some examples CD341ow/-, CD133+ cells, can be isolated from a source using any conventional means known in the art such as, without limitation, positive selection against stem cell markers, negative selection against lineage markers, size exclusion, detection of metabolic differences in the cells, detection of differences in clearance or accumulation of a substance by the cell, adhesion differences, direct culturing of buffy coat under conditions exclusively supportive for stem cells. The source of CD34+ cells for use in the methods of the present invention can contain a number of sub-species of hematopoietic progenitor cells including, without limitation, one or more of CD34+ hematopoietic progenitors; CD34+ early hematopoietic progenitors and/or stem cells; and/or CD133+ early hematopoietic progenitors and/or stem cells.

2. Maintaining HSCs in culture

[0101] CD34+ cells derived from the sources described above are cultured in any of the cell culture media described herein. These media maintain and enhance the hematopoietic stem cell phenotype. Furthermore, the addition of one or more agents that bind a tetraspanin augments these effects. Specifically, use of a tetraspanin binding agent in the culture media increases the rate of expansion of HSCs while maintaining (and usually improving) all measured stem cell markers (such as, but not limited to CD133 and CD90 positive cells). These improvements can be seen after as little as 3 days of culture. In some embodiments, the tetraspanin binding agent used in the culture is one or more antibodies to tetraspanin 29 (CD9).

[0102] In particular, source cells cultured in any of the cell culture media described herein exhibit increased numbers of CD 133+ and/or CD90+ positive cells compared to source cells that are not cultured in any of the media described herein after about any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 25, 30, 35, 40, 45, or 50 days or more in culture. Specifically, source cells cultured in the media described herein using the methods disclosed herein exhibited around 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 7.5, or 10 or more times the number of CD 133+ and/or CD90+ positive cells compared to source cells that are not cultured in any of the media described herein after about any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 days or more in culture.

[0103] Source cells cultured in the cell culture media described herein also exhibit increased number of CD90+/CD38 low/- cells compared to source cells that are not cultured in any of the media described herein after about any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 25, 30, 35, 40, 45, or 50 days or more in culture. Specifically, source cells cultured in the media described herein using the methods disclosed herein exhibited around 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 7.5, 10, 12.5, 15, 17.5, or 20 or more times the number of CD90+/CD38 low/- cells compared to source cells that are not cultured in any of the media described herein after about any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 days or more in culture.

[0104] In some embodiments, source cells cultured in the cell culture media described herein exhibit increased numbers of CD90+/CD38 low/- cells compared to source cells that are not cultured in any of the media described herein after about 7 or 13 days in culture.

Specifically, source cells cultured in media comprising a base medium or feed medium, one or more agents that bind a tetraspanin, a retinoic acid receptor (RA ) inhibitor, and a PTEN inhibitor exhibited at least 5, 10, 15, or 20 or more times the number of CD90+/CD38 low/- cells compared to source cells that are not cultured in any of the media described herein after about 7 or 13 days in culture.

[0105] In some embodiments, source cells cultured in the cell culture media described herein exhibit increased numbers of CD90+/CD38 low/- cells compared to source cells that are not cultured in any of the media described herein after about 7 or 13 days in culture. Specifically, source cells cultured in media comprising a base medium or feed medium, one or more agents that bind a tetraspanin, SF1670, and a ER50891 exhibited at least 5, 10, 15, or 20 or more times the number of CD90+/CD38 low/- cells compared to source cells that are not cultured in any of the media described herein after about 7 or 13 days in culture.

[0106] The cell culture methods disclosed herein include culturing cells under low oxygen conditions. As used herein, the phrase "low oxygen conditions" refers to an atmosphere to which the cultured cells are exposed having less than about 5% oxygen, such as any of about 4.5%, 4%, 3.5%, 3%, 2.75%, 2.5%, 2.25%, 2%, 1.75%, 1.5%%, 1.25%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% or less oxygen. "Low oxygen conditions" can also refer to an atmosphere to which the culture cells are exposed to having 10%, 9.5, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, or 5% or lower amounts of oxygen. "Low oxygen conditions" can also refer to any range in between 0.5% and 10% oxygen. Control of atmospheric oxygen in cell culture can be performed by any means known in the art, such as by addition of nitrogen.

[0107] The invention also contemplates populations of cells that made by the methods described herein. Populations of cells containing HSCs provided herein confer the advantages found in cord blood. A person of skill in the art would readily recognize the characteristics of stem cells from cord blood and the advantageous properties therein. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the populations of cells containing HSCs provided herein are expanded HSC cells. In some embodiments, the expanded HSC cells in the populations of cells have retained their stem cell phenotype for an extended period of time. For example, in some embodiments, populations of cells containing HSCs include expanded HSC cells with cell surface phenotypes that include CD45+, CD34+, CD133+, CD90+, CD45RA-, and/or CD38 low/- and have been cultured in vitro for at least 3, 7, 10, 13, 14, 20, 25, 30, 40, or 50 or more days. In some embodiments, populations of cells containing HSCs include expanded HSC cells with cell surface phenotypes that includes CD 133+ and/or CD90+ and have been cultured in vitro for at least 3, 7, 10, 13, 14 or more days.

B. Methods of treatment

[0108] Provided herein are methods for treating an individual in need of hematopoietic reconstitution. The method involves administering to the individual a therapeutic agent containing any of the cultured HSCs derived according to the methods of the present invention.

[0109] One of ordinary skill in the art may readily determine the appropriate

concentration, or dose of the cultured HSCs disclosed herein for therapeutic administration. The ordinary artisan will recognize that a preferred dose is one that produces a therapeutic effect, such as preventing, treating and/or reducing diseases, disorders and injuries, in a patient in need thereof. Of course, proper doses of the cells will require empirical determination at time of use based on several variables including but not limited to the severity and type of disease, injury, disorder or condition being treated; patient age, weight, sex, health; other medications and treatments being administered to the patient; and the like.

[0110] An effective amount of cells may be administered in one dose, but is not restricted to one dose. Thus, the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of pharmaceutical composition. Where there is more than one administration of a therapeutic agent in the present methods, the administrations can be spaced by time intervals of one minute, two minutes, three, four, five, six, seven, eight, nine, ten, or more minutes, by intervals of about one hour, two hours, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. In the context of hours, the term "about" means plus or minus any time interval within 30 minutes. The administrations can also be spaced by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The invention is not limited to dosing intervals that are spaced equally in time, but encompass doses at non-equal intervals.

[0111] A dosing schedule of, for example, once/week, twice/week, three times/week, four times/week, five times/week, six times/week, seven times/week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, and the like, is available for the invention. The dosing schedules encompass dosing for a total period of time of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, and twelve months.

[0112] Provided are cycles of the above dosing schedules. The cycle can be repeated about, e.g., every seven days; every 14 days; every 21 days; every 28 days; every 35 days; 42 days; every 49 days; every 56 days; every 63 days; every 70 days; and the like. An interval of non-dosing can occur between a cycle, where the interval can be about, e.g., seven days; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63 days; 70 days; and the like. In this context, the term "about" means plus or minus one day, plus or minus two days, plus or minus three days, plus or minus four days, plus or minus five days, plus or minus six days, or plus or minus seven days.

[0113] Cells derived from the methods of the present invention may be formulated for administration according to any of the methods disclosed herein in any conventional manner using one or more physiologically acceptable carriers optionally comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen. The compositions may also be administered to the individual in one or more physiologically acceptable carriers. Carriers for cells may include, but are not limited to, solutions of normal saline, phosphate buffered saline (PBS), lactated Ringer's solution containing a mixture of salts in physiologic concentrations, or cell culture medium.

[0114] The HSC populations of the invention and therapeutic agents comprising the same can be used to augment or replace bone marrow cells in bone marrow transplantation. Human autologous and allogenic bone marrow transplantation are currently used as therapies for diseases such as leukemia, lymphoma and other life-threatening disorders. The drawback of these procedures, however, is that a large amount of donor bone marrow must be removed to insure that there are enough cells for engraftment.

[0115] The HSC populations of the invention and therapeutic agents comprising the same can provide stem cells and progenitor cells that would reduce the need for large bone marrow donation. It would also be, according to the methods of the invention, to obtain a small marrow donation and then expand the number of stem cells and progenitor cells culturing and expanding in the placenta before infusion or transplantation into a recipient. Alternatively, sufficient numbers of HSCs can be obtained according to the methods of the present invention using only non-mobilized peripheral blood, thereby completely eliminating the need for bone marrow donation altogether.

[0116] Compositions and methods of the present invention are useful in the expansion of stem cells. I some embodiments, the expansion can be rapid compared to traditional methods of expansion. In some embodiments, expansion may occur in the course of hours, days, or weeks (e.g., selective expansion can occur for about 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, one day, two days, three days, four days, five days, six days, seven days, nine days, ten days, 11 days, 12 days, 13 days, two weeks, three weeks, four weeks, or more. In some embodiments, a stem cell population may be expanded in terms of total cell count by two-fold, three-fold, four-fold, five-fold, 6~fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more. In some embodiments, a stem cell population may be expanded in terms of the relative number of cells with a stem cell phenotype in a broader cell population (e.g. cells with a stem cell phenotype may make up about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 98%, 99%, or 100% of a cell population). Expansion may be measured by a number of metrics including by doubling time for example, by the amount of time it takes for a total cell number to double (e.g., from 500 cells to 1,000 cells), or the time it takes for a relative percentage of the population to double (e.g., from 10% stem cells to 20% stem cells).

[0117] In another embodiment, the HSC populations of the invention and therapeutic agents comprising the same can be used in a supplemental treatment in addition to

chemotherapy. Most chemotherapy agents used to target and destroy cancer cells act by killing all proliferating cells, i.e., cells going through cell division. Since bone marrow is one of the most actively proliferating tissues in the body, hematopoietic stem cells are frequently damaged or destroyed by chemotherapy agents and in consequence, blood cell production is diminishes or ceases. Chemotherapy must be terminated at intervals to allow the patient's hematopoietic system to replenish the blood cell supply before resuming chemotherapy. It may take a month or more for the formerly quiescent stem cells to proliferate and increase the white blood cell count to acceptable levels so that chemotherapy may resume (when again, the bone marrow stem cells are destroyed). [0118] While the blood cells regenerate between chemotherapy treatments, however, the cancer has time to grow and possibly become more resistant to the chemotherapy drugs due to natural selection. Therefore, the longer chemotherapy is given and the shorter the duration between treatments, the greater the odds of successfully killing the cancer. To shorten the time between chemotherapy treatments, the HSC populations of the invention and therapeutic agents comprising the same cultured according to the methods of the invention could be introduced into the individual. Such treatment would reduce the time the individual would exhibit a low blood cell count, and would therefore permit earlier resumption of the chemotherapy treatment.

C. Methods for producing a cell culture medium

[0119] Further provided herein are methods for producing a cell culture medium (such as any of the cell culture media disclosed herein) for culturing hematopoietic stem cells (HSC). The method involves combining a base or a feed medium; and one or more agents that bind a tetraspanin (including any of the tetraspanin binding agents disclosed herein, such as, CD9 antibodies, e.g., H19 and/or M-L13). In additional embodiments, the method also includes combining one or both of stem cell factor (SCF) and/or thrombopoietin (TPO). The method can also include combining one or more of a caspase inhibitor, a DNA methylation inhibitor, a p38 MAPK inhibitor, a GSK3 inhibitor, an RAR receptor antagonist, a PTEN inhibitor, an inhibitor of the JAK/STAT pathway, and/or FBS (such as, heat inactivated FBS).

[0120] A "base medium," as used herein, is a medium used for culturing cells which is, itself, directly used to culture the cells and is not used as an additive to other media, although various components may be added to a base medium. Examples of base media include, without limitation, DMEM medium, IMDM medium, 199/109 medium, HamF10/F12 medium, McCoy's 5 A medium, Alpha MEM medium (without and with phenol red), and RPMI 1640 medium. A base medium may be modified, for example by the addition of salts, glucose, or other additives.

[0121] A "feed medium" is a medium used as a feed in a culture of a source of CD34+ cells (e.g. bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood cells). A feed medium, like a base medium, is designed with regard to the needs of the particular cells being cultured. Thus, a base medium can be used as a basis for designing a feed medium. A feed medium can have higher concentrations of most, but not all, components of a base culture medium. For example, some components, such as salts, maybe kept at about IX of the base medium concentration, as one would want to keep the feed isotonic with the base medium. Thus, in some embodiments, various components are added to keep the feed medium physiologic and others are added because they replenish nutrients to the cell culture. Other components, for example, nutrients, maybe at about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X or more of their normal concentrations in a base medium.

V. Systems and Kits

[0122] Also provided herein are systems for maintaining and/or enhancing the expansion of hematopoietic stem cells in culture. This system includes a source of CD34+ cells in culture (such as a CD34+ cells from one or more of bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood) and any of the cell culture media compositions described herein. In a particular embodiment, the system of the present invention maintains low oxygen culturing conditions. As such, the system provides an atmosphere to which the cultured cells are exposed having less than about 5% oxygen, such as any of about 4.5%, 4%, 3.5%, 3%, 2.75%, 2.5%, 2.25%, 2%, 1.75%, 1.5%%, 1.25%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% or less oxygen. In some embodiments the system provides an atmosphere to which the culture cells are exposed to having 10%, 9.5, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, or 5% or lower amounts of oxygen. In some embodiments, the system provides an atmosphere to which the culture cells are exposed having any range in between 0.5% and 10% oxygen. Control of atmospheric oxygen in the system can be accomplished by any means known in the art, such as by addition of nitrogen.

[0123] In additional aspects, the invention disclosed herein provides one or more kits. These kits can include either a base medium or a feed medium (such as, but not limited to, DMEM medium, DM medium, 199/109 medium, HamF10/F12 medium, McCoy's 5 A medium, Alpha MEM medium (without and with phenol red), and RPMI 1640 medium) as well as one or more agents that bind a tetraspanin (including, without limitation, one or more of an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, a small molecule chemical compound or any combination thereof). In some

embodiments, the one or more agents that bind a tetraspanin is an antibody or functional binding fragment thereof (such as, one or more antibodies to TSPAN39 (CD9)). In other embodiments, the tetraspanin binding agents are the antibodies HI9a and M-L13.

[0124] The kit can also include written instructions for maintaining and/or enhancing the expansion of HSCs in culture by culturing the cells using the kit's cell culture media

components. The kit can also include additional components for inclusion into the cell culture media, such as one or more of thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10, laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OAC1), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fins-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and

ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS) (including heat-inactivated FBS).

[0125] In some embodiments, the kit includes also thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fins-related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS). In some embodiments, the kit also includes a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor. In some embodiments, the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670.

[0126] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0127] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting. EXAMPLES

Example 1: Isolation, enrichment, enhancement, and stabilization of hematopoietic stem cells derived from non-mobilized peripheral blood

[0128] This Example describes the isolation and culturing of hematopoietic stem cells derived from non-mobilized peripheral blood.

Materials and Methods

[0129] CD34+ cells were isolated from donor peripheral blood. Whole blood was centrifuged at 1750g (the speed of centriguation may vary) for 20 minutes. Plasma was drawn off using a pipette or syringe. Concentrated red blood cell (RBC) layer was drawn off with a pipette or syringe. Residual RBCs were lysed using standard RBC lysis protocols. White blood cell (WBC) layer was washed in PBS and spun down several times at 300g for 7 minutes to remove platelets and some debris. Cells were pelleted and incubated with unlabeled CD64 antibody. Cells then undergo negative depletion using biotinylated CD2, CD3, CD4, CD5, CD8, CD1 lb, CD14, CD16, CD19, CD20, CD45RA, CD56, CD235 (in some examples CD15, CD25 and other lineage specific antibodies may also be used). Cells which bind these antibodies are depleted using streptavidin beads. The resultant progenitor enriched cell pool were cultured directly (residual mature cells and late progenitors will be killed off over time in culture) or cell pool can undergo a CD34 or CD133 positive selection to further enrich for the desired cells before culturing.

[0130] Isolated CD34+ cells were incubated in an in vitro culture media described in Table 1, designated C5-3H1B.

Table 1:

[0131] Cultures were incubated at 1.75% oxygen (controlled by nitrogen) and 5% C0₂.

[0132] Stem cell properties verified by flow cytometry, purification of CD133+, CD90+ cells from 2-week old culture followed by 28-day single cell methylcellulose assays. Following ~2 week culture, CD90+, CD133+ cells were triple sorted using flow cytometry (BD FACS ARIA II) to ensure purity. Purified CD90+, CD 133+ cells were then diluted such that, on average, a single cell occupies its own well in a culture dish (for example, a 96 well plate). Following 14 days in complete methylcellulose formulation (from RD Systems) wells that expanded were passaged into their own well in a larger format (for example, a 12-well dish) for at least another 14 days. Only wells that form colonies (clusters of cells exceeding 20) in the passaged and unpassaged wells count as likely originating from an HSC. Results

[0133] Using the media and culture conditions described above, hematopoietic stem cells stem cells were cultured, maintained, and expanded for greater than 2 weeks.

[0134] Continuous culture in C5-3H1B with the addition of anti-CD9 antibody leads to a change in the adherence properties of the cells. They appear more likely to grow in tight colonies as opposed to loosely associated colonies or single cells. The cells are also more likely to alternate between a semi-adherent state and suspended state on plastic culture dish (FIG. 1).

[0135] For handling, manipulation, analysis and therapeutic uses the cells may need to be effectively dissociated into single cells. However, manual, mechanical dissociation may not be entirely effective without harm to the cells. Alternating between C5-3H1B conditions with and without addition of CD9 antibody can either prevent clustering or colony formation or reverse this effect for downstream applications.

[0136] To minimize the effect of anti-CD9 on adherence and homing behaviors of the stem cells and early progenitors, at the end of the culture term, a "washout phase" can be beneficial. This is done by removing CD9 from the culture and putting the cells in C5-3H1B or other maintenance or supportive proliferative conditions until turnover of the CD9 protein has occurred sufficiently to restore a more normal phenotype to the cells. Detection of bound CD9 on the surface of the cells and migration assays using cytokine gradients such as G-CSF or SDF-1 can confirm normalized properties of the cells.

Example 2: C5-3H1B enriches cultures for stem cells and early progenitors, but late progenitors are eliminated

[0137] This Examples shows that cells cultured using the media and methods described in Example 1 are enriched for stem cells and early progenitor cells.

Materials and Methods

[0138] Small molecule components are added separately and fresh each time the media needs to be refreshed. Cytokines were stored together. Antibodies solutions were depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal occurred at least every few days. Recipe follows the respective component charts in this document.

Results

[0139] Flow cytometric analysis of three and seven day cells cultured in C5-3H1B medium demonstrates that CD34+ and CD133- cell counts progressively decline over time in the same cultures both with and without the addition of anti-CD9 antibodies (FIG. 2). CD 133+ cells measured as a percentage of CD34+ cells show an increase over time in the same cultures both with and without the addition of anti-CD9 antibodies (FIG. 3). This demonstrates that the phenotype enriched for hematopoietic stem cells is maintained much better than later, less potent progenitors. Of note is that CD34+, CD133- cells are dying off as CD34+, CD 133+ cells are simultaneously maintained and enhanced, with some expansion".

Example 3: Manipulation of tetraspanin class surface proteins in cultured HSCs

[0140] This Example shows that addition of anti-human CD9 to C5-3H1B augments the maintenance and enhancement of the hematopoietic stem cell phenotype.

Materials and Methods

[0141] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines were stored together. Antibodies solutions were depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days. Recipe follows the respective component charts in this document.

Results

[0142] Use of anti-CD9 in culture increased the rate of expansion and maintained (and usually improved) all measured hematopoietic stem cell markers. Improvements were seen after 3 and 7 days of culture (FIG. 4A-C). Cultures were initiated with the same number of CD34+, CD133+ cells. Fluorescence intensities shown below were calculated using the same cell populations shown in FIG. 4A. Example 4: High osmolality CaCl¾ enhances the effects of manipulation of tetraspanin class surface proteins in cultured HSCs

[0143] This Example describes that addition of high osmolality CaCl₂ further augments the effects of anti-human CD9 on the maintenance and enhancement of the hematopoietic stem cell phenotype

Materials and Methods

[0144] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days. Recipe follows the respective component charts in this document.

Results

[0145] Use of anti-CD9 in culture increased the rate of expansion. This effect was enhanced by the presence of high osmolality CaCl₂. CaCl₂ at a final osmolarity of 340mOsm was added to alpha MEM culture conditions (shown in FIG. 5 as CaCl). After 3 days, cells grown in high osmolarity CaCl₂ showed increased cell number when compared to those cells with and without anti-human CD9 in a culture media comprising a 50:50 mix of alpha MEM (constituents shown below in Table 2, available from ThermoFisher Scientific, Carlsbad, CA, Cat. No.

41061029) with 10% FBS and Peprotech's hESC media (Cat. No. BM-hESC, Peprotech, Rocky Hills, NJ) (shown in FIG. 5 as ESC/MEM or E/M). The 50:50 ESC/MEM media contains CaCl₂ at 300mOsm. The result was surprising as 340mOsm CaCl₂ is far outside the normal

physiological range for calcium

Table 2: Components of MEM

o um yruvate . . .

Example 5: Addition of anti IL-3 has an positive expansive effect

[0146] Antibodies to IL-3 and antibodies to CD9 in culture have an positive effect to the expansion and enhancement of hematopoietic stem cells derived from blood. Using methods described herein for the selection of multipotent stem cells from blood, these cells are expanded and enhanced in vitro by culture conditions such as those described in Examples 2, 3, or 4 above with the addition of an anti-IL-3 antibody. The anti-IL-3 antibody is added at a concentration of 2μg/mL or less. Anti-IL-3 antibody further selects against non-stem cells, while allowing the expansion and enhancement of multipotent cells in culture.

Example 6: Cyclical expansion and enhancement of multipotent cell populations.

[0147] It has been observed that the compositions and methods of the present invention described above produce a cell population with phenotypic similarities to cord blood.

Enhancement of multipotent cell properties using methods of the present invention produces cell populations that can later differentiate as do cord blood cells. By utilizing the differentiating capabilities and cord blood like phenotype of the cell populations, increased expansion and enhancement of cell populations are made. This extended enhancement and expansion need not require maintenance in a multipotent state, but rather an initial enhancement and expansion of selected cells from blood isolation. Following an initial expansion and enhancement cells are allowed, or gently encouraged, toward differentiation. These cells are allowed to expand and can then be brought back from a differentiated, or near differentiated state, by following the methods of the invention utilized in the initial expansion/enhancement step. This expansion of differentiated, or nearly differentiated cells which can then be returned to a multipotent state occurs over multiple cycles.

Example 7: Addition of VEGF-C has an positive expansive effect

[0148] VEGF-C and antibodies to CD9 in culture have a positive effect to the expansion and enhancement of hematopoietic stem cells derived from blood. Using methods described hereinabove for the selection of multipotent stem cells from blood, these cells are expanded and enhanced in vitro by culture conditions such as those described in Examples 2, 3, or 4 above with the addition of VEGF-C. The VEGF-C is added at a concentration of 500ng/mL. VEGF-C acts to enhance the selective expansion and enhancement of multipotent cells in culture, with minimal effect on the non-stem cells.

Example 8: Addition of SF1670 to cultures including a tetraspanin have a positive expansive effect

[0149] This Example describes that addition of SF1670 to cultures including a tetraspanin further augments the effects of anti-human CD9 on the maintenance and

enhancement of the hematopoietic stem cell phenotype.

Materials and Methods

CD34+ cells were isolated from donor peripheral blood as described in Example 1. Isolated CD34+ cells were incubated in an in vitro culture media of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetal bovine serum (FBS); Base, +CD9, and +CD9+SF further included the components described in Table 3. Each condition tested also included an antibiotic solution that includes penicillin, streptomycin, and amphotericin B to avoid contamination.

Table 3: Additional Components included in the culture media of Base, +CD9, and +CD9+SF conditions.

[0150] Cultures were incubated at 5% oxygen and 5% C0₂.

[0151] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days. [0152] On the days indicated one-half of the volume of the cell culture was removed for data analysis (flow cytometry). The culture volume was replenished with fresh media according to the conditions tested. The data reported accounts for the dilution factor introduced in this procedure.

Results

[0153] Flow cytometic analysis of seven and 14 day cell cultures as described above demonstrates that addition of SF1670 to cultures including a tetraspanin improves the

maintenance and enhancement of hematopotentic stem cells (FIG. 6A-E). In particular, FIG. 6B-D illustrate the increase in CD34+ cells, CD 133+ cells (6C), and CD90+ (6D) at days 7 and 14 when culture medium includes SF1670 (+CD9+SF) as compared to Conditions 1 and 2. FIG. 6E shows the increase in CD90+/CD38 low/- cells at days 7 and 14 when culture medium includes SF1670 (+CD9+SF) as compared to +CD9 and base conditions. CD90+/CD38 low/- is a measure for the most primitive HSC cells.

Example 9: Addition of SF1670 and ER50891 to cultures including a tetraspanin have a positive expansive effect

[0154] This Example describes that addition of SF1670 and ER50891 together in culture further augments the effects of anti-human CD9 on the maintenance and enhancement of the hematopoietic stem cell phenotype. In particular, the addition of SF1670 and ER50891 together greatly improve expansion of the primitive HSC cell surface phenotype, CD90+/CD38 low/-.

Materials and Methods

CD34+ cells were isolated from donor peripheral blood as described in Example 1. Isolated CD34+ cells were incubated in an in vitro culture media of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetal bovine serum (FBS); Base Conditions, +SF Conditions, and +SF+ER conditions further included the components described in Table 4. Each condition tested also included an antibiotic solution that includes penicillin, streptomycin, and

amphotericin B to avoid contamination.

Table 4: Additional Components included in the culture media of Base Conditions, +SF Conditions, and +SF+ER Conditions.

[0155] Cultures were incubated at 5% oxygen and 5% C0₂. [0156] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days.

[0157] On the days indicated one-half of the volume of the cell culture was removed for data analysis (flow cytometry). The culture volume was replenished with fresh media according to the conditions tested. The data reported accounts for the dilution factor introduced in this procedure.

Results

[0158] Flow cytometic analysis of four and seven day cell cultures as described above demonstrates that addition of SF1670 and ER50891 to cultures including a tetraspanin improves the maintenance and enhancement of hematopotentic stem cells (FIG. 7A-E). In particular, FIG. 7E shows the increase in CD90+/CD38 low/- cells at days 7 and 14 when culture medium includes SF1670 and ER50891 as compared to base conditions and +SF conditions.

CD90+/CD38 low/- is a measure for the most primitive HSC cells.

Example 10: Addition of SF1670 and ER50891to cultures including a tetraspanin has a positive expansive effect

[0159] This Example describes that addition of SF1670 and ER50891 together in culture further augments the effects of anti-human CD9 on the maintenance and enhancement of the hematopoietic stem cell phenotype. In particular, the addition of SF1670 and ER50891 together greatly improve expansion of the primitive HSC cell surface phenotype CD90+/CD38 low/-. This example further demonstrates that the effect is not dependent on oxygen levels when culturing the cells.

Materials and Methods

CD34+ cells were isolated from donor peripheral blood as described in Example 1. Isolated CD34+ cells were incubated in an in vitro culture media of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetal bovine serum (FBS); "Base" Conditions, +SF Conditions, and +SF+ER conditions further included the components described in Table 5. Each condition tested also included an antibiotic solution that includes penicillin, streptomycin, and amphotericin B to avoid contamination.

Table 5: Additional Components included in the culture media of "Base" Conditions, +SF

Conditions, and +SF+ER Conditions.

[0160] Cultures were incubated at atmospheric oxygen and 5% C0₂.

[0161] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days.

[0162] On the days indicated one-half of the volume of the cell culture was removed for data analysis (flow cytometry). The culture volume was replenished with fresh media according to the conditions tested. The data reported accounts for the dilution factor introduced in this procedure.

Results

[0163] Flow cytometic analysis of seven and 13 day cell cultures as described above demonstrates that addition of SF1670 and ER50891 to cultures including a tetraspanin improves the maintenance and enhancement of hematopotentic stem cells (FIG. 8A-E). In particular, FIG. 8E shows the increase in CD90+/CD38 low/- cells at days 7 and 14 when culture medium includes SF1670 and ER50891 as compared to base conditions and +SF conditions.

CD90+/CD38 low/- is a measure for the most primitive HSC cells. Examples 9 and 10 demonstrate that the effect is independent of oxygen level during cell incubation, and Example 10 demonstrates that hematopotentic stem cells may be maintained and enhanced in culture for at least 13 days.

Example 11: Stem Cell Preserving effects of anti-CD9 antibodies

[0164] This Example demonstrates the stem cell preserving effects of using anti-CD9 antibodies when culturing hematopoietic stem cells derived from non-mobilized peripheral blood.

Materials and Methods

CD34+ cells were isolated from donor peripheral blood as described in Example 1. Isolated CD34+ cells were incubated in an in vitro culture media of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetal bovine serum (FBS). The cultures included three additional components shown in Table 6. The two conditions tested were with anti-CD9 antibodies (+CD9) and without anti-CD9 antibodies (-CD9). In antibody "+" conditions (with antibody), the concentration of antibody present was 500ng/mL, and the antibodies used were HI9a and M- L13. Both conditions included an antibiotic solution that includes penicillin, streptomycin, and amphotericin B to avoid contamination.

Table 6: Additional Components included in the culture media:

[0165] Cultures were incubated at 5% oxygen and 5% C0₂.

[0166] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days.

[0167] On the days indicated one-half of the volume of the cell culture was removed for data analysis (flow cytometry). The culture volume was replenished with fresh media according to the conditions tested. The data reported accounts for the dilution factor introduced in this procedure.

Results

[0168] The amount of CD90+ and CD 133+ cells in culture were measured over 14 days of incubation at the conditions indicated (+ anti-human CD9 and - anti-human CD9) (FIG. 9A and FIG. 9B). The results are shown as a percentage of cells as compared to the +anti-CD9 condition. Over the course of incubation, the relative amount of CD90+ and CD 133+ cells decreased when cultured without anti-CD9 antibodies.

Example 12: Addition of particular cytokines and growth factors do not improve HSC expansion or maintenance under tested conditions [0169] This Example shows that addition of pleiotriphin, for example, does not improve maintenance or enhancement of hematopoietic stem cells under the tested conditions.

Materials and Methods

[0170] CD34+ cells were isolated from donor peripheral blood as described in Example 1. Isolated CD34+ cells were incubated in an in vitro culture media of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetal bovine serum (FBS). SF Conditions, SF+H+I Conditions, +PTN conditions, and +EXTRA Conditions further included the components described in Table 7. Each condition tested also included an antibiotic solution that includes penicillin, streptomycin, and amphotericin B to avoid contamination.

Table 7: Additional Components included in the culture media of SF Conditions, SF+H+I Conditions, +PTN conditions, and +EXTRA Conditions.

Clones: HI9a, M-Ll 3)

[0171] Cultures were incubated at atmospheric oxygen and 5% C0₂.

[0172] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days.

[0173] On the days indicated one-half of the volume of the cell culture was removed for data analysis (flow cytometry). The culture volume was replenished with fresh media according to the conditions tested. The data reported accounts for the dilution factor introduced in this procedure.

Results

[0174] Under the conditions tested, addition of pleitrophin (+PTN conditions) did not improve the expansion or maintenance of any of the cell surface hematopoietic stem cell phenotypes tested CD45+ (FIG. 10A), CD34+ (FIG. 10B), CD133+ (FIG. IOC), CD90+ (FIG. 10D), or CD90+/CD38 low/- (FIG. 10E) as compared to SF or SF+H+I conditions. This suggests the PTN has no effect or may damage hematopoietic cells when culturing under the provided conditions. +EXTRA conditions, which include cytokines and growth factors such as EGF, ANG1, VEGF165, and bFGF, also do not significantly alter the expansion or maintenance of an of the cell surface hematopoietic stem cell phenotypes tested (FIGs. 10A-E) as compared to SF or SF+H+I conditions. Collectively, these data suggest that hematopoietic stem cells can be effectively enhanced and maintained without PTN or other growth factors/cytokines included in +EXTRA conditions.

Example 13: Stem Cell Preserving effects of anti-tetraspanin antibodies

[0175] This Example demonstrates the stem cell preserving effects of using anti- tetraspanin antibodies when culturing hematopoietic stem cells derived from non-mobilized peripheral blood. In particular, this Example demonstrates the effects of using anti-CD9 antibodies and anti-CD81 antibodies

[0176] Materials and Methods

[0177] CD34+ cells were isolated from donor peripheral blood as described in Example 1. Isolated CD34+ cells were incubated in an in vitro culture media of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetal bovine serum (FBS). Each culture included three additional components shown in Table 8. The effect of two different tetraspanin antibodies were determined (CD9 and CD81). Thus, four conditions were tested. (1) With anti-CD9 antibodies (+CD9), (2) without anti-CD9 antibodies (-CD9), (3) with anti-CD81 antibodies (+CD81), and (4) without anti-CD81 antibodies (-CD81). In antibody "+" conditions (with antibody), the concentration of antibody present was 500ng/mL. The anti-CD9 antibodies used were PA5- 11556 and C-4. The anti-CD81 antibodies were 1D6 and NBP2 -20564. All conditions included an antibiotic solution that includes penicillin, streptomycin, and amphotericin B to avoid contamination.

Table 8: Additional Components included in the culture media:

[0178] Cultures were incubated at atmospheric oxygen and 5% C0₂.

[0179] Small molecule components were added separately and fresh each time the media needs to be refreshed. Cytokines can be stored together. Antibodies solutions are depleted of sodium azide and added fresh to the culture each time the media is replenished at the moment. Media renewal should occur at least every few days.

[0180] On the days indicated one-half of the volume of the cell culture was removed for data analysis (flow cytometry). The culture volume was replenished with fresh media according to the conditions tested. The data reported accounts for the dilution factor introduced in this procedure.

Results

[0181] The addition of anti-tetraspanin antibodies (anti-CD9 or anti-CD81) improves the maintenance of hematopotentic stem cell phenotypes (FIG. 11 A, FIG. 1 IB; FIG. 12A & FIG. 12B). The results in the referenced figures are shown as a percentage of cells as compared to the +CD9 condition. Over the course of incubation, the relative amount of CD34+ and CD 133+ cells decreased in samples cultured without anti-CD9 antibodies or anti-CD81 antibodies.

Claims

CLAIMS We claim:

1. A method for expanding hematopoietic stem cells in culture, the method comprising contacting a source of CD34+ cells in culture with an effective amount of one or more agents that bind a tetraspanin, thereby expanding hematopoietic stem cells in the culture.

2. The method of claim 1, wherein the source of CD34+ cells is selected from the group consisting of bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood.

3. The method of claim 1, wherein the source of CD34+ cells is non-mobilized peripheral blood.

4. The method of claim 2 or claim 3, wherein the source of CD34+ cells comprises one or more of (a) CD34+ hematopoietic progenitors; (b) CD34+ early hematopoietic progenitors and/or stem cells; and/or (c) CD133+ early hematopoietic progenitors and/or stem cells.

5. The method of any one of claims 1-4, wherein the one or more agents that bind a tetraspanin are one or more agents selected from the group consisting of an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and a small molecule chemical compound.

6. The method of claim 5, wherein the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof.

7. The method of claim 6, wherein the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof.

8. The method of any one of claims 1-7, wherein the tetraspanin is tetraspanin 29

(TSPAN29).

9. The method of any one of claims 1-8, wherein the tetraspanin is tetraspanin 28

(TSPAN28).

10. The method of any one of claims 1-9, wherein the method further comprises culturing the cells under low oxygen conditions.

11. The method of claim 11, wherein low oxygen conditions comprise an atmosphere containing about 5% oxygen or less.

12. The method of any one of claims 1-11, wherein the method further comprises contacting the cells with one or more agents selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), hepatocyte growth factor (HGF), P38 MAPK inhibitor, retinoic acid receptor (RAR) inhibitors or modulators, epidermal growth factor (EGF), JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors.

13. The method of any one of claims 1-11, wherein the method further comprises contacting the cells with thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L).

14. The method of any one of claims 1-11 or claim 13, wherein the method further comprises contacting the cells with a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor.

15. The method of claim 14, wherein the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670.

16. The method of any one of claims 1-15, wherein said method stabilizes the hematopoietic stem cell phenotype.

17. The method of claim 16, wherein the hematopoietic stem cell phenotype comprises CD45+, CD34+, CD133+, CD90+, CD45RA-, CD38 low/-, and negative for major

hematopoietic lineage markers including CD2, CD3, CD4, CD5, CD8, CD14, CD16, CD19, CD20, CD56.

18. The method of any one of claims 1-17, wherein CD133+ and/or CD90+ positive cells are increased compared to cells in culture that are not contacted with one or more agents that bind a tetraspanin.

19. The method of claim 18, wherein the cells exhibit at least about two times the number of CD 133+ and/or CD90+ positive cells compared to cells in culture that are not contacted with one or more agents that bind a tetraspanin.

20. The method of any one of claims 1-19, wherein the source of the CD34+ cells is a human being.

21. A medium for expanding hematopoietic stem cells in culture comprising:

(a) (i) a base medium or (ii) a feed medium; and

(b) one or more agents that bind a tetraspanin.

22. The medium of claim 21, wherein the one or more agents that bind a tetraspanin are one or more agents selected from the group consisting of an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and a small molecule chemical compound.

23. The medium of claim 22, wherein the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof.

24. The medium of claim 23, wherein the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof.

25. The medium of any one of claims 22-24, wherein the tetraspanin is tetraspanin 29 (TSPAN29).

26. The medium of any one of claims 22-25, wherein the tetraspanin is tetraspanin 28 (TSPAN28).

27. The medium of any one of claims 22-25, wherein the medium further comprises (c) one or more agents selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10, laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OACl), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fms-related tyrosine kinase 3 ligand

(FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS).

28. The medium of any one of claims 22-25, wherein the medium further comprises (c) thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS).

29. The medium of any one of claims 22-25, or claim 28, further comprising a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor.

30. The medium of claim 29, wherein the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670.

31. The medium of any one of claims 27-30, wherein the FBS is heat inactivated.

32. The medium of any one of claims 22-28, wherein the base medium is a base salt medium.

33. The medium of claim 31, wherein the base salt medium is alpha MEM.

34. The medium of claim 32, wherein the base salt medium comprises 320-380mOsm CaCl₂.

35. A method for expanding hematopoietic stem cells in culture, the method comprising contacting a source of CD34+ cells in culture with the medium of any one of claims 21-34, thereby expanding hematopoietic stem cells in the culture.

36. A system for expanding hematopoietic stem cells in culture, the system comprising (a) a source of CD34+ cells in culture; and (b) the medium of any one claims 21-34.

37. The system of claim 36, wherein the source of CD34+ cells is selected from the group consisting of bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood.

38. The system of claim 37, wherein the source of CD34+ cells is non-mobilized peripheral blood.

39. The system of claim 37 or claim 38, wherein the source of CD34+ cells comprises one or more of (a) CD34+ hematopoietic progenitors; (b) CD34+ early hematopoietic progenitors and/or stem cells; and/or (c) CD133+ early hematopoietic progenitors and/or stem cells.

40. The system of any one of claims 36-39, further comprising (c) an atmosphere containing low oxygen.

41. The system of claim 40, wherein the atmosphere contains about 5% oxygen or less.

42. The system of any one of claims 36-41, wherein the source of CD34+ cells is a human being.

43. A kit comprising:

(a) (i) a base medium or (ii) a feed medium; and

(b) one or more agents that bind a tetraspanin.

44. The kit of claim 43, further comprising (c) written instructions for maintaining and/or expanding hematopoietic stem cells in culture.

45. The kit of claim 43 or claim 44, wherein the one or more agents that bind a tetraspanin are one or more agents selected from the group consisting of an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and a small molecule chemical compound.

46. The kit of claim 45, wherein the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof.

47. The kit of claim 46, wherein the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof.

48. The kit of any one of claims 43-47, wherein the tetraspanin is tetraspanin 29 (TSPAN29).

49. The method of any one of claims 43-48, wherein the tetraspanin is tetraspanin 28

(TSPAN28).

50. The kit of any one of claims 43-46, further comprising (d) one or more agents selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANGl), VEGF165, IL-10, laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OACl), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fins-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and

ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS).

51. The kit of any one of claims 43-46, further comprising (d) thrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fins- related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS).

52. The kit of any one of claims 43-46, or claim 51, further comprising a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor.

53. The kit of claim 52, wherein the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670.

54. The kit of any one of claims 50-53, wherein the FBS is heat inactivated.

55. The kit of any one of claims 43-54, wherein the base medium is a base salt medium.

The kit of claim 55, wherein the base salt medium is alpha MEM.

57. The kit of claim 55, wherein the base salt medium comprises 320-380mOsm CaCl₂.

58. A population of hematopoietic stem cells produced by the method of any one of claims 1- 20 or 35.

59. A therapeutic agent comprising the population of hematopoietic stem cells of claim 58.

60. A method of treating an individual in need of hematopoietic reconstitution, comprising administering to said individual the therapeutic agent of claim 59.

61. The method of claim 60, wherein the individual is a bone marrow donor or recipient.

62. The method of claim 61, wherein the individual is diagnosed with cancer.

63. The method of claim 62, wherein the method is used as a supplemental treatment in addition to chemotherapy.

64. The method of claim 63, wherein the method is used to shorten the time between chemotherapy treatments.

65. The method of claim 60, wherein the individual is diagnosed with an autoimmune disease.

66. A method for producing a cell culture media for culturing hematopoietic stem cells (HSC), the method comprising: combining (a) a base or a feed medium; and (b) one or more agents that bind a tetraspanin.

67. The method of claim 66, further comprising combining (c) thrombopoietin (TPO) and/or stem cell factor (SCF).

68. The method of claim 66 or claim 67, further comprising (d) combining one or more of insulin-like growth factor 1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10, laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OACl), P38 MAPK inhibitor JAK/STAT inhibitors, anti-IL3, phosphatase and tensin homolog (PTEN) inhibitors, human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS).

69. The method of claim 66 or claim 67, further comprising (d) insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L), and fetal bovine serum (FBS).

70. The method of claim 66, claim 67, or claim 69, further comprising a retinoic acid receptor (RAR) inhibitor or modulator and/or a phosphatase and tensin homolog (PTEN) inhibitor.

71. The method of claim 70, wherein the RAR inhibitor or modulator is ER50891, and the PTEN inhibitor is SF1670.

72. The method of any one of claims 68-71, wherein the FBS is heat-inactivated FBS.

73. The method of any one of claims 66-72, wherein the one or more agents that bind a tetraspanin are one or more agents selected from the group consisting of an antibody or functional binding fragment thereof, a non-antibody binding polypeptide, an aptamer, and a small molecule chemical compound.

74. The method of claim 73, wherein the one or more agents that bind a tetraspanin is one or more antibodies or functional binding fragments thereof.

75. The method of claim 74, wherein the one or more agents that bind a tetraspanin are two or more antibodies or functional binding fragments thereof.

76. The method of any one of claims 66-75, wherein the tetraspanin is tetraspanin 29

(TSPAN29).

77. The method of any one of claims 66-76, wherein the tetraspanin is tetraspanin 28

(TSPAN28).

78. The method of claim 76, wherein the base or feed medium is Alpha MEM. The method of claim 78, comprising combining the following ingredients using the

The method of claim 78, comprising combining the following ingredients using the

- Ingredients - - Concentration -

Cytokines/Growth Factors TPO 150ng/ml

SCF lOOng/ml

FLT3L lOOng/ml

IGF1 250ng/ml

HGH 250ng/ml

Small Molecules

SF1670 (PTEN inhibitor) 250nM

ER50891 (RAR receptor ΙΟΟηΜ antagonist)

Other

Heat Inactivated FBS 10% v/v

Anti-human CD9 0^g/ml

(Dual antibodies used.

Clones: HI9a, M-L13)

A medium for expanding hematopoietic stem cells in culture comprising:

SF1670 (PTEN inhibitor) 500nM

Tofacitinib (JAK/STAT 500nM inhibitor)

Other

Heat Inactivated FBS 10% v/v

Anti-human CD9 5μg/ml

(Dual antibodies used.

Clones: HI9a, M-L13)

A medium for expanding hematopoietic stem cells in culture comprising: