Methods and compositions relating to hematopoietic stem cell expansion, enrichment, and maintenance

Abstract

To provide compositions and methods for ex vivo production, expansion, enrichment, and / or maintenance of hematopoietic stem cells.SOLUTION: There is provided a method of producing an expanded population of hematopoietic stem cells ex vivo. The method comprises contacting a population of hematopoietic stem cells with an agent of (a) a histone demethylase inhibitor selected from a particular group; (b) a TGFβ receptor inhibitor selected from a particular group; and (c) a histone deacetylase inhibitor selected from a particular group. The agent is present in an amount sufficient to produce an expanded population of hematopoietic stem cells.SELECTED DRAWING: Figure 48

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 184,599, filed June 25, 2015, and U.S. Provisional Patent Application No. 62 / 207,136, filed August 19, 2015, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. This ASCII copy was created on June 13, 2016, is named 701039-084082-PCT_SL.txt, and is 3,946 bytes in size.

[0003] Technical Field The technology described herein relates to compositions and methods for the ex vivo expansion, enrichment, and enrichment of hematopoietic stem cells. [Background technology]

[0004] background Hematopoietic stem cells have considerable therapeutic potential, but a limitation that has hindered their use in the clinic has been the difficulty associated with obtaining sufficient numbers of these cells. In particular, hematopoietic stem cells are difficult to maintain, proliferate, and expand ex vivo. Another challenge that must be overcome to further develop the use of hematopoietic stem cells (HSCs) as a therapeutic is the loss of pluripotency that can occur when these cells are cultured ex vivo. Currently, there is a need for compositions and methods for the ex vivo maintenance, proliferation, and expansion of HSCs that preserve the pluripotency and hematopoietic function of these cells. The inventors have discovered a number of compounds that enable the ex vivo maintenance, proliferation, expansion, and enrichment of hematopoietic stem cells. The compositions and methods of the present invention address the challenges posed by conventional HSC therapies by providing strategies for maintaining, proliferating, and expanding these cells, and by enriching heterogeneous cell populations for HSCs while preserving the ability of ex vivo cultured cells to self-renew and differentiate into multiple different blood cell types in vitro and when transplanted into a recipient. Summary of the Invention

[0005] The present invention relates to compositions and methods for the expansion, enrichment, and maintenance of hematopoietic stem cells during ex vivo culture. The compositions and methods of the present invention can be used to generate expanded hematopoietic stem cell populations that retain the ability to differentiate into multiple cell types within the hematopoietic lineage. Furthermore, the present invention provides methods for introducing polynucleotides into hematopoietic stem cells during ex vivo expansion and for modulating the expression of polynucleotides in hematopoietic stem cells during ex vivo expansion. Hematopoietic stem cells or their progeny, or cells derived from these hematopoietic stem cells, produced according to the compositions and methods of the present invention can be infused into recipients, such as human patients, to treat various pathologies. The present invention also provides media for culturing hematopoietic stem cells containing agents useful for the expansion, enrichment, and maintenance of these cells. Furthermore, the present invention provides kits comprising the compositions of the present invention described herein.

[0006] In a first aspect, the present invention provides a method for manufacturing a semiconductor device comprising: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation In some embodiments, the one or more agents together exhibit one or more effects selected from the group consisting of: (a) a cytotoxic effect of one or more of the one or more cytotoxic effects ...

[0007] In a second aspect, the present invention provides a method for producing a cellular membrane comprising: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation In some embodiments, the one or more agents together exhibit one or more effects selected from the group consisting of: (a) a hematopoietic cell population enriched for hematopoietic stem cells; (b) a hematopoietic cell population enriched for hematopoietic stem cells; (c) a hematopoietic cell population enriched for hematopoietic stem cells; (d) a hematopoietic cell population enriched for hematopoietic stem cells; (e) a hematopoietic cell population enriched for hematopoietic stem cells; (f) a hematopoietic cell population enriched for hematopoietic stem cells; (g) a hematopoietic cell population enriched for hematopoietic stem cells; (g) a hematopoietic cell population enriched for hematopoietic stem cells; (h) a hematopoietic cell population enriched for hematopoietic stem cells; (i) a hematopoietic cell population enriched for hematopoietic stem cells; (j) a hematopoietic cell population enriched for hematopoietic stem cells; (j) a hematopoietic cell population enriched for hematopoietic stem cells; (k ...

[0008] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and (c) maintaining hematopoietic stem cell functional potential of a hematopoietic stem cell population ex vivo for at least two days by contacting a first hematopoietic stem cell population with one or more agents, such that after two days or more, the hematopoietic stem cell population contacted with the one or more agents that together exhibit one or more effects selected from the group consisting of: (a) a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but not contacted with the one or more agents. In some embodiments, the one or more agents together exhibit two or more effects selected from the group above.

[0009] In embodiments of the above aspects of the present invention, the modulation of histone methylation is activation of histone methylation, maintenance of histone methylation, or inhibition of histone demethylation. In further embodiments, the modulation of histone acetylation is activation of histone acetylation, maintenance of histone acetylation, or inhibition of histone deacetylation. In certain embodiments of the present invention, the one or more agents comprise a compound that activates histone methylation, a compound that maintains histone methylation, or a compound that inhibits histone demethylation, and a compound that inhibits TGFβ signaling.

[0010] In some embodiments, the method comprises contacting the hematopoietic stem cell population with one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11. In some embodiments, the one or more compounds listed in Table 11 comprise UM171.

[0011] In certain embodiments of the present invention, the compound that activates histone methylation, maintains histone methylation, or inhibits histone demethylation is a histone demethylase inhibitor, and the compound that inhibits TGFβ signaling is a TGFβ receptor inhibitor. The one or more agents that inhibit TGFβ signaling may include a TGFβ receptor inhibitor. In some embodiments, the TGFβ receptor inhibitor is selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01. In some embodiments, the TGFβ receptor inhibitor is A83-01.

[0012] In certain cases, the histone demethylase inhibitor is an LSD1 inhibitor. For example, the LSD1 inhibitor can be LSD1 inhibitor IV RN-1 or tranylcypromine, and the TGFβ receptor inhibitor can be ALK5 inhibitor II (E-616452). In some embodiments, the method includes contacting a hematopoietic stem cell population with one or more agents that regulate histone methylation. In some embodiments, the one or more agents that regulate histone methylation activate histone methylation, maintain histone methylation, or inhibit histone demethylation. For example, the one or more agents that regulate histone methylation can include a histone demethylase inhibitor. In some embodiments, the histone demethylase inhibitor is an LSD1 inhibitor, e.g., an LSD1 inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. In some embodiments, the LSD1 inhibitor is tranylcypromine.

[0013] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase In some embodiments, the one or more agents that together inhibit the action of one or more proteins selected from the group consisting of: (a) a hematopoietic stem cell population; (b) a hematopoietic stem cell population; (c) a hematopoietic stem cell population; (d) a hematopoietic stem cell population; (e) a hematopoietic stem cell population; (f) a hematopoietic stem cell population; (g) a hematopoietic stem cell population; (h) a hematopoietic stem cell population; (i) a hematopoietic stem cell population; (ii) a hematopoietic stem cell population; (iii) a hematopoietic stem cell population; (iv) a hematopoietic stem cell population; (v) a hematopoietic stem cell population; (vi ...

[0014] A further aspect of the present invention is a method for producing a pharmaceutical composition comprising the steps of: a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase In some embodiments, the one or more agents together inhibit the action of two or more proteins selected from the group consisting of:

[0015] In another aspect, the present invention provides a method for producing a hematopoietic stem cell population comprising culturing a first hematopoietic stem cell population after two days or more under the same conditions and for the same time as the first hematopoietic stem cell population. a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase and (c) a hematopoietic stem cell functional capacity that is greater than the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has not been contacted with one or more agents that together inhibit the action of one or more proteins selected from the group consisting of: (i) a hematopoietic stem cell functional capacity that is greater than the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has not been contacted with one or more agents that together inhibit the action of one or more proteins selected from the group consisting of: (ii) a hematopoietic stem cell functional capacity that is greater than the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has not been contacted with one or more agents that together inhibit the action of one or more proteins selected from the group consisting of: (iii) a hematopoietic stem cell functional capacity that is greater than the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has not been contacted with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: (iv) a hematopoietic stem cell functional capacity that is greater than the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has not been contacted with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: (v) a hematopoietic stem cell functional capacity that is greater than the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has not been contacted with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: (vi ...

[0016] In some embodiments, the method includes contacting a hematopoietic stem cell population with one or more agents that modulate histone acetylation. In some embodiments, the one or more agents that modulate histone acetylation activate histone acetylation, maintain histone acetylation, or inhibit histone deacetylation. In some embodiments, the one or more agents that modulate histone acetylation include a histone deacetylase inhibitor, such as a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. In some embodiments, the histone deacetylase inhibitor is trichostatin A.

[0017] In certain embodiments of the above aspects of the invention, the one or more agents comprise a combination of agents selected from the combinations of agents in Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6.

[0018] In certain embodiments of the above aspects of the present invention, the histone demethylase is LSD1. In further embodiments, the one or more agents comprise a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. In some cases, the protein transducing TGFβ signaling is a TGFβ receptor. In further embodiments, the one or more agents comprise a compound that inhibits a protein transducing TGFβ signaling selected from the group consisting of ALK5 inhibitor II (E-616452), LY364947, A83-01, and DMH1. In certain cases, the one or more agents comprise a compound that inhibits a protein transducing p38 signaling, and the compound is SB203580. Additionally or alternatively, the one or more agents include a compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2 (also called FGF basic, e.g., recombinant mouse FGF2). In yet further cases, the one or more agents include a compound that inhibits histone deacetylase, selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and isodax (romidespin). In other embodiments, the one or more agents together inhibit the action of a histone demethylase and a protein that transduces TGFβ signaling. In certain embodiments, the histone demethylase is LSD1. In further embodiments, the protein that transduces TGFβ signaling is a TGFβ receptor. In certain cases, the one or more agents include LSD1 inhibitor IV RN-1 and ALK5 inhibitor II (E-616452). In further embodiments, the one or more agents comprise a compound that inhibits p38 signaling. In still other cases, the one or more agents comprise a compound that inhibits histone deacetylase.In other embodiments, the one or more agents further comprise a compound that inhibits BMP signaling. In certain embodiments, the one or more agents comprise a combination of inhibitors or other agents specified in any one of Tables 1-10.

[0019] In a further aspect, the present invention provides a method of generating an expanded hematopoietic stem cell population ex vivo by contacting a hematopoietic stem cell population with (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II (E-616452), LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax (romidespin).

[0020] In another aspect, the present invention relates to a method of enriching a cell population for hematopoietic stem cells ex vivo by contacting a hematopoietic cell population containing one or more hematopoietic stem cells with (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II (E-616452), LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax (romidespin).

[0021] In another aspect, the present invention provides a method for treating a first population of hematopoietic stem cells with a first agent selected from the group consisting of: (a) LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; and (b) ALK5 inhibitor II (E-616452), LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax. (romidespin), wherein the first hematopoietic stem cell population exhibits, after two days or more, a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but not contacted with the first and second agents.

[0022] In embodiments of any of the above aspects of the invention, the one or more agents can be a combination of agents selected from the combinations of agents in Table 7, Table 8, Table 9, and Table 10. In certain embodiments of the above aspects of the invention, the one or more agents are present in an amount sufficient to stimulate expansion of the hematopoietic stem cell population by 10% or more of the hematopoietic stem cells not contacted with the one or more agents on day 7 of culture or later (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture). In further embodiments, the one or more agents are present in an amount sufficient to stimulate an expansion of the hematopoietic stem cell population by 10% or more on day 7 or later of culture (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture) compared to a hematopoietic stem cell population contacted with a substance that inhibits aryl hydrocarbon receptor signaling (e.g., Stem Regenin 1, also known as SR1, or an analog thereof), UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof. In certain cases, the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more on culture day 7 or later (e.g., on culture day 7, 10, 12, 14, 15, 20, or later) relative to a hematopoietic stem cell population not contacted with the one or more agents. In yet other embodiments, the one or more agents are present on culture day 7 or later (e.g., on culture day 7, 10, 12, 14, 15, 20, or later) in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more relative to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling (e.g., SR1 or an analog thereof), UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof.

[0023] In some embodiments, the method comprises contacting the hematopoietic stem cell population with one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11. In some embodiments, the one or more compounds listed in Table 11 comprise UM171.

[0024] In some embodiments, the method further comprises contacting the hematopoietic stem cell population with one or more agents that inhibit TGFβ signaling. The one or more agents that inhibit TGFβ signaling may comprise a TGFβ receptor inhibitor. In some embodiments, the TGFβ receptor inhibitor is selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01. In some embodiments, the TGFβ receptor inhibitor is A83-01.

[0025] In some embodiments, the method includes contacting the hematopoietic stem cell population with one or more agents that modulate histone methylation. In some embodiments, the one or more agents that modulate histone methylation activate histone methylation, maintain histone methylation, or inhibit histone demethylation. For example, the one or more agents that modulate histone methylation may include a histone demethylase inhibitor. In some embodiments, the histone demethylase inhibitor is an LSD1 inhibitor, such as an LSD1 inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. In some embodiments, the LSD1 inhibitor is tranylcypromine.

[0026] In some embodiments, the method includes contacting a hematopoietic stem cell population with one or more agents that modulate histone acetylation. In some embodiments, the one or more agents that modulate histone acetylation activate histone acetylation, maintain histone acetylation, or inhibit histone deacetylation. In some embodiments, the one or more agents that modulate histone acetylation include a histone deacetylase inhibitor, such as a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. In some embodiments, the histone deacetylase inhibitor is trichostatin A.

[0027] In some embodiments, the method comprises: a. Inhibition of p38 signaling; and b. Activation of canonical Wnt signaling The method comprises contacting the subject with one or more agents that exhibit one or more effects selected from the group consisting of:

[0028] In some embodiments, the methods comprise contacting the hematopoietic stem cell population with one or more agents that inhibit aryl hydrocarbon receptor signaling, eg, SR1.

[0029] In another aspect, the present invention provides a method for treating a hematopoietic stem cell population with one or more compounds listed in Table 11, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; e. Regulation of histone acetylation; and f. Inhibition of aryl hydrocarbon receptor signaling and one or more agents that exhibit one or more effects selected from the group consisting of:

[0030] In another aspect, the present invention provides a hematopoietic cell population containing one or more hematopoietic stem cells, comprising one or more compounds listed in Table 11, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; e. Regulation of histone acetylation; and f. Inhibition of aryl hydrocarbon receptor signaling and one or more agents that exhibit one or more effects selected from the group consisting of:

[0031] In another aspect, the present invention provides a method for treating a first population of hematopoietic stem cells with one or more compounds listed in Table 11, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; e. Regulation of histone acetylation; and f. Inhibition of aryl hydrocarbon receptor signaling and one or more compounds and one or more agents that exhibit one or more effects selected from the group consisting of: wherein after two days or more, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional capacity that is superior to the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has been cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but that has not been contacted with the one or more compounds and one or more agents.

[0032] In another aspect, the present invention provides a method for treating a hematopoietic stem cell population with one or more agents that inhibit aryl hydrocarbon receptor signaling, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and (c) contacting a subject with one or more agents that exhibit one or more effects selected from the group consisting of:

[0033] In another aspect, the present invention provides a method for treating a hematopoietic cell population containing one or more hematopoietic stem cells with one or more agents that inhibit aryl hydrocarbon receptor signaling, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation with one or more agents that exhibit one or more effects selected from the group consisting of:

[0034] In another aspect, the present invention provides a method for treating a first hematopoietic stem cell population with one or more agents that inhibit aryl hydrocarbon receptor signaling; a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation with one or more agents that exhibit one or more effects selected from the group consisting of: wherein after two days or more, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional capacity that is superior to the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population that has been cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but has not been contacted with the one or more agents.

[0035] In some embodiments, the method comprises contacting the hematopoietic stem cell population with one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11.

[0036] In some embodiments, the method comprises contacting the hematopoietic stem cell population with a TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01.

[0037] In some embodiments, the method includes contacting the hematopoietic stem cell population with a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine.

[0038] In some embodiments, the method comprises contacting the hematopoietic stem cell population with a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax.

[0039] In some embodiments, the method comprises: a. Compounds that inhibit proteins that mediate p38 signaling, including SB203580; and b. A compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. The method comprises contacting the subject with one or more agents selected from the group consisting of:

[0040] In some embodiments, the methods include contacting the hematopoietic stem cell population with one or more agents that inhibit the aryl hydrocarbon receptor, eg, SR1.

[0041] In some embodiments, the methods comprise contacting the hematopoietic stem cell population with a compound that inhibits BMP signaling.

[0042] In another aspect, the present invention provides a method for treating a hematopoietic stem cell population with one or more compounds listed in Table 11, and a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. Proteins that promote β-catenin degradation; e. histone deacetylase; and f. aryl hydrocarbon receptor with one or more agents that inhibit the action of one or more proteins selected from the group consisting of:

[0043] In another aspect, the present invention provides a hematopoietic cell population containing one or more hematopoietic stem cells, comprising one or more compounds listed in Table 11, and a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. Proteins that promote β-catenin degradation; e. histone deacetylase; and f. aryl hydrocarbon receptor with one or more agents that inhibit the action of one or more proteins selected from the group consisting of:

[0044] In another aspect, the present invention provides a method for treating a first population of hematopoietic stem cells with one or more compounds listed in Table 11, and a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. Proteins that promote β-catenin degradation; e. histone deacetylase; and f. aryl hydrocarbon receptor with one or more agents that inhibit the action of one or more proteins selected from the group consisting of: wherein after two days or more, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional capacity that is superior to the hematopoietic stem cell functional capacity of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but that has not been contacted with the one or more compounds and the one or more agents.

[0045] In another aspect, the present invention provides a method for treating a hematopoietic stem cell population with one or more agents that inhibit the aryl hydrocarbon receptor, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation with one or more agents that inhibit the action of one or more proteins selected from the group consisting of:

[0046] In another aspect, the present invention provides a method for treating a hematopoietic cell population containing one or more hematopoietic stem cells with one or more agents that inhibit the aryl hydrocarbon receptor, and a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation with one or more agents that inhibit the action of one or more proteins selected from the group consisting of:

[0047] In another aspect, the present invention provides a method for treating a first population of hematopoietic stem cells with one or more agents that inhibit the aryl hydrocarbon receptor; a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation with one or more agents that inhibit the action of one or more proteins selected from the group consisting of: wherein after two days or more, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but that has not been contacted with the one or more agents.

[0048] In some embodiments, the method further comprises contacting the hematopoietic stem cell population with one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11.

[0049] In some embodiments, the method further comprises contacting the hematopoietic stem cell population with a TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01.

[0050] In some embodiments, the method includes contacting the hematopoietic stem cell population with a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine.

[0051] In some embodiments, the method comprises contacting the hematopoietic stem cell population with a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax.

[0052] In some embodiments, the method comprises: a. Compounds that inhibit proteins that mediate p38 signaling, including SB203580; and b. A compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. The method comprises contacting the compound with one or more compounds selected from the group consisting of:

[0053] In some embodiments, the method comprises contacting the hematopoietic stem cell population with SR1.

[0054] In another aspect, the present invention provides a method for treating a hematopoietic stem cell population with one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11, and a. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. Compounds that inhibit proteins that transmit p38 signaling, including SB203580; d. A compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2; e. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax; and f. Aryl hydrocarbon receptor inhibitors, including SR1 wherein the one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11, and the one or more agents are present in an amount sufficient to generate the expanded hematopoietic stem cell population.

[0055] In another aspect, the present invention provides a hematopoietic cell population containing one or more hematopoietic stem cells, comprising UM171, and a. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. Compounds that inhibit proteins that transmit p38 signaling, including SB203580; d. A compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2; e. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax; and f. Aryl hydrocarbon receptor inhibitors, including SR1 and one or more agents selected from the group consisting of: wherein UM171 and the one or more agents are present in an amount sufficient to produce a cell population enriched for hematopoietic stem cells.

[0056] In another aspect, the present invention provides a method for treating a first population of hematopoietic stem cells with one or more compounds selected from UM171, structural analogs thereof, and the compounds listed in Table 11; a. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. Compounds that inhibit proteins that transmit p38 signaling, including SB203580; d. A compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2; e. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax; and f. Aryl hydrocarbon receptor inhibitors, including SR1 and one or more agents selected from the group consisting of: wherein after two days or more, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but that has not been contacted with UM171 and the one or more agents.

[0057] In another aspect, the present invention provides a hematopoietic stem cell population comprising SR1, and a. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. Compounds that inhibit proteins that transmit p38 signaling, including SB203580; d. A compound that inhibits a protein that promotes beta-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2; and e. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax and one or more agents selected from the group consisting of: wherein SR1 and the one or more agents are present in an amount sufficient to generate the expanded hematopoietic stem cell population.

[0058] In another aspect, the present invention provides a hematopoietic cell population containing one or more hematopoietic stem cells, comprising SR1, and a. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. Compounds that inhibit proteins that transmit p38 signaling, including SB203580; d. A compound that inhibits a protein that promotes beta-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2; and e. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax and one or more agents selected from the group consisting of: wherein SR1 and the one or more agents are present in amounts sufficient to produce a cell population enriched for hematopoietic stem cells.

[0059] In another aspect, the present invention provides a method for identifying a first hematopoietic stem cell population comprising: a. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. Compounds that inhibit proteins that transmit p38 signaling, including SB203580; d. A compound that inhibits a protein that promotes beta-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2; and e. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax and one or more agents selected from the group consisting of: wherein after two days or more, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but that has not been contacted with SR1 and the one or more agents.

[0060] In some embodiments, the one or more agents or compounds are present in an amount sufficient to stimulate an expansion of the cell population by 10% or more on day 7 of culture or later (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture) relative to a hematopoietic stem cell population not contacted with the one or more agents or compounds.

[0061] In some embodiments, the one or more agents or compounds are present in an amount sufficient to stimulate an expansion of the cell population by 10% or more compared to a hematopoietic stem cell population contacted with a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture).

[0062] In some embodiments, the one or more agents or compounds are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more relative to a hematopoietic stem cell population not contacted with the one or more agents or compounds on day 7 or later of culture (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture).

[0063] In some embodiments, the one or more agents or compounds are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more compared to a hematopoietic stem cell population contacted with a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture).

[0064] In some embodiments, the first hematopoietic stem cell population exhibits hematopoietic stem cell functional capacity that is superior to the hematopoietic stem cell functional capacity of the control hematopoietic stem cell population on day 3 or later of culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later).

[0065] In some embodiments, the hematopoietic stem cells are mammalian cells, such as human cells. In some embodiments, the hematopoietic stem cells are CD34+ cells. In some embodiments, at least 10% of the CD34+ cells are CD34+ cells, CD34+CD38- cells, CD34+CD38-CD90+ cells, CD34+CD38-CD90+CD45RA- cells, or CD34+CD38-CD90+CD45RA-CD49F+ cells. In some embodiments, the hematopoietic stem cells are derived from human umbilical cord blood. In some embodiments, the hematopoietic stem cells are derived from human mobilized peripheral blood. In some embodiments, the hematopoietic stem cells are derived from human bone marrow. In some embodiments, the hematopoietic stem cells are freshly isolated from a human. In some embodiments, the hematopoietic stem cells have been previously cryopreserved. In some embodiments, the mammalian cells are mouse cells.

[0066] In embodiments of the above methods for maintaining hematopoietic stem cell functional potential of a hematopoietic stem cell population, the hematopoietic stem cell population exhibits hematopoietic stem cell functional potential on day 2 or later in culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later) that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population.

[0067] In embodiments of any of the above aspects of the present invention, the hematopoietic stem cells are mammalian cells. In certain cases, the mammalian cells are human cells. In certain embodiments, the human cells are CD34+ cells. In certain cases, at least 10% of the CD34+ cells are CD34+CD38- cells, CD34+CD38-CD90+ cells, CD34+CD38-CD90+CD45RA- cells, or CD34+CD38-CD90+CD45RA-CD49F+ cells. In certain embodiments, the hematopoietic stem cells are derived from human umbilical cord blood. In other embodiments, the hematopoietic stem cells are derived from human mobilized peripheral blood. In alternative embodiments, the hematopoietic stem cells are derived from human bone marrow. In certain cases, the hematopoietic stem cells are freshly isolated from a human. In other cases, the hematopoietic stem cells have been previously cryopreserved. In certain embodiments of the present invention, the mammalian cells are mouse cells.

[0068] In further embodiments of any of the above methods of the present invention, the hematopoietic stem cells are cultured for two or more days (e.g., 3 days, 5 days, 7 days, 10 days, 12 days, 14 days, 15 days, 20 days, or more). In certain cases, the hematopoietic stem cells are contacted with one or more agents for two or more days (e.g., 3 days, 5 days, 7 days, 10 days, 12 days, 14 days, 15 days, 20 days, or more). In some cases, the hematopoietic stem cells are contacted with one or more agents simultaneously. In other embodiments, the hematopoietic stem cells are contacted with one or more agents at different times.

[0069] In certain embodiments of the above methods of the present invention, the hematopoietic stem cells maintain hematopoietic stem cell functional potential at day 2 in culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). In certain cases, the hematopoietic stem cells maintain hematopoietic stem cell functional potential after transplantation at day 2 or later in culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). In other embodiments, the hematopoietic stem cells maintain long-term engraftment potential at day 2 in culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). In certain cases, when hematopoietic stem cells are transplanted into a patient, they induce the recovery of a cell population selected from the group consisting of neutrophils, platelets, erythrocytes, monocytes, macrophages, antigen-presenting cells, microglia, osteoclasts, dendritic cells, and lymphocytes. In certain embodiments, the lymphocytes are selected from the group consisting of natural killer (NK) cells, T cells (e.g., CD4+ cells or CD8+ cells), and B cells. In certain cases, the hematopoietic stem cells can localize to hematopoietic tissues in the transplanted recipient to reestablish productive hematopoiesis.

[0070] In further embodiments of the present invention, hematopoietic stem cells are cultured on a plastic surface or on a support comprising vitronectin, fibronectin, or Matrigel. In certain cases, hematopoietic stem cells are cultured in the presence of 2-20% oxygen, 2-12% oxygen, or about 5% oxygen. In some embodiments, hematopoietic stem cells are cultured in the presence of 2-12% oxygen. In some embodiments, hematopoietic stem cells are cultured in the presence of about 5% oxygen.

[0071] In further embodiments of the present invention, the hematopoietic stem cells are originally present in a mononuclear cell fraction prior to treatment with one or more agents. In certain cases, the hematopoietic stem cells are originally present in a CD34+, CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA-, or CD34+CD38-CD90+CD45RA-CD49F+ enriched cell fraction prior to contact with one or more agents. In some embodiments, the hematopoietic stem cells are originally present in a CD34+, CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA-, or CD34+CD38-CD90+CD45RA-CD49F+, or CD34+CD38-CD90+CD45RA-CD49F+EPCR+ enriched cell fraction prior to contact with one or more agents or compounds. In certain cases, the hematopoietic stem cells are originally present in a non-enriched cell fraction prior to contact with one or more agents.

[0072] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. inserting one or more polynucleotides into a hematopoietic stem cell population; and b. Expanding or maintaining a hematopoietic stem cell population according to the above method of the present invention. The present invention provides a method for introducing one or more polynucleotides and / or modulating expression of one or more polynucleotides in a hematopoietic stem cell population, comprising:

[0073] In certain instances, (a) precedes (b). In other embodiments, (b) precedes (a).

[0074] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. inserting a polynucleotide into a hematopoietic stem cell population; and b. Expanding a hematopoietic stem cell population according to the method of any one of the above embodiments, or maintaining the hematopoietic stem cell functional potential of a hematopoietic stem cell population according to the method of any one of the above embodiments. The present invention provides a method for introducing a polynucleotide into a hematopoietic stem cell population, comprising:

[0075] In some embodiments, step (a) above precedes step (b). In some embodiments, step (b) precedes step (a).

[0076] In further embodiments, the method further comprises introducing one or more reagents that cleave nucleic acids in the cell. In certain cases, the one or more reagents that cleave nucleic acids in the cell comprise zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated proteins, or meganucleases.

[0077] In certain embodiments of the invention, one or more polynucleotides are introduced into hematopoietic stem cells by contacting the hematopoietic stem cells with a viral vector (e.g., a retrovirus, an adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), a parvovirus (e.g., adeno-associated virus), a coronavirus, a negative-strand RNA virus, e.g., an orthomyxovirus (e.g., influenza virus), a rhabdovirus (e.g., rabies and vesicular stomatitis virus), a paramyxovirus (e.g., measles and Sendai), a positive-strand RNA virus, e.g., a picornavirus and an alphavirus, and a double-stranded DNA virus, including a herpesvirus (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and a poxvirus (e.g., vaccinia, modified vaccinia Ankara, fowlpox, and canarypox)). In other cases, one or more polynucleotides are introduced into hematopoietic stem cells by contacting the hematopoietic stem cells with a vector encoding a transposable element (e.g., a piggybac transposon or a sleeping beauty transposon).

[0078] In some embodiments, the method includes contacting hematopoietic stem cells with a vector selected from the group consisting of a viral vector (e.g., a retrovirus, adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, or poxvirus) and a transposable element (e.g., a piggyBac transposon or a Sleeping Beauty transposon).

[0079] In certain embodiments of the invention, one or more polynucleotides are introduced into hematopoietic stem cells by electroporation, Nucleofection™, or squeeze-poration.

[0080] In some embodiments, the method includes contacting the cells with a transforming agent selected from the group consisting of a cationic polymer (e.g., diethylaminoethyl-dextran), a cationic lipid, calcium phosphate, an activated dendrimer, and a magnetic bead.

[0081] In some embodiments, the method comprises introducing the polynucleotide into the hematopoietic stem cells by microinjection or laserfection.

[0082] In some embodiments, the polynucleotide comprises a regulatory sequence selected from the group consisting of a promoter sequence, an enhancer sequence, or a silencer sequence.

[0083] In some embodiments, the polynucleotide encodes a molecule selected from the group consisting of a protein and RNA (mRNA, tRNA, siRNA, miRNA, shRNA). In some embodiments, the polynucleotide is chemically modified RNA.

[0084] In certain embodiments of the invention, one or more polynucleotides are introduced into hematopoietic stem cells by contacting the hematopoietic stem cells with cationic lipids, cationic polymers such as diethylaminoethyl (DEAE)-dextran, calcium phosphate, activated dendrimers, magnetic beads, or by microinjection or laserfection of the hematopoietic stem cells.

[0085] In further embodiments, the one or more polynucleotides are introduced into the hematopoietic stem cells by contacting the hematopoietic stem cells with nanoparticles containing the one or more polynucleotides. In certain embodiments, the one or more polynucleotides are introduced into the hematopoietic stem cells by contacting the hematopoietic stem cells with one or more VSV-G-induced microvesicles (also called gesicles).

[0086] In certain cases, one or more polynucleotides introduced into hematopoietic stem cell populations contain gene regulatory elements, such as promoters, enhancers, or silencers.In other cases, one or more polynucleotides encode proteins or RNA molecules (e.g., mRNA, tRNA, siRNA, miRNA, or shRNA).In certain embodiments, one or more polynucleotides introduced into hematopoietic stem cell populations are chemically modified RNA molecules.

[0087] In a further embodiment, the method further comprises introducing the expanded hematopoietic stem cell population, or progeny thereof, into a recipient, into which the polynucleotide has been inserted.

[0088] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. providing a population of hematopoietic stem cells; b. Expanding the hematopoietic stem cell population according to the above method of the present invention; c. Optionally, differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocytic progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, lymphocytes, NK cells, B cells, and / or T cells; and d. Introducing the expanded hematopoietic stem cell population or its progeny into a recipient The present invention provides a method for treating a recipient with hematopoietic stem cells or their progeny, comprising:

[0089] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. providing a population of hematopoietic stem cells; b. Enriching a hematopoietic stem cell population according to the above method of the present invention; c. Optionally, differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocytic progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, lymphocytes, NK cells, B cells, and / or T cells; and d. Introducing the cell population enriched for hematopoietic stem cells or their progeny into a recipient The present invention relates to a method of treating a recipient with hematopoietic stem cells or their progeny, comprising:

[0090] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. providing a population of hematopoietic stem cells; b. maintaining the hematopoietic stem cell functional potential of the hematopoietic stem cell population according to the above method of the present invention; c. Optionally, differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocytic progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, and lymphocytes, e.g., NK cells, B cells, and / or T cells; and d. Introducing the hematopoietic stem cell population or its progeny into a recipient The present invention provides a method for treating a recipient with hematopoietic stem cells or their progeny, comprising:

[0091] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. providing a hematopoietic stem cell population generated by the above method of the present invention; b. optionally differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocytic progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, lymphocytes, NK cells, B cells, and / or T cells; and c. Introducing the hematopoietic stem cell population or its progeny into a recipient The present invention provides a method for treating a recipient with hematopoietic stem cells or their progeny, comprising:

[0092] In some embodiments of the above-mentioned methods of the present invention, the recipient is a human. In certain embodiments, the hematopoietic stem cells are derived from one or more hematopoietic stem cells isolated from a human donor. In certain cases, the hematopoietic stem cells are derived from the donor's mobilized peripheral blood. In further embodiments, the donor has previously been administered one or more mobilizing agents selected from the group consisting of a CXCR4 antagonist (e.g., AMD3100), GCSF, and GROβ.

[0093] In some embodiments of any of the above methods of the present invention, hematopoietic stem cells are further contacted with a substance that inhibits aryl hydrocarbon receptor signaling.For example, the substance that inhibits aryl hydrocarbon receptor signaling can be SR1 or its analog.In a further embodiment of the present invention, hematopoietic stem cells are further contacted with UM171 or its analog.In yet another embodiment, hematopoietic stem cells are further contacted with a prostaglandin, such as dmPGE2 or its analog.In certain cases, hematopoietic stem cells can be contacted with a Notch signaling agonist.In a further embodiment, hematopoietic stem cells can be contacted with a SIRT1 inhibitor, such as nicotinamide, cambinol, or its analog.

[0094] In some embodiments, the hematopoietic stem cells are further contacted with a Notch signaling agonist.

[0095] In some embodiments, the hematopoietic stem cells are further contacted with a SIRT1 inhibitor, hi some embodiments, the inhibitor or SIRT1 is selected from the group consisting of nicotinamide, cambinol, and analogs thereof.

[0096] In some embodiments of the methods of the present invention, the recipient is a patient with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), myelodysplastic syndrome (MDS), multiple myeloma, aplastic anemia, bone marrow failure, myeloproliferative disorders such as myelofibrosis, essential thrombocytopenia, or polycythemia vera, Fanconi anemia, dyskeratosis congenita, common variable immune deficiency (CVID, e.g., CVID1, CVID2, CVID3, CVID4, CVID5, and CVID6), human immunodeficiency virus (HIV), hemophagocytic lymphohistiocytosis, lymphohistiocystosis), amyloidosis, solid tumors such as neuroblastoma, germ cell tumors, breast cancer, Wilms' tumor, medulloblastoma, and neuroectodermal tumors, autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, systemic lupus erythematosus, and type I diabetes, or protein deficiency diseases such as adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), hemophilia A and B, Hurler syndrome, Hunter syndrome, Fabry disease, Gaucher disease, epidermolysis bullosa, globoid cell leukodystrophy, Sanfillipo syndrome, and Morquio syndrome.

[0097] In some embodiments, the recipient is diagnosed with sickle cell anemia, alpha thalassemia, beta thalassemia, delta thalassemia, hemoglobin E / thalassemia, hemoglobin S / thalassemia, hemoglobin C / thalassemia, hemoglobin D / thalassemia, chronic granulomatous disease (X-linked chronic granulomatous disease, autosomal recessive (AR) chronic granulomatous disease, chronic granulomatous disease AR I NCF1, chronic granulomatous disease AR CYBA, chronic granulomatous disease AR II NCF2, chronic granulomatous disease AR III NCF4), X-linked severe combined immunodeficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID, Rag1 / Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, congenital granulocytopenia, congenital granulocytopenia-congenital neutropenia-SCN1, congenital granulocytopenia-congenital neutropenia-SCN2, familial hemophagocytic lymphohistiocytosis (FHL), familial hemophagocytic lymphohistiocytosis type 2 (FHL2, perforin mutation), agammaglobulinemia (X-linked agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, hemolytic anemia due to erythrocyte pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, X-linked adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative disorder, unicentric Castleman's disease, multicentric Castleman's disease, congenital amegakaryocytic thrombocytopenia thrombocytopenia (CAMT) type I, reticular dysplasia, Fanconi anemia, acquired idiopathic sideroblastic anemia, systemic mastocytosis, von Willebrand disease (VWD), congenital dyserythroid anemia type 2, cartilage-hair hypoplasia syndrome, hereditary spherocytosis, Blackvan-Diamond syndrome, Shwachman-Diamond syndrome, thrombocytopenia-radial coloboma syndrome, osteopetrosis, childhood osteopetrosis, mucopolysaccharidoses, Lesch-Nyhan syndrome, glycogen storage disease, congenital mastocytosis, Omenn syndrome, X-linked immunodysregulation polyendocrinopathy enteropathy,and enteropathy (IPEX), IPEX characterized by FOXP3 mutations, X-linked syndrome of polyendocrinopathy, immune dysfunction, and diarrhea (XPID), X-linked autoimmunity-allergic dysregulation syndrome (XLAAD), IPEX-like syndrome, hyper IgM type 1, hyper IgM type 2, hyper IgM type 3, hyper IgM type 4, hyper IgM A human patient suffering from an inherited blood disorder selected from the group consisting of type 5, X-linked hyperimmunoglobulin M, bare lymphocyte syndrome type I, and bare lymphocyte syndrome type II (bare lymphocyte syndrome type II, MHC class I deficiency; bare lymphocyte syndrome type II, complementation group A; bare lymphocyte syndrome type II, complementation group C; bare lymphocyte syndrome type II, complementation group D; bare lymphocyte syndrome type II, complementation group E).

[0098] In some embodiments, the recipient is a human patient suffering from a hematolymphoid malignancy, a non-hematolymphoid malignancy, or a protein deficiency, or is a tissue or cell transplant recipient (e.g., to induce tolerance to the transplanted tissue or cells).

[0099] Hematopoietic stem cell populations and their progeny expanded, enriched, or maintained by the compositions and / or methods of the invention can also be used to treat patients (e.g., human patients) suffering from hematolymphoid malignancies, non-hematolymphoid malignancies, or protein deficiencies. In other embodiments, the patient may be the recipient of a tissue or cell transplant, and the hematopoietic stem cells or their progeny are administered to induce tolerance to the transplanted tissue or cells.

[0100] In some embodiments of the above methods of treating a patient with hematopoietic stem cells or their progeny, the hematopoietic stem cells are autologous or syngeneic. Alternatively, the hematopoietic stem cells can be allogeneic.

[0101] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation The present invention provides a composition comprising one or more agents that together exhibit one or more effects selected from the group consisting of:

[0102] In some embodiments, the one or more agents together exhibit two or more effects selected from the group above.

[0103] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. one or more compounds listed in Table 11, and b. one or more agents that inhibit TGFβ signaling; c. one or more agents that modulate histone methylation; d. one or more agents that modulate histone acetylation; and e. One or more agents that inhibit aryl hydrocarbon receptor signaling The present invention provides a composition comprising one or more of:

[0104] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. one or more agents that inhibit aryl hydrocarbon receptor signaling, and b. one or more agents that inhibit TGFβ signaling; c. one or more agents that modulate histone methylation; and d. one or more agents that modulate histone acetylation The present invention provides a composition comprising one or more of:

[0105] In some embodiments, the compound listed in Table 11 is UM171.

[0106] In some embodiments, the modulation of histone methylation is the activation of histone methylation, the maintenance of histone methylation, or the inhibition of histone demethylation. In further embodiments, the modulation of histone acetylation is the activation of histone acetylation, the maintenance of histone acetylation, or the inhibition of histone deacetylation. In certain embodiments, one or more agents include a compound that activates histone methylation, a compound that maintains histone methylation, or a compound that inhibits histone demethylation, and a compound that inhibits TGFβ signaling. In certain cases, the compound that activates histone methylation, a compound that maintains histone methylation, or a compound that inhibits histone demethylation is a histone demethylase inhibitor, and the compound that inhibits TGFβ signaling is a TGFβ receptor inhibitor. In certain embodiments, the histone demethylase inhibitor is an LSD1 inhibitor. In further embodiments, the LSD1 inhibitor is LSD1 inhibitor IV RN-1, and the TGFβ receptor inhibitor is ALK5 inhibitor II (E-616452).

[0107] In some embodiments, the aryl hydrocarbon receptor inhibitor is SR1.

[0108] In some embodiments, the composition comprises a compound that inhibits BMP signaling.

[0109] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase The present invention provides a composition comprising one or more agents that together inhibit the action of one or more proteins selected from the group consisting of:

[0110] In some embodiments, the one or more agents together inhibit the action of two or more proteins selected from the group above.

[0111] In some embodiments of this aspect of the present invention, the histone demethylase is LSD1. In further embodiments, the one or more agents comprise a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. In certain cases, the protein transducing TGFβ signaling is a TGFβ receptor. In some embodiments, the one or more agents comprise a compound that inhibits a protein transducing TGFβ signaling selected from the group consisting of ALK5 inhibitor II (E-616452), LY364947, A83-01, and DMH1. In certain cases, the one or more agents comprise a compound that inhibits a protein transducing p38 signaling, and the compound is SB203580. In further embodiments, the one or more agents comprise a compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2 (e.g., recombinant mouse FGF2). In yet other embodiments, the one or more agents comprise a compound that inhibits histone deacetylase, selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and isodax (romidespin). In certain cases, the one or more agents together inhibit the action of a histone demethylase and a protein that transduces TGFβ signaling. In certain embodiments, the histone demethylase is LSD1. In further embodiments, the protein that transduces TGFβ signaling is a TGFβ receptor. In certain cases, the one or more agents comprise LSD1 inhibitor IV RN-1 and ALK5 inhibitor II (E-616452). In further embodiments, the one or more agents comprise a compound that inhibits p38 signaling. In other embodiments, the one or more agents comprise a compound that inhibits histone deacetylase. In certain cases, the one or more agents comprise a compound that inhibits BMP signaling.

[0112] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. The compounds listed in Table 11, and b.TGFβ receptor inhibitors; c. histone demethylase inhibitors; d. histone deacetylase inhibitors; and e. Aryl hydrocarbon receptor inhibitors The present invention provides a composition comprising one or more of:

[0113] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. aryl hydrocarbon receptor inhibitors, and b.TGFβ receptor inhibitors; c. Compounds listed in Table 11; d. histone demethylase inhibitors; and e. histone deacetylase inhibitors The present invention provides a composition comprising one or more of:

[0114] In some embodiments, the composition comprises: a. Compounds that inhibit proteins that mediate p38 signaling, including SB203580; and b. A compound that inhibits a protein that promotes β-catenin degradation, selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. The composition comprises one or more agents selected from the group consisting of:

[0115] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a. UM171, a structural analog thereof, or a compound listed in Table 11, and b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. A histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; d. a histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax; and e.SR1 The present invention provides a composition comprising one or more of:

[0116] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a.SR1, and b. A TGFβ receptor inhibitor selected from the group consisting of ALK5 inhibitor II, LY364947, DMH1, and A83-01; c. a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; and d. A histone deacetylase inhibitor selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. The present invention provides a composition comprising one or more of:

[0117] In another aspect, the present invention provides a composition comprising: (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; and (b) a second agent selected from the group consisting of ALK5 inhibitor II (E-616452), LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax (romidespin). In certain cases, the one or more agents are present in an amount sufficient to generate an expanded hematopoietic stem cell population. In further embodiments, the one or more agents are present in an amount sufficient to generate a cell population enriched for hematopoietic stem cells. In yet other embodiments, the one or more agents are present in an amount sufficient to maintain hematopoietic stem cell functional potential of the hematopoietic stem cell population for at least two days. In certain embodiments, the one or more agents are present in an aqueous solution. In other embodiments, the one or more agents are present as a lyophilized solid.

[0118] In some embodiments of the above-described compositions of the present invention, the one or more agents are present in an amount sufficient to stimulate an expansion of the cell population by 10% or more on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture) compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling (e.g., SR1 or an analog thereof), UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof. In certain cases, the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling (e.g., SR1 or an analog thereof), UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, such as nicotinamide, cambinol, or an analog thereof, on the 7th or subsequent day of culture (e.g., on the 7th, 10th, 12th, 14th, 15th, 20th, or subsequent day of culture). In certain embodiments, the one or more agents are present in an amount sufficient to maintain the long-term engraftment potential of hematopoietic stem cells after contact with the cells in culture for 2 or more days (e.g., 3, 5, 7, 10, 12, 14, 15, 20, or more days) following transplantation.

[0119] In a further aspect, the present invention provides a composition comprising a multi-component combination specified in any one of Tables 1-10.

[0120] In another aspect, the present invention provides a cell culture medium comprising any of the above compositions of the present invention. In certain cases, the cell culture medium is substantially serum-free. In some embodiments, cytokines may be added to the cell culture medium of the present invention, for example, to further stimulate the proliferation of hematopoietic stem cells or to induce the differentiation of hematopoietic stem cells into desired blood cell populations.

[0121] In the above-described composition embodiments of the invention, the composition may further comprise a population of hematopoietic stem cells in contact with one or more agents. In certain cases, the hematopoietic stem cells have been cultured in the presence of the one or more agents for 2 or more days (e.g., 3 days, 5 days, 7 days, 10 days, 12 days, 14 days, 15 days, 20 days, or more).

[0122] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and one or more substances selected from the group consisting of a substance that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, are present in amounts together sufficient to generate the expanded hematopoietic stem cell population, wherein the cells are further contacted with the one or more substances.

[0123] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and one or more substances selected from the group consisting of a substance that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, are present in sufficient amounts together to produce a cell population enriched for hematopoietic stem cells, wherein the cells are further contacted with the one or more substances.

[0124] In a further aspect, the present invention provides a method for isolating a first population of hematopoietic stem cells by: a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and (c) maintaining hematopoietic stem cell functional capacity of a hematopoietic stem cell population ex vivo for at least two days by contacting the first hematopoietic stem cell population with one or more agents that together exhibit one or more effects selected from the group consisting of:

[0125] In yet another aspect, the present invention provides a population of hematopoietic stem cells produced by any of the above methods of the present invention. In other cases, the present invention provides a cell population derived from hematopoietic stem cells produced by any of the above methods of the present invention.

[0126] In a further aspect, the present invention provides a kit comprising any of the above-described compositions of the present invention in addition to a package insert. In certain cases, the package insert instructs the user of the kit to expand, enrich, or maintain a hematopoietic stem cell population ex vivo. In other cases, the package insert instructs the user to express a polynucleotide in hematopoietic stem cells. In a further embodiment, the package insert instructs the user to administer the hematopoietic stem cells to a recipient.

[0127] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in the description of particular embodiments and are not intended to limit the invention, the scope of which is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and the definition of a term provided herein, the definition provided herein shall prevail.

[0128] As used herein, the term "expanded population" of hematopoietic stem cells or hematopoietic progenitor cells refers to a cell population comprising at least one more hematopoietic stem cell such that the amount of hematopoietic stem cells in the population is greater (e.g., at least 10% greater, at least 20% greater, at least 30% greater) than the number of HSCs before administration of one or more agents as described herein (e.g., one or more agents that together exhibit two or more effects selected from the group consisting of modulating histone methylation, inhibiting TGFβ signaling, inhibiting p38 signaling, activating canonical Wnt signaling, and modulating histone acetylation). In some embodiments, the one or more agents modulate TGFβ signaling, modulate lysine methylation, modulate p38 signaling, modulate canonical Wnt signaling, modulate histone methylation, or modulate histone acetylation.

[0129] As used herein, an agent that inhibits histone demethylation refers to a substance or composition (e.g., a small molecule, protein, interfering RNA, messenger RNA, or other natural or synthetic compound, or composition, e.g., viral or other material comprised of multiple substances) that can attenuate or prevent the activity of histone demethylase or other enzymes that catalyze the formation of intermediates leading to histone demethylation. Inhibition can occur through direct interaction or through indirect means, for example, by reducing the amount of histone demethylase produced in the cell or by inhibiting the interaction of histone demethylase with methylated histone substrates. Histone demethylases include lysine-specific demethylases, e.g., LSD1 and LSD2, as well as other FAD-dependent histone demethylases. Histone demethylases can also be inhibited by ferrous iron (Fe 2+Also included are dioxygenases, such as AlkB, and Jumonji C (JmjC) domain-containing histone demethylases, such as JHDM1, JHDM2, and members of the JMJD2 superfamily of histone demethylases, which catalyze the oxidative demethylation of histone residues using a methylated histone demethylase (JmjC). Other enzymes that convert methylated histone residues to reactive intermediates that subsequently undergo oxidative demethylation include monoamine oxidases. Histone demethylation inhibitors may bind directly to histone demethylases and compete with methylated histone substrates for binding at the enzyme active site. Alternatively, agents that inhibit histone demethylation may bind to histone demethylases at a location distant from the active site, disrupting or preventing the interaction of the enzyme with the methylated histone substrate, for example, by inducing a conformational change in the enzyme, or disrupting or preventing the catalytic cycle, for example, by inactivating or displacing an enzyme cofactor.

[0130] Agents that inhibit histone demethylation exhibit a half-maximal inhibitory concentration (IC) of 100 μM or less (e.g., 1 nM to 100 μM) as determined from histone demethylation assays known in the art or described herein. 50 ) can attenuate or prevent the formation of demethylated histone residues. Exemplary assays that can be used to elucidate the biological activity of histone demethylation inhibitors include, but are not limited to, cell-based growth inhibition assays and dissociation-enhanced lanthanide fluorescence assays as described in U.S. Pat. No. 8,735,622, time-resolved fluorescence resonance energy transfer assays as described in WO 2014 / 151945, and mass spectrometry-based assays and coupled-enzyme formaldehyde dehydrogenase assays as described in WO 2010 / 043866, among others.

[0131] As used herein, an agent that inhibits the TGFβ signaling pathway refers to a substance or composition (e.g., a small molecule, protein, interfering RNA, messenger RNA, or other natural or synthetic compound, or composition, e.g., a viral or other material composed of multiple substances) that can attenuate or prevent the transcription of one or more genes transcribed by the action of SMAD transcriptional coactivator proteins. An agent that inhibits the TGFβ signaling pathway may disrupt the signal transduction cascade that leads to SMAD-induced gene transcription at one or more points within this pathway. For example, a TGFβ signaling pathway inhibitor may disrupt or prevent TGFβ or TGFβ superfamily ligands, such as activin, Nodal, bone morphogenetic protein (BMP), growth differentiation factor (GDF), or Müllerian inhibitory factor (MIF), from binding to their endogenous receptors, thereby inhibiting the phosphorylation and activation of receptor-associated SMAD proteins. TGFβ signaling pathway inhibitors may function by, for example, binding to SMAD proteins and preventing or disrupting the interaction of SMAD proteins with nucleoporins, thereby preventing the translocation of one or more SMAD proteins to the nucleus. TGFβ signaling pathway inhibitors may stabilize the interaction of one or more SMAD proteins with SMAD Anchor for Receptor Activation (SARA). This stabilization sequesters the SMAD proteins in the cytoplasm and orchestrates their translocation to the nucleus. Other examples of TGFβ signaling pathway inhibitors include substances that bind to SMAD proteins and sequester them from DNA-binding transcription factors, thereby preventing target gene transcription, such as neurogenin. Alternative TGFβ signaling pathway inhibitors include substances that promote ubiquitination of one or more SMAD proteins, thereby marking them for degradation by the proteasome and preventing target gene transcription.

[0132] Exemplary assays that can be used to confirm the inhibitory activity of TGFβ signaling pathway inhibitors include, but are not limited to, electrophoretic mobility shift analysis, antibody supershift assays, and TGFβ-inducible gene reporter assays, as described in WO2006 / 012954, among others.

[0133] As used herein, an agent that inhibits the p38 signaling pathway refers to a substance or composition (e.g., a small molecule, protein, interfering RNA, messenger RNA, or other natural or synthetic compound, or composition, e.g., a viral or other material composed of multiple substances) that can attenuate or block the activity of p38 mitogen-activated protein kinase (MAPK, e.g., p38α, p38β, p38γ, or p38δ) or any protein directly or indirectly involved in the activation of one or more of these enzymes. Agents that inhibit the p38 signaling pathway may include substances such as monoclonal antibodies that bind to cytokine receptors, such as IL-1R, and block p38 MAP kinase activation via this receptor. Alternatively, p38 signaling pathway inhibitors may directly bind to p38 protein and attenuate or block phosphorylation of the p38 activation loop by MAP kinase. Alternatively, agents that inhibit the p38 signaling pathway may disrupt the formation of polyubiquitin chains at lysine residues of TNF receptor-associated factors (TRAFs), which serve as scaffolds for MAPK complexes. Other inhibitors of the p38 signaling pathway include those that promote phosphorylation of MAP kinases at sites distant from the activation loop, preventing their association with p38, and those that acetylate MAP kinases within the activation loop, thus preventing their phosphorylation and concomitant activation.

[0134] Exemplary assays that can be used to confirm the inhibitory activity of agents that inhibit the p38 signaling pathway include, but are not limited to, a fluorescence anisotropy competitive binding assay, as well as a time-resolved fluorescence resonance energy transfer assay, as described in WO2006 / 012954, among others.

[0135] As used herein, the agent that inhibits histone deacetylase refers to a substance or composition (such as small molecule, protein, interference RNA, messenger RNA, or other natural or synthetic compounds, or composition, such as viral material or other materials that are composed of multiple substances) that can weaken or inhibit the activity of histone deacetylase, more specifically, its enzymatic activity, by direct interaction or indirect means, for example, by reducing the amount of histone deacetylase produced in cells, or by inhibiting the interaction between histone deacetylase and acetylated histone substrate.Inhibiting histone deacetylase enzymatic activity means reducing the ability of histone deacetylase to catalyze the removal of acetyl groups from histone residues (for example, monomethylated, dimethylated, or trimethylated lysine residues in histone proteins; monomethylated arginine residues, or symmetrical / asymmetrical dimethylated arginine residues). Preferably, such inhibition is specific, such that an agent that inhibits histone deacetylation reduces the ability of histone deacetylase to remove acetyl groups from histone residues at a concentration of the inhibitor that is lower than the concentration of the inhibitor required to produce another, unrelated biological effect.

[0136] As used herein, the terms "histone deacetylase" and "HDAC" refer to any member of a family of enzymes that catalyze the removal of acetyl groups from the ε-amino group of lysine residues at the N-terminus of histones.Unless otherwise specified by context, the term "histone" is intended to refer to any histone protein, including HI, H2A, H2B, H3, H4, and H5, from any species.Human HDAC proteins or gene products include, but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, and HDAC-11.

[0137] As used herein, the agent that inhibits the protein that promotes β-catenin degradation includes the agent that inhibits β-catenin phosphorylation or ubiquitination.These agents can be any substance (for example, small molecule, protein, interfering RNA, messenger RNA, or other natural or synthetic compounds, or compositions, such as viral materials or other materials composed of multiple substances) that can reduce the rate or extent of β-catenin degradation, for example, by weakening the catalytic activity of phosphorylation of serine and / or threonine residues (for example, at residues Ser33, Ser37, and / or Thr41), which makes β-catenin a substrate for ubiquitination and proteasome-mediated degradation.By extending the half-life of functional β-catenin, these agents promote the simultaneous increase in the rate or extent of transcription of genes that are transcribed by the action of β-catenin transcriptional coactivators. Exemplary agents that inhibit β-catenin phosphorylation include agonists of the canonical β-catenin / Wnt signaling pathway, a signaling cascade that mediates the inhibition of glycogen synthase kinase 3 (GSK3) by providing substrates that compete with β-catenin for phosphorylation.

[0138] As used herein, "Wnt signaling agonist" refers to an agonist of the canonical Wnt signaling pathway. Agonists of this pathway also include other compounds that directly bind to Wnt proteins or Frizzled and LRP5 / 6 co-receptor proteins to promote an increase in the concentration of β-catenin in the nucleus of mammalian cells. Alternatively, β-catenin / Wnt pathway agonists may function by inhibiting one or more secreted Frizzled-related proteins (SFRPs) or Wnt inhibitory proteins (WIFs), which bind to Wnt proteins and sequester them from endogenous Wnt co-receptors.

[0139] Exemplary methods that can be used to confirm the activity of β-catenin / Wnt pathway agonists include, but are not limited to, monitoring the expression of reporter genes under the control of TCF / LEF family transcription factors, as well as TOPFlash luciferase reporter assays, as described in US2014 / 0044763.

[0140] As used herein, compounds that inhibit aryl hydrocarbon receptor signaling include agents that inhibit the signal transduction cascade mediated by the binding of aryl hydrocarbon receptor with its activating ligand.Aryl hydrocarbon receptor is a cytosolic ligand-inducible transcription factor that translocates to the nucleus upon binding to an agonist ligand and promotes the transcription of target genes containing specific sequence motifs, such as genes encoding cytochrome P450A1 enzymes containing upstream dioxin response elements.Examples of agents that inhibit aryl hydrocarbon receptor signaling include aryl hydrocarbon receptor inhibitors, which may include compounds that directly bind to the aryl hydrocarbon receptor and thus compete with aryl hydrocarbon receptor ligands for binding to this receptor, such as SR1.Further examples of agents that inhibit aryl hydrocarbon receptor signaling include agents that prevent the translocation of active aryl hydrocarbon receptor to the nucleus and agents that inhibit the interaction of aryl hydrocarbon receptor with the DNA of target genes (for example, promoter regions containing XRE sites).

[0141] As used herein, Notch signaling agonist refers to the substance that promotes the activation of Notch pathway function.As used herein, the term " Notch pathway function " refers to the function that is mediated by Notch signaling pathway, including but not limited to the nuclear translocation of Notch intracellular domain, the nuclear translocation of RBP-Jκ or its Drosophila homologue Suppressor of Hairless, the activation of bHLH gene of Enhancer of Split complex, for example Mastermind, the activation of HES-1 gene or KBF2 (also called CBF1) gene, the inhibition of Drosophila neuroblast segregation, and the binding of Notch with Delta protein, Jagged / Serrate protein, Fringe, Deltex, or RBP-Jκ / Suppressor of Hairless, or its homologue or analogue. The phenotypes caused by Notch signal transduction cascade and Notch signal transduction are described, for example, in Kopan et al., Cell 137:216 (2009) and Jarriault, et al., Mol. Cell. Biol. 18:7423 (1998).The disclosures of each of these are incorporated herein by reference.Examples of Notch agonists are described, for example, in US2014 / 0369973 and US7,399,633.The disclosures of each of these are incorporated herein by reference. Exemplary Notch agonists include, but are not limited to, Notch proteins, and analogs, derivatives, and fragments thereof; other proteins that transduce the Notch signaling pathway, and analogs, derivatives, and fragments thereof; activating antibodies that stimulate Notch receptor activity, and antigen-binding fragments thereof that retain agonist activity; nucleic acids that encode proteins that enhance Notch signaling; and proteins, derivatives, and analogs thereof that bind to or otherwise interact with Notch proteins or other proteins in the Notch pathway such that Notch pathway activity is promoted.Such agonists include, but are not limited to, Notch proteins and their derivatives containing the Notch intracellular domain, Notch nucleic acids encoding the foregoing, and proteins that contact the Notch interaction domain of a Notch ligand (e.g., the extracellular domain of Delta or Serrate). Other agonists include, but are not limited to, RBPJκ / Suppressor of Hairless or Deltex. Furthermore, Fringe can be used, for example, with Delta protein to enhance Notch activity. These proteins, their fragments, and derivatives can be recombinantly expressed and isolated, or chemically synthesized using peptide and protein synthesis methods known in the art.

[0142] As used herein, the term "inhibitor" refers to any natural or synthetic compound that can reduce the activity of a target protein or signaling pathway. Inhibitors may be, for example, peptides, proteins, antibodies, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic compounds, or inorganic compounds. Inhibitors may directly or indirectly attenuate or block the activity of a target protein. Direct inhibition can be achieved, for example, by binding to a protein and preventing it from interacting with endogenous molecules, such as enzymes, substrates, or other binding partners, thereby reducing the activity of the protein. For example, an inhibitor may bind to an enzyme active site and sterically hinder the binding of endogenous substrates at this site, thus reducing the enzymatic activity of the protein. Alternatively, indirect inhibition can be achieved, for example, by binding to a protein that promotes the activity of the target protein, inducing a conformational change, or catalyzing a chemical modification of the target protein. For example, indirect inhibition of a target protein may be achieved by binding to and inactivating a kinase that catalyzes the phosphorylation of, and thus activates, the target protein.

[0143] As used herein, the term "hematopoietic stem cell" (or "HSC") refers to an immature blood cell that has the capacity to self-renew and differentiate into mature blood cells comprising a variety of lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells express CD34 + It is known in the art that the CD34 cells may or may not be present. +HSCs are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are thought to comprise a subpopulation of cells with the stem cell characteristics defined above, whereas in mice, HSCs are CD34-. Furthermore, HSCs are also referred to as long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on functional capacity and cell surface marker expression. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers, including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra). Furthermore, ST-HSCs are less quiescent (i.e., more active) and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSCs have a high self-renewal capacity (i.e., they survive throughout adulthood and can be serially transplanted through successive recipients), whereas ST-HSCs have a poor self-renewal capacity (i.e., they survive for only a limited period of time and do not have serial transplantation capacity). Any of these HSCs can be used in all of the methods described herein. Optionally, ST-HSCs are useful because they are highly proliferative and can therefore rapidly generate differentiated progeny.

[0144] As used herein, the phrase "hematopoietic stem cell functional potential" refers to functional properties of hematopoietic stem cells, including: (1) multipotency (referring to the ability to differentiate into multiple different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells); (2) self-renewal (referring to the ability of hematopoietic stem cells to give rise to daughter cells with equivalent capabilities to the parent cell, and further, this ability occurs repeatedly throughout an individual's life without being exhausted); and (3) the ability of hematopoietic stem cells or their progeny to be reintroduced into a transplant recipient and thereafter home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis.

[0145] Hematopoietic stem cells are optionally obtained from blood products. Blood products include products obtained from the body or from bodily organs that contain cells of hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, placenta, peripheral blood, or mobilized peripheral blood. All of the above crude or unfractionated blood products can be enriched for cells with hematopoietic stem cell characteristics in a number of ways. For example, more mature differentiated cells are selected for removal by cell surface molecules they express. Optionally, the blood product is enriched for cells with hematopoietic stem cell characteristics, such as CD34. + Cells are fractionated by positive selection against CD34 + The cells are capable of self-renewal, multipotency, and contain a subpopulation of hematopoietic stem cells that, when reintroduced into a transplant recipient, can home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis. Such selection can be accomplished, for example, using commercially available magnetic anti-CD34 beads (Dynal, Lake Success, NY). Unfractionated blood products are optionally obtained directly from donors or retrieved from cryopreserved storage. Hematopoietic stem cells can also optionally be obtained from differentiated embryonic stem cells, differentiated induced pluripotent stem cells, or other reprogrammed adult cell types.

[0146] As used herein, the term "stem cell" or "undifferentiated cell" refers to a cell in an undifferentiated or partially differentiated state that has the property of self-renewal and the developmental potential to differentiate into multiple cell types. Stem cells can proliferate and give rise to more such stem cells while maintaining their functional capacity. Stem cells can divide asymmetrically, known as obligatory asymmetric differentiation, in which one daughter cell retains the functional capacity of the parent stem cell and the other daughter cell expresses some other specific function, phenotype, and / or developmental potential that differs from the parent cell. The daughter cell can itself be induced to proliferate and give rise to progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with the developmental potential of the parent. Differentiated cells may themselves be derived from pluripotent cells, which are themselves derived from pluripotent cells, or a portion of the stem cells in a population can divide symmetrically into two stem cells. Thus, the term "stem cell" refers to any subset of cells that, under certain circumstances, have the developmental potential to differentiate into a more specialized or differentiated phenotype than a stem cell, and, under certain circumstances, retain the ability to proliferate without substantial differentiation. In some embodiments, the term stem cell generally refers to a natural parent cell whose descendants (progeny cells) specialize by differentiation, often in various directions, by acquiring entirely different characteristics, as occurs, for example, in the gradual diversification of embryonic cells and tissues. Some differentiated cells have the ability to give rise to cells with greater developmental potential. Such ability may be natural or may be artificially induced by treatment with various factors. Cells that began as stem cells may progress to a differentiated phenotype, but can also be induced to "revert" and then re-express the stem cell phenotype. This term is often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "retrodifferentiation."

[0147] As used herein, "homing ability" refers to the ability of a stem cell to localize to an in vivo site capable of supporting productive hematopoiesis, such as the bone marrow.

[0148] As used herein, a cell population "enriched" for a particular cell type refers to a population in which the relative proportion of cells of a particular type is increased compared to a previous cell population (e.g., compared to a cell population prior to treatment with one or more agents that exhibit two or more actions combined selected from the group consisting of modulation of histone methylation, inhibition of TGFβ signaling, inhibition of p38 signaling, activation of canonical Wnt signaling, and modulation of histone acetylation).

[0149] The term "pluripotent," when used with reference to "pluripotent cells," refers to cells that have the developmental potential to differentiate into multiple different hematopoietic cell types. Hematopoietic stem cells are pluripotent and can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but cannot form neurons.

[0150] As used herein, the phrase " preserve pluripotency " or " maintain pluripotency " refers to the process of preserving the degree of pluripotency of cell population over a period of time.The degree of pluripotency of cell population represents the number and identity of differentiated cell types that cell population can differentiate into.For example, the cell population that exhibits pluripotency that is maintained ex vivo (for example, in culture) for 2 days can differentiate into at least the same number of different cell types as it was able to differentiate at the beginning of cell culture period.

[0151] As used herein, " mobilizing agent " refers to an agent that can induce the migration of hematopoietic stem cells from the bone marrow to peripheral blood of a subject.Exemplary mobilizing agents include CXCR4 antagonists, such as AMD3100, as well as GCSF and GROβ.

[0152] As used herein, the step of "contacting" a cell population with one or more agents can be accomplished in a variety of ways. For example, hematopoietic stem cell populations may be contacted with one or more agents that together exhibit two or more effects selected from the group consisting of modulation of histone methylation, inhibition of TGFβ signaling, inhibition of p38 signaling, activation of canonical Wnt signaling, and modulation of histone acetylation (e.g., LSD1 inhibitor IV RN-1; LSD1 inhibitor II S2101; LSD1 inhibitor LSD1-C76; LSD1 inhibitor III CBB1007; LSD1 inhibitor I; ALK5 inhibitor II (E-616452); LY364947; A83-01; trichostatin A; tranylcypromine; SB203580; CHIR99021; DMH1; sodium acetate; and istodax) by culturing the hematopoietic stem cells in the presence of these agents for a period of time, e.g., two days or more. When multiple kinds of active substances are contacted with cell population, multiple kinds of active substances can be present together in cell culture medium, so that cells are simultaneously exposed to one or more kinds of active substances.Or, one or more kinds of active substances can be added to cell culture medium successively.For example, one or more kinds of active substances can be added to the cell population in culture according to a specific regimen, for example, different active substances are added to culture medium at different times during the culture period.

[0153] As used herein, the term "engraftment potential" refers to the ability of hematopoietic stem and progenitor cells to repopulate tissues, regardless of whether they are naturally circulating or provided by transplantation. This term encompasses all events surrounding or underlying engraftment, such as cell tissue homing and colonization within the tissue of interest. Engraftment efficiency or rate can be assessed or quantified using any clinically accepted parameter known to those skilled in the art, including, for example, assessment of competitive repopulating units (CRUs); marker incorporation or expression in the tissue to which the stem cells have homed, colonized, or engrafted; or assessment of the subject's progress by disease progression, hematopoietic and progenitor cell survival, or recipient survival. In one embodiment, engraftment is confirmed by measuring peripheral blood white blood cell counts in the post-transplant period. Alternatively, engraftment can be assessed by measuring bone marrow cell recovery by donor cells in a bone marrow aspirate sample.

[0154] As used herein, the term "self-renewal" refers to the ability of a stem cell to generate daughter stem cells that have the same phenotype, characteristics, and functional capabilities as the original stem cell. In particular, as used herein, self-renewal is defined as the ability to continue to proliferate while maintaining an undifferentiated pluripotent stem cell state.

[0155] As used herein, a cell population that is "freshly isolated" from a donor refers to a cell population that is isolated from a donor without being cryopreserved and thawed prior to infusion. The cell population may be isolated from a donor and separated into two intermediate populations, one of which may be infused into a patient, and the other may be cryopreserved. In this case, the intermediate population infused into a patient is considered freshly isolated. A cell population is considered freshly isolated from a donor if it is cultured ex vivo before being infused into a patient. For example, this culture step may be performed to expand, enrich, and / or maintain a hematopoietic stem cell population before the resulting cells are administered to a patient. In these cases, the resulting cells are considered freshly isolated from a donor if the cells administered to a patient are not cryopreserved and thawed prior to being infused into a patient.

[0156] As used herein, the term "ZsGr" refers to the fluorescent cassette ZsGreen, and references to "Fdg5·ZsGr" indicate that the fluorescent ZsGreen reporter cassette has been knocked in-frame into the endogenous Fgd5 locus. In these cases, ZsGreen expression is under the control of the Fgd5 promoter. Previously, we demonstrated that Fgd5 is expressed exclusively in hematopoietic stem cells of the mouse hematopoietic system. Furthermore, we demonstrated that highly pure hematopoietic stem cells can be isolated using Fdg5·ZsGreen mice based on monochromatic ZsGreen fluorescence. Hematopoietic stem cells isolated from such mice are referred to as ZsGr+ or Fgd5·ZsGr+. This reporter construct is further detailed in Gazit R, Mandal PK, Ebina W, Ben-Zvi A, Nombela-Arrieta C, Silberstein LE, Rossi DJ. Journal of Experimental Medicine. 211(7):1315-31 (2014). This disclosure is incorporated herein by reference.

[0157] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce," "reduce," "reduce," or "inhibit" typically refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a particular treatment), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater. As used herein, "reduce" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" refers to 100% inhibition compared to a reference level.

[0158] As used herein, "CRU (competitive reconstituting unit)" refers to a measure of long-term engrafting stem cells that can be detected after in vivo transplantation.

[0159] As used herein, the term "modulate" refers to altering or inducing a change in a specific biological activity. Modulation includes, but is not limited to, stimulating or inhibiting activity (e.g., by activating a receptor so that it does not transduce a signaling pathway to initiate a signaling cascade, by activating an endogenous inhibitor that attenuates a biological activity, or by inhibiting the activity of a protein that inhibits a specific biological function). Modulating a protein that transduces a specific signaling pathway can result in an increase or decrease in activity (e.g., histone methylation, TGFβ signaling, p38 signaling, Wnt signaling, or histone acetylation), a change in the affinity of a protein in the pathway for another protein, or another change in the structural, functional, or immunological properties associated with the pathway or the activity of a protein within the pathway.

[0160] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase of up to or including 100%, or an increase of 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or an increase of 2-fold to 10-fold or more compared to a reference level. In the context of a marker, an "increase" is a statistically significant increase in such level.

[0161] As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0162] As used herein, a "recipient" is a patient who receives a transplant, such as a transplant containing a population of hematopoietic stem cells or differentiated cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0163] As used herein, a "donor" is a human or animal from which one or more cells are isolated before the one or more cells or their progeny are administered to a recipient. The one or more cells may be, for example, a hematopoietic stem cell population that is expanded, enriched, or maintained according to the methods of the invention before the one or more cells or their progeny are administered to a recipient.

[0164] Preferably, the subject is a mammal.The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow.A non-human mammal can be advantageously used as a subject corresponding to an animal model of disease and / or treatment.The subject may be male or female.

[0165] As used herein, the terms "protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues joined together by peptide bonds between the α-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated amino acids, glycated amino acids, glycosylated amino acids, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides. However, the usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0166] As used herein, the terms "polynucleotide," "nucleic acid," or "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, a nucleic acid can be DNA. In another aspect, a nucleic acid can be RNA. In another aspect, a nucleic acid can be chemically modified RNA. For example, a nucleic acid can be chemically modified messenger RNA. In another aspect, a nucleic acid can be RNA synthesized using natural or synthetic nucleotide analogs. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA, tRNA, siRNA, miRNA, or shRNA.

[0167] As used herein, the term " siRNA " refers to the double-stranded nucleic acid molecule that can RNA interference or " RNAi ", as disclosed in, for example, Bass, Nature 411: 428-429 (2001); Elbashir et al., Nature 411: 494-498 (2001); WO2000 / 044895; WO2001 / 036646; WO1999 / 032619; WO2000 / 001846; WO2001 / 029058; WO1999 / 007409; and WO2000 / 044914.Each of these disclosures is incorporated herein by reference.As used herein, siRNA molecule is not limited to the molecule that contains only RNA, but also includes chemically modified nucleotide and non-nucleotide that has RNAi ability or activity.

[0168] As used herein, the term "miRNA" refers to a class of small, non-coding, single-stranded RNAs, typically 18-23 nucleotides in length. miRNA molecules can regulate gene expression by modulating the stability and translation of mRNAs that encode specific proteins. miRNAs also influence other nuclear processes, such as heterochromatin formation and genome reorganization.

[0169] As used herein, the term "shRNA" (short hairpin RNA) refers to an RNA duplex containing an siRNA, a portion of which has a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion located between the two sequences forming the duplex. The loop may vary in length. For example, the loop may be 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also contain a 3' overhang portion or a 5' overhang portion. For example, the overhang may be a 3' overhang or a 5' overhang, and may be 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0170] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0171] As used herein, the term "administering" refers to placing a compound, cell, or cell population disclosed herein in a subject by a method or route that results in at least partial delivery of the agent at the desired site. Pharmaceutical compositions containing the compounds or cells disclosed herein can be administered by any suitable route that results in effective treatment in the subject.

[0172] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2SD) or more than 2SD.

[0173] Except in the working examples, or unless otherwise specified, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." As used herein, the term "about" indicates a deviation of ±10%.

[0174] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and each component thereof that is essential to the method or composition, but permits the inclusion of elements not specified, whether essential or non-essential.

[0175] The term "consisting of" refers to the compositions, methods, and each component thereof described herein, excluding any element not recited in the description of the embodiment.

[0176] As used herein, the term "consisting essentially of" refers to elements required for a particular embodiment. The term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment.

[0177] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0178] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methods, protocols, and reagents described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4. thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in and Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0179] As used herein, " LSD1 inhibitor II S2101 " refers to a compound having the structure of formula I or its pharmaceutically acceptable salt.The synthesis of LSD1 inhibitor II S2101 and the structure and synthesis of related compounds, for example, derivatives, including compounds with similar biological activity, are known in the art.See, for example, WO1998 / 009625; WO2003 / 016309; WO2004 / 087646; WO2005 / 033066; WO2006 / 034769; WO2007 / 068330; WO2009 / 147217; WO2014 / 194280; US2013 / 0178520; US5,426,196; EP764640; and EP764632.The disclosures of each of these are incorporated herein by reference. TIFF2025116082000002.tif43128

[0180] As used herein, "LSD1 inhibitor IV RN-1" refers to a compound having the structure of Formula II or a pharmaceutically acceptable salt thereof. The synthesis of LSD1 inhibitor IV RN-1, as well as the structures and syntheses of related compounds, e.g., derivatives, including compounds with similar biological activity, are known in the art. See, e.g., WO2015 / 003643; WO2014 / 205266; WO2014 / 19428; WO2012 / 122405; WO2014 / 018375; WO2009 / 147217; WO2007 / 068330; WO2006 / 034769; WO2005 / 033066; WO2004 / 087646; WO2003 / 016309; WO1998 / 009625; WO1997 / 010822; WO1997 / 010825; WO1995 / 017408; and US2013 / 0178520, each of which is incorporated herein by reference in its entirety. TIFF2025116082000003.tif25128

[0181] As used herein, "LSD1 inhibitor LSD1-C76" refers to a compound having the structure of Formula III or a pharmaceutically acceptable salt thereof. The synthesis of the LSD1 inhibitor LSD1-C76, as well as the structures and syntheses of related compounds, e.g., derivatives, including compounds with similar biological activity, are known in the art. TIFF2025116082000004.tif23128

[0182] As used herein, "LSD1 inhibitor III CBB1007" refers to a compound having the structure of Formula IV or a pharmaceutically acceptable salt thereof. The synthesis of LSD1 inhibitor III CBB1007, as well as the structures and syntheses of related compounds, e.g., derivatives, including compounds with similar biological activity, are known in the art. TIFF2025116082000005.tif29128

[0183] As used herein, "LSD1 inhibitor I" refers to a compound having the structure of Formula V or a pharmaceutically acceptable salt thereof. LSD1 inhibitor I is also referred to in the art as BHC110 inhibitor I, histone lysine demethylase inhibitor III, and / or KDM1 inhibitor I. The synthesis of LSD1 inhibitor I, as well as the structures and syntheses of related compounds, e.g., derivatives, including compounds with similar biological activity, are known in the art. TIFF2025116082000006.tif21128

[0184] As used herein, "ALK5 inhibitor II" (also referred to as "RepSox" or "E-616452") refers to a compound having the structure of Formula VI or a pharmaceutically acceptable salt thereof. ALK5 inhibitor II (E-616452) is also known in the art as transforming growth factor-b type I receptor kinase inhibitor II or Repsox. TIFF2025116082000007.tif33128

[0185] As used herein, " LY364947 " refers to the compound having the structure of formula VII or its pharmaceutically acceptable salt.LY364947 is also known in the art as ALK5 inhibitor I TbR-I inhibitor transforming growth factor-b type I receptor kinase inhibitor.The synthesis of LY364947 and ALK5 inhibitor II, and the structure and synthesis of related compounds, such as derivatives, including compounds with similar biological activity, are known in the art.See, for example, US6,028,072;WO2002 / 062794;WO2004 / 026302;WO2004 / 026306;WO2004 / 072033;WO2007 / 088651;WO2007 / 070866;WO2007 / 039151;and WO2007 / 052943. each of which is incorporated herein by reference in its entirety. TIFF2025116082000008.tif39128

[0186] As used herein, "A83-01" refers to a compound having the structure of Formula VIII or its pharmaceutically acceptable salt.The synthesis of A83-01 and the structure and synthesis of related compounds, including compounds with similar biological activity, for example, derivatives, are known in the art.See, for example, US2003 / 0064997;US2003 / 0064997;US2003 / 0064997;US5,777,097;US5,871,934;GB2306108;WO1993 / 014081;WO1995 / 003297;WO1997 / 33883;WO1997 / 35855;and WO1993 / 014081.Each of these is incorporated herein by reference in its entirety. TIFF2025116082000009.tif35128

[0187] As used herein, "trichostatin A" refers to a compound having the structure of Formula IX or a pharmaceutically acceptable salt thereof. The synthesis of trichostatin A and the structure and synthesis of related compounds, e.g., derivatives, including compounds with similar biological activity, are known in the art. See, for example, US 4,690,918; US 4,946,999; EP 0827946; JP 07206670; and JP 60149520. Each of these is incorporated herein by reference in its entirety. TIFF2025116082000010.tif37128

[0188] As used herein, " tranylcypromine " refers to a compound having the structure of formula X or its pharmaceutically acceptable salt.The synthesis of tranylcypromine and the structure and synthesis of related compounds, such as derivatives, including compounds with similar biological activity, are known in the art.See, for example, US2,993,931; US2,997,422; US3,011,945; US3,079,403; US3,134,676; and US3,244,596.Each of these is incorporated herein by reference in its entirety. TIFF2025116082000011.tif21128

[0189] As used herein, "SB203580" refers to the compound having the structure of formula XI or its pharmaceutically acceptable salt.The synthesis of SB203580 and the structure and synthesis of related compounds, such as derivatives, including compounds with similar biological activity, are known in the art.See, for example, WO2007 / 070866;WO2008 / 022182;WO2010 / 065917;WO2010 / 077955;and WO2010 / 102267.Each of these is incorporated herein by reference in its entirety. TIFF2025116082000012.tif40128

[0190] As used herein, "CHIR99021" refers to a compound having the structure of formula XII or its pharmaceutically acceptable salt.The synthesis of CHIR99021 and the structure and synthesis of related compounds, such as derivatives, including compounds with similar biological activity, are known in the art.See, for example, WO1999 / 065897;WO2002 / 020495;WO2005 / 003948;WO2006 / 001863;WO2006 / 117212;WO2007 / 016485;WO2007 / 075911;WO2007 / 083978;And US2002 / 0156087.Each of these is incorporated herein by reference in its entirety. TIFF2025116082000013.tif34128

[0191] As used herein, " DMH1 " refers to a compound having the structure of formula XIII or its pharmaceutically acceptable salt.The synthesis of DMH1 and the structure and synthesis of related compounds, for example, derivatives, including compounds with similar biological activity, are known in the art.See, for example, WO2012 / 115120; WO2013 / 016452; WO2013 / 163228; WO2013 / 166488; WO2014 / 138088; WO2014 / 176606; WO2014 / 200115; WO2014 / 062138; US2014 / 0248696; and US8,822,684.Each of these is incorporated herein by reference in its entirety. TIFF2025116082000014.tif56128

[0192] As used herein, " istodax " or " romidespine " refers to a compound having the structure of formula XIV or its pharmaceutically acceptable salt. The synthesis of istodax and the structure and synthesis of related compounds, such as derivatives, including compounds with similar biological activity, are known in the art. For example, see WO14 / 102731; WO12 / 009336; WO13 / 106696; WO02 / 20817; US4,977,138; US7,611,721; US7,608,280; and US2012 / 046442; and J. Am. Chem. Soc. 118:7237-7238, 1996. Each of these is incorporated herein by reference in its entirety. TIFF2025116082000015.tif59128

[0193] Other terms are defined herein in the description of various aspects of the present invention. [Brief explanation of the drawings]

[0194] [Figure 1] This shows that HSC potency is rapidly lost upon culture. Peripheral blood analysis after transplantation of freshly isolated murine HSCs or HSCs cultured ex vivo for 12 days in S-clone+IL12 / SCF / TPO+0.75% BSA into lethally irradiated hosts. Note that cultured cells do not retain the ability to generate multilineage (B cell, T cell, myeloid, granulocyte) peripheral blood donor chimerism, whereas freshly transplanted HSCs generate robust levels of donor peripheral blood chimerism comprised of all analyzed blood lineages, including B cells, T cells, myeloid cells, and granulocytes. [Figure 2]We demonstrate that engineered Fgd5-ZsGreen reporter mice faithfully label HSCs. Bone marrow cells derived from Fgd5-ZsGreen reporter mice were enriched for cKit+ cells using magnetic beads and analyzed by flow cytometry for ZsGreen expression and a panel of markers (lineage (Ter119), CD3, CD4, CD8, B220, Mac1, Gr1, Il7RA), cKit, Sca1, CD150, and CD48). ZsGreen expression was demonstrated for the indicated stem / progenitor subpopulations. Further details regarding the construction, characterization, and specificity of the Fgd5-ZsGreen HSC reporter mice can be found in Gazit R, Mandal PK, Ebina W, Ben-Zvi A, Nombela-Arrieta C, Silberstein LE, Rossi DJ. Journal of Experimental Medicine, 211(7):1315-31 (2014). [Figure 3A] Figure 3A shows that Fgd5·ZsGr+ marks HSC potential during ex vivo culture of HSCs. Figure 3A shows an overview. Fgd5·ZsGr+ HSCs were isolated from reporter mice and cultured in S-clone+IL12 / SCF / TPO+0.75% BSA for 4 days. On day 4, 300 ZsGr+ cells and 300 ZsGr- cells were selected and competitively transplanted into lethally irradiated mice. This demonstrates that the ZsGr+ fraction retained all HSC activity, as detailed in Figure 3B. [Figure 3B] Figure 3B shows that Fgd5·ZsGr+ marks HSC potential during ex vivo culture of HSCs. Figure 3B illustrates peripheral blood chimerism and peripheral blood granulocyte chimerism in mice transplanted with ZsGr+ and ZsGr- cells. Lineage contribution of ZsGr+ and ZsGr- cells. The data indicate that only ZsGr+ cells retain HSC activity. [Figure 4]A schematic diagram of small molecule screening for compounds supporting ex vivo HSC maintenance and expansion is shown. 1) ZsGr+ HSCs were isolated using bone marrow of Fgd5·ZsGr+ HSC reporter mice. 2) Isolation of HSCs labeled with the ZsGr reporter. 3) Development of an assay in which hits were identified by sustained HSC reporter expression in cultured HSCs over DMSO controls after 6 days of ex vivo culture. 4) Hit compounds were then functionally validated in in vitro and in vivo transplantation assays. [Figure 5] Figures 5A and 5B show the development of a sensitive assay. Figure 5A shows that a total of 200 ZsGr+ HSCs (derived from Fgd5 ZsGreen HSC reporter mice) and ZsGr- HSCs (derived from wild-type mice lacking the Fgd5 Zs+ reporter) were seeded per well at various ratios (1:0; 1:1; 1:10; 1:20, 1:100, 0:1; indicated as ZsGr+ percentage) and imaged using Operetta (Perkin Elmer) after 2 days of ex vivo culture. Individual cells were plotted above or below the threshold for ZsGreen detection. Figure 5B shows that the percentage of ZsGreen+ cells was determined after 2 days of culture. This confirmed the analytical parameters and sensitivity for robust ZsGr+ signal detection after 2 days of culture. [Figure 6] A breakdown of pathways targeted in the primary small molecule screen is shown. [Figure 7] Figures 7A and 7B show initial screening results for various small molecule and growth factor library screens. Figure 7A shows the number of screened compounds, initial hits (shown as dose responses), and validated hits (by flow cytometry to quantify ZSGr+) from each of four different libraries: small molecules targeting kinases, epigenetic regulators, and GPCRs, as well as a growth factor peptide library. Figure 7B illustrates representative results from a six-point dose response (10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM). * indicates a hit. [Figure 8] Ex vivo culture of 20 mouse HSCs (cell line—Sca1+ckit+CD34-Flk2-CD150+Fgd5·ZsGr+) for 7 or 14 days in the presence of DMSO, LSD1 inhibitor IV (LS), Tgfβ inhibitor (RepSox), or a combination of both (C2) is shown. Images were taken at 4x magnification. Note that cultures in the presence of LS and C2 are more homogeneous and less differentiated. [Figure 9] Figures 9A and 9B show the expansion of phenotypic mouse HSCs over 14 days ex vivo. Twenty mouse HSCs (cell lineage—Sca1+ckit+CD34-Flk2-CD150+Fgd5·ZsGr+) were cultured for 14 days in the presence of DMSO, LSD1 inhibitor IV (LS), TGFβ inhibitor RepSox (RS), and a combination of both (C2). This method supports the maintenance and expansion of ZsGr+ HSCs. Figure 9A shows a representative FACS plot of HSCs cultured ex vivo for 14 days in the presence of DMSO and a combination of LSD1 inhibitor (LS) and TGFβ inhibitor RepSox (RS) (C2). It can be seen that the presence of C2 increased the level of phenotypic HSCs. Figure 9B illustrates the number of cell lineage-Sca1+ckit+CD34-Flk2-CD150+Fgd5·ZsGr+HSCs in each condition after 14 days of ex vivo culture. [Figure 10] Figures 10A-10C show that the LSD1 inhibitor (LS), the Tgfβ inhibitor RepSox (RS), and a combination of both (C2) support the maintenance and expansion of ZsGr+ mouse HSCs. Figure 10A illustrates bright-field and ZsGr images of 20 Fgd5·ZsGr+ HSCs cultured ex vivo for 4.5 days in the presence of the LSD1 inhibitor IV (LS), the Tgfβ inhibitor RepSox (RS), and a combination of both (C2). Figure 10B shows the frequency of ZsGr+ cells remaining after 4.5 days of ex vivo culture. Figure 10C illustrates the number of ZsGr+ and ZsGr- cells after 4.5 days of culture. [Figure 11]Figures 11A and 11B show the results of a colony formation assay of purified mouse HSCs exposed to DMSO, LSD1 inhibitor (LS), Tgfβ inhibitor RepSox (RS), and a combination of both (C2) for 14 days of culture. HSCs were cultured in the presence of DMSO, LSD1 inhibitor IV (LS), Tgfβ inhibitor RepSox (RS), and a combination of both (C2) for 14 days, and then 1 / 300 of the culture was plated into 1 ml of MethoCult3434. Ten days after plating, resulting colonies were picked and placed into methylcellulose. Figure 11A illustrates the number and composition of colonies. Figure 11B illustrates the total number of cells generated by HSCs cultured for 14 days. [Figure 12] Figures 12A-12D show the results of competitive transplantation of purified mouse HSCs (cell line—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) exposed to DMSO, LSD1 inhibitor (LS), TGFβ inhibitor RepSox (RS), and a combination of both (C2) for 14 days. Cultures derived from 100 starting mouse HSCs were cultured ex vivo for 14 days in the presence of DMSO, LSD1 inhibitor IV (LS), TGFβ inhibitor RepSox (RS), and a combination of both (C2) before competitive transplantation into lethally irradiated recipients. Figure 12A shows a graph of peripheral blood donor chimerism. Figure 12B shows a graph of peripheral blood donor (CD45.2) granulocyte chimerism. Figure 12C shows a graph of individual recipient mouse donor chimerism. FIG. 12D depicts a graph of lineage contribution of donor reconstitution showing B cells (B220+), T cells (CD3+), and myeloid cells (Mac1+) at 24 weeks post-transplant. [Figure 13A]Figure 13A shows the results of a limiting dilution assay of purified mouse HSCs (cell line—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) cultured for 14 days with a combination (C2) of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS). Cultures generated from 1, 5, 20, 50, or 200 starting mouse HSCs were cultured ex vivo in the presence of C2 for 14 days prior to competitive transplantation. Figure 13A illustrates peripheral blood donor chimerism. [Figure 13B] Figure 13B shows the results of a limiting dilution assay of purified mouse HSCs (cell line—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) cultured for 14 days with a combination (C2) of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS). Cultures generated from 1, 5, 20, 50, or 200 starting mouse HSCs were cultured ex vivo in the presence of C2 for 14 days prior to competitive transplantation. Figure 13B illustrates peripheral blood donor (CD45.2) granulocyte chimerism. [Figure 13C] Figure 13C shows the results of a limiting dilution assay of purified mouse HSCs (cell lineage—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) cultured for 14 days with a combination (C2) of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS). Cultures generated from 1, 5, 20, 50, or 200 starting mouse HSCs were cultured ex vivo in the presence of C2 for 14 days prior to competitive transplantation. Figure 13C illustrates the lineage contribution of donor reconstitution, showing B cells (B220+), T cells (CD3+), and myeloid cells (Mac1+). [Figure 13D]Figure 13D shows the results of a limiting dilution assay of purified mouse HSCs (cell line—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) cultured for 14 days with a combination (C2) of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS). Cultures generated from 1, 5, 20, 50, or 200 starting mouse HSCs were cultured ex vivo in the presence of C2 for 14 days before competitive transplantation. Figure 13D illustrates donor chimerism for individual recipients. [Figure 13E] Figure 13E shows the results of a limiting dilution assay of purified mouse HSCs (cell line—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) cultured for 14 days with a combination (C2) of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS). Cultures generated from 1, 5, 20, 50, or 200 starting mouse HSCs were cultured ex vivo in the presence of C2 for 14 days prior to competitive transplantation. Figure 13E illustrates the granulocyte chimerism of individual recipients 20 weeks after transplantation. [Figure 13F] Figure 13F shows the results of a limiting dilution assay of purified mouse HSCs (cell line—Sca1+ckit+CD150+CD48-CD34-Fgd5ZsGr+) cultured for 14 days with a combination (C2) of the LSD1 inhibitor IV (LS) and the TGFβ inhibitor RepSox (RS). Cultures generated from 1, 5, 20, 50, or 200 starting mouse HSCs were cultured ex vivo in the presence of C2 for 14 days prior to competitive transplantation. Figure 13F illustrates the calculation of limiting dilution frequencies. bioinf.wehi.edu.au / software / elda / Hu, Y, and Smyth, GK (2009). ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. Journal of Immunological Methods 347, 70-78 [Figure 14]Figures 14A and 14B show secondary small molecule screens for compounds that synergize with C2 (LSD1 inhibitor IV and Tgfβ inhibitor) to support HSC ex vivo maintenance and expansion. Schematic representation of the primary screen (Figure 14A) and secondary screen (Figure 14B). In the secondary screen, 124 potential hit compounds identified in the primary screen were rescreened in the presence of C2 (screening 2). [Figure 15] Figures 15A-15C show potential hits from secondary screening, targeting pathways of interest including Tgfβ, histone methylation, histone acetylation, p38 signaling, and Wnt signaling. Figure 15A illustrates an outline of two strategies used to identify hits in secondary small molecule screening (C2 is a combination of LSD1 inhibitor IV and Tgfβ inhibitor RepSox). Figure 15B illustrates hits discovered by following a strategy based on ZsGreen+ HSC percentage (Strategy 1). Figure 15C illustrates hits discovered by following a strategy based on ZsGreen+ HSC number (Strategy 2). [Figure 16] Figures 16A and 16B show hits from a secondary screen of compounds identified in the primary screen and then rescreened in the presence of C2 (a combination of the LSD1 inhibitor IV and the Tgfβ inhibitor RepSox). Figure 16A illustrates hits discovered by following a strategy based on the percentage of ZsGreen+ HSCs. Figure 16B illustrates hits discovered by following a strategy based on the number of ZsGreen+ HSCs. [Figure 17]These images show the results of an experiment testing compounds previously reported to maintain mouse HSCs. ZsGreen-positive HSCs were cultured for 6 days in the presence of dmPGE2 (North, Zon, Nature. 2007), BIO (Ko et al., Stem Cells. 2011), p38 inhibitor (Wang et al., Stem Cells Dev. 2011), and DMSO (negative control). D2 represents ZsGr+ HSCs maintained ex vivo for 2 days. The threshold for cells identified as ZsGreen+ (i.e., HSCs) is indicated by an arrow. [Figure 18] Figures 18A and 18B show a hypothesis-driven strategy for tuning candidate pathways for ex vivo HSC maintenance and expansion. Figure 18A illustrates the selection of candidate target pathways by comparing intestinal stem cell and hematopoietic stem cell maintenance and proliferation signals. Figure 18B illustrates the selection of agents / pathway modulators. [Figure 19] A schematic diagram for assessing the activity of pathway modulators on HSC maintenance and expansion is shown. Fgd5-ZsGreen+ immunophenotypic HSCs (cell line - cKit+Sca1+CD150+CD48-Fgd5·ZsGreen+) were sorted and cultured in the presence of cytokines alone (standard medium) or in the additional presence of seven candidate pathway modulators (W7 medium). The seven pathway-modulating compounds were A83-01, tranylcypromine, trichostatin A, SB203580, CHIR99021, DMH1, and sodium acetate (referred to here as supplements). After 14 days of culture, multiparametric analysis of cell immunophenotypes was performed by flow cytometry. [Figure 20] We demonstrate that combinatorial modulation of seven candidate pathways maintains and expands immunophenotypic HSCs during ex vivo culture. Fifty mouse HSCs were cultured in serum-free medium supplemented with SCF, TPO, and IL-12 with or without seven candidate pathway modulators. Flow cytometry analysis was performed on day 15. The seven pathway-modulating compounds were A83-01, tranylcypromine, trichostatin A, SB203580, CHIR99021, DMH1, and sodium acetate. [Figure 21A] Figure 21A shows the contribution of each compound / pathway to the ability to maintain and expand HSC phenotype during ex vivo culture. Fifty mouse HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured in the absence of compound and the presence of cytokines only (SCF, TPO, and IL-12) (control), or with all seven compounds (W7) or with or without one of each compound (A83-01 (A), tranylcypromine (TC), trichostatin A (TSA), SB203580 (p38i), CHIR99021 (Chir), DMH1 (DMH), and sodium acetate (OAC)). Flow cytometry analysis was performed on day 14. (Figure 21A) shows differentiation into lineage+ cells (stained with an antibody cocktail against B cell, T cell, myeloid cell, erythroid, and granulocyte antigens). In this case, the cell line positive is to the right of the dashed line. [Figure 21B] Figure 21B shows the contribution of each compound / pathway to the ability to maintain and expand phenotypic HSCs during ex vivo culture. Fifty mouse HSCs (cell line-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured in the absence of compound and the presence of cytokines only (SCF, TPO, and IL12) (control), or with all seven compounds (W7) or with or without one of each compound (A83-01 (A), tranylcypromine (TC), trichostatin A (TSA), SB203580 (p38i), CHIR99021 (Chir), DMH1 (DMH), and sodium acetate (OAC)). Flow cytometry analysis was performed on day 14. Figure 21B illustrates the absolute number of HSCs after 14 days of culture from 50 starting HSCs under the indicated conditions. [Figure 22]Figures 22A and 22B show that structurally distinct HDAC inhibitors function equally well to maintain immunophenotypic HSCs. One hundred mouse HSCs (cell line-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured in the absence of compound and the presence of cytokines alone (SCF, TPO, and IL12) (control), or supplemented with a cocktail of compounds (lithium chloride, nicotinamide, N-acetylcysteine, ascorbic acid, A83-01, and SB203580) and the structurally distinct HDAC inhibitors valproic acid (VPA) or trichostatin A (TSA). Figure 22A illustrates flow cytometry analysis on day 7. Figure 22B illustrates the percentage of Fgd5-ZsGreen+Sca1+ cells for each replicate experiment. [Figure 23] This figure shows that the addition of additional compounds during ex vivo culture reduces the heterogeneity of Fgd5·ZsGr+ HSC cells with respect to CD48 and Sca1 expression. Forty mouse HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured for 12 days in the presence of cytokines (SCF, TPO, and IL-12) and a compound cocktail (lithium chloride, nicotinamide, N-acetylcysteine, ascorbic acid, A83-01, and SB203580, trichostatin A) containing either a DNA methyltransferase inhibitor (RG108) or a G9a inhibitor (UNC0638), or both. Flow cytometry plots of Fgd5+ lineage-cells from the indicated culture conditions are shown. Histograms indicate the percentages of the indicated subpopulations. [Figure 24]This figure shows the in vivo function of mouse HSCs cultured for 14 days in the presence of DMSO (standard) or a combination of seven pathway-targeting compounds: the TGFβ inhibitor A83-01, the Lsd1 inhibitor tranylcypromine, the HDAC inhibitor trichostatin A, the p38 kinase inhibitor SB203580, the BMP inhibitor DMH1, the Gsk3β inhibitor Chir99021, and sodium acetate. Ten HSCs were cultured for 14 days under the indicated conditions and then competitively transplanted in vivo (against 2 x 10 congenitally labeled bone marrow cells) into lethally irradiated hosts. Peripheral blood donor chimerism was demonstrated at the indicated times posttransplant. [Figure 25A] Figure 25A shows the in vivo function of mouse HSCs cultured for 14 days in the presence of DMSO (S: standard) or seven pathway-targeting compounds (W7: Tgfβ inhibitor A83-01, Lsd1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A, p38 kinase inhibitor SB203580, BMP inhibitor DMH1, Gsk3β inhibitor Chir99021, and sodium acetate). One hundred HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured under the indicated conditions for 14 days and then competitively transplanted in vivo (against 2 × 105 congenically labeled bone marrow cells). Figure 25A illustrates peripheral blood donor chimerism at the indicated time points after transplantation. [Figure 25B] Figure 25B shows the in vivo function of mouse HSCs cultured for 14 days in the presence of DMSO (S: standard) or seven pathway-targeting compounds (W7: Tgfβ inhibitor A83-01, Lsd1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A, p38 kinase inhibitor SB203580, BMP inhibitor DMH1, Gsk3β inhibitor Chir99021, and sodium acetate). One hundred HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured for 14 days under the indicated conditions and then competitively transplanted in vivo (against 2 × 105 congenically labeled bone marrow cells). Figure 25B illustrates granulocyte donor chimerism at the indicated time points after transplantation. [Figure 25C] Figure 25C shows the in vivo function of mouse HSCs cultured for 14 days in the presence of DMSO (S: standard) or seven pathway-targeting compounds (W7: Tgfβ inhibitor A83-01, Lsd1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A, p38 kinase inhibitor SB203580, BMP inhibitor DMH1, Gsk3β inhibitor Chir99021, and sodium acetate). One hundred HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured for 14 days under the indicated conditions and then competitively transplanted in vivo (against 2 × 105 congenically labeled bone marrow cells). Figure 25C illustrates donor HSC chimerism in the bone marrow of transplant recipients transplanted with HSCs cultured for 14 days under the indicated conditions. [Figure 26] We demonstrate that modulation of four pathways is sufficient to maintain / expand the immunophenotype of mouse HSCs. Fifty HSCs (lineage-, ckit+, Sca1+, CD150+, CD48-, Fgd5ZsGr+) were cultured for 14 days in serum-free medium supplemented with cytokines in the presence of DMSO or four pathway-targeting compounds (W4: Tgfβ inhibitor A83-01, Lsd1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A, p38 kinase inhibitor SB203580) identified from an initial set of seven compounds (Tgfβ inhibitor A83-01, Lsd1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A, p38 kinase inhibitor SB203580, BMP inhibitor DMH1, Gsk3β inhibitor Chir99021, and sodium acetate). The immunophenotype of HSCs (cell lineage - cKit+Sca1+CD48-CD150+Fgd5ZsGreen+CD41-) was analyzed by flow cytometry. [Figure 27] Figure 1 shows a sorting strategy for obtaining primary human HSCs from CD34+ enriched cord blood. Sorted HSCs have the immunophenotype of CD34+ lineage-CD38-CD45RA-CD90+. [Figure 28]Figures 28A and 28B show that the combination (C2) of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS) can maintain and expand phenotypic human cord blood (CB) HSCs after 14 days of ex vivo culture. Figure 28A illustrates a schematic of the experimental design. Figure 28B illustrates flow cytometry of cultured cord blood HSCs (CD34+CD45RA-CD90+CD38-, initially seeded at 50 cells per well) after 14 days of plating in the presence of DMSO, stemregenin 1 (SR1), the LSD1 inhibitor (LS), the Tgfβ inhibitor RepSox (RS), and the combination (C2) of the LSD1 inhibitor IV and RepSox. The percentage of CD34+CD45RA- cells (contour plot) and the percentage of CD90+ cells (histogram) (+ / - standard deviation) are shown. [Figure 29] Figures 29A and 29B show that the combination (C2) of LSD1 inhibitor IV (LS) and Tgfβ inhibitor RepSox (RS) can maintain and expand phenotypically human cord blood HSCs after 14 days of ex vivo culture. Figure 29A illustrates the percentage of HSCs (defined as CD34+CD45RA-CD90+) relative to total viable cells. Figure 29B illustrates the absolute number of HSCs after culturing 50 cord blood HSCs (CD34+CD45RA-CD90+CD38-) in the presence of DMSO, stemregenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfβ inhibitor RepSox (RS), and the combination (C2) of LSD1 IV inhibitor and RepSox. *p<0.05 unpaired t-test. [Figure 30]Figures 30A and 30B show that the combination of the LSD1 inhibitor IV (LS) and the Tgfβ inhibitor RepSox (RS) (C2) maintains and enhances the primitive in vitro colony-forming ability of human cord blood HSCs after 14 days of ex vivo culture. Figure 30A illustrates the number and composition of colonies adjusted to represent the entire well after 14 days of ex vivo culture (Note: Only a portion of the well was added to methocult for colony formation). Figure 30B illustrates the frequency of myeloid colony types from 14-day cord blood HSC cultures. Note the increased frequency of the most primitive GEMM colonies (colonies composed of granulocyte, macrophage, erythroid, and megakaryocytic lineages) after culture in RS, LS, or C2. [Figure 31] Figures 31A and 31B show that the combination (C2) of LSD1 inhibitor IV (LS) and Tgfβ inhibitor RepSox (RS) can maintain and expand phenotypically human bone marrow HSCs after 14 days of ex vivo culture. Figure 31A depicts flow cytometry of cultured bone marrow-derived HSCs (FACS-purified as CD34+CD45RA-CD90+CD38- cells, 80 cells per well) after plating for 14 days in the presence of DMSO, stemregenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfβ inhibitor RepSox (RS), and the combination (C2) of LSD1 inhibitor IV and RepSox. The percentage of CD34+CD45RA- cells (dot plot) and the percentage of CD90+ cells (histogram) (+ / - standard deviation) are shown. Figure 31B illustrates the percentage of immunophenotyped HSCs (CD34+CD45RA-CD90+CD38-) after culture in the presence of DMSO, stemregenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfβ inhibitor RepSox (RS), and a combination of LSD1 inhibitor IV and RepSox (C2). *p<0.05, **p<0.005 unpaired t-test. [Figure 32A]Figure 32A shows that the combination (C2) of LSD1 inhibitor IV (LS) and Tgfβ inhibitor RepSox (RS) can maintain and expand phenotypically human mobilized peripheral blood HSCs after 14 days of ex vivo culture. Figure 32A depicts flow cytometry of cultured mobilized peripheral blood HSCs (FACS-purified as CD34+CD45RA-CD90+CD38-, 50 cells per well) after plating for 14 days in the presence of DMSO, stemregenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfβ inhibitor RepSox (RS), and the combination (C2) of LSD1 inhibitor IV and RepSox. The percentage of CD34+CD45RA- cells (contour plot) and the percentage of CD90+ cells (histogram) (+ / - standard deviation) are shown. [Figure 32B] Figure 32B shows that the combination (C2) of LSD1 inhibitor IV (LS) and Tgfβ inhibitor RepSox (RS) can maintain and expand phenotypic human mobilized peripheral blood HSCs after 14 days of ex vivo culture. Figure 32B illustrates the percentage of immunophenotypic HSCs (CD34+CD45RA-CD90+) and stem and progenitor cells (CD34+CD45RA-) after 14 days of culture in the presence of DMSO, stemregenin 1 (SR1), LSD1 inhibitor IV (LS), Tgfβ inhibitor RepSox (RS), and the combination (C2) of LSD1 inhibitor IV and RepSox. [Figure 33]Figures 33A and 33B show that immunophenotypes of cord blood HSCs can be maintained and expanded using compounds targeting seven pathways identified in a mouse system. Two hundred cord blood HSCs (FACS-purified as cell lineages—CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of DMSO or in the presence of seven pathway-targeting compounds (combination: Tgfβ inhibitor A83-01, Lsd1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A, p38 kinase inhibitor SB203580, BMP inhibitor DMH1, Gsk3β inhibitor Chir99021, and sodium acetate). (Figure 33A) shows the immunophenotype of the cells after culture, analyzed by flow cytometry. (FIG. 33B) shows quantification of immunophenotype HSC (cell lineage—CD34+CD45RA-CD38-CD90+). [Figure 34] Figures 34A and 34B show that targeting three pathways is sufficient to maintain and expand immunophenotyped human cord blood HSCs. Two hundred cord blood HSCs (FACS-purified as cell lineage-CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated chemical combinations (Tgfβ inhibitor (A, A83-01), HDAC inhibitor (TSA, trichostatin A), and LSD1 inhibitor (TC, tranylcypromine)) and analyzed by flow cytometry. Figure 34A illustrates the immunophenotype of the cells after culture. Figure 34B illustrates the quantification of immunophenotyped HSCs (cell lineage-CD34+CD45RA-CD38-CD90+) cultured under the indicated conditions. [Figure 35]Figures 35A and 35B show that the addition of a minimal combination of chemicals and a p38 inhibitor improves the yield of human cord blood HSCs. Two hundred cord blood HSCs (FACS-purified as cell lines—CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated chemical combinations (Tgfβ inhibitor (A, A83-01), HDAC inhibitor (TSA, trichostatin A), and LSD1 inhibitor (TC, tranylcypromine)) or with the addition of a p38 inhibitor (p38i, SB203580) and analyzed by flow cytometry. Figure 35A illustrates the immunophenotype of the cells after culture. FIG. 35B illustrates the quantification of immunophenotyped HSCs (cell lineage—CD34+CD45RA−CD38−CD90+CD49F+) cultured under the indicated conditions. [Figure 36] We demonstrate that culturing under hypoxic conditions improves the yield of human umbilical cord blood HSCs. Two hundred umbilical cord blood HSCs (FACS-purified as lineage-CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) and three pathway-targeting compounds (W3: TGFβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)) in a standard tissue culture incubator (atmospheric oxygen, 21% O2) or a hypoxic incubator (5% O2). Immunophenotypes of HSCs (lineage-CD34+CD45RA-CD38-CD90+) cultured under the indicated conditions were quantified after culture. [Figure 37A]Figure 37A shows a comparison of the chemical combination with compounds previously reported to expand cord blood HSCs. Two hundred cord blood HSCs (FACS-purified as cell lines—CD34+CD45RA-CD90+CD38-) were cultured in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of stemregenin 1 (SR1), UM171, or a chemical combination (W3: Tgfβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)) for 12 days and analyzed by flow cytometry. Figure 37A illustrates the immunophenotype of the cells after culture. [Figure 37B] Figure 37B shows a comparison of the chemical combination with compounds previously reported to expand cord blood HSCs. 200 cord blood HSCs (FACS-purified as cell lines—CD34+CD45RA-CD90+CD38-) were cultured in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of stemregenin 1 (SR1), UM171, or a chemical combination (W3: Tgfβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)) for 12 days and analyzed by flow cytometry. Figure 37B illustrates the percentages of the indicated populations. [Figure 37C] Figure 37C shows a comparison of the chemical combinations with compounds previously reported to expand cord blood HSCs. Two hundred cord blood HSCs (FACS-purified as cell lineages—CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of stemregenin 1 (SR1), UM171, or a chemical combination (W3: Tgfβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)) and analyzed by flow cytometry. Figure 37C illustrates the quantification of immunophenotyped HSCs (cell lineages—CD34+CD45RA-CD38-CD90+) after 12 days of culture under the indicated conditions. [Figure 38A]Figure 38A shows the ex vivo maintenance and expansion of human mobilized peripheral blood CD34+ cells using compounds identified using mouse cells. 3,000 CD34+-enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated individual or combinations of chemicals (Tgfβ inhibitor (A, A83-01), HDAC inhibitor (TSA, trichostatin A), LSD1 inhibitor (TC, tranylcypromine), and p38 inhibitor (p38i, SB203580)) and analyzed by flow cytometry. Figure 38A illustrates the immunophenotype of the cells after culture. [Figure 38B] Figure 38B shows the ex vivo maintenance and expansion of human mobilized peripheral blood CD34+ cells using compounds identified using mouse cells. 3,000 CD34+ enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated individual or combinations of chemicals (Tgfβ inhibitor (A, A83-01), HDAC inhibitor (TSA, trichostatin A), LSD1 inhibitor (TC, tranylcypromine), and p38 inhibitor (p38i, SB203580)) and analyzed by flow cytometry. Figure 38B illustrates the percentages of the indicated populations. [Figure 38C]Figure 38C shows the ex vivo maintenance and expansion of human mobilized peripheral blood CD34+ cells using compounds identified using mouse cells. 3000 CD34+ enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated individual or combinations of chemicals (Tgfβ inhibitor (A, A83-01), HDAC inhibitor (TSA, trichostatin A), LSD1 inhibitor (TC, tranylcypromine), and p38 inhibitor (p38i, SB203580)) and analyzed by flow cytometry. Figure 38C illustrates quantification of immunophenotyped HSCs (cell lines—CD34+CD45RA-CD38-CD90+CD49F+) after 7 days of culture in the indicated conditions (W7: A83-01(A), tranylcypromine (TC), trichostatin A (TSA), SB203580 (p38i), CHIR99021 (Chir), DMH1 (DMH), sodium acetate (OAC), and W3: A83-01(A), tranylcypromine (TC), trichostatin A (TSA)). [Figure 39] Ex vivo culture of human mobilized peripheral blood CD34+ cells with a combination of chemicals enriches for immunophenotyped HSCs. 3000 CD34+-enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of DMSO, the indicated individual chemicals (stemregenin (SR1), UM171), or a combination of four compounds (W4: TGFβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), LSD1 inhibitor (tranylcypromine), and p38 inhibitor (SB203580)). Analysis was performed by flow cytometry. The immunophenotyped HSC (cell lineage-CD34+CD45RA-CD38-CD90+) fraction in CD34+-enriched mobilized peripheral blood was quantified before ex vivo culture (no culture) or after 7 days of culture under the indicated conditions. After culture, an 8-fold enrichment of HSCs is demonstrated. [Figure 40A]Figure 40A shows a comparison of the chemical combinations' ability to maintain / expand human mobilized peripheral blood HSCs with compounds previously reported to expand umbilical cord blood HSCs. Fifty mobilized peripheral blood HSCs (FACS-purified as cell lines—CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of stemregenin 1 (SR1), UM171, or a chemical combination (W3: TGFβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)), or with the addition of a p38 inhibitor (p38i, SB203580), and analyzed by flow cytometry. Figure 40A illustrates the immunophenotype of the cells after culture. [Figure 40B] Figure 40B shows a comparison of the chemical combinations' ability to maintain / expand human mobilized peripheral blood HSCs with compounds previously reported to expand umbilical cord blood HSCs. Fifty mobilized peripheral blood HSCs (FACS-purified as cell lineages—CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of stemregenin 1 (SR1), UM171, or a chemical combination (W3: TGFβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)), or with the addition of a p38 inhibitor (p38i, SB203580), and analyzed by flow cytometry. Figure 40B illustrates the percentages of the indicated populations. [Figure 40C]Figure 40C shows a comparison of the chemical combinations' ability to maintain / expand human mobilized peripheral blood HSCs with compounds previously reported to expand umbilical cord blood HSCs. Fifty mobilized peripheral blood HSCs (FACS-purified as cell lineages—CD34+CD45RA-CD90+CD38-) were cultured for 12 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of stemregenin 1 (SR1), UM171, or a chemical combination (W3: Tgfβ inhibitor (A83-01), HDAC inhibitor (trichostatin A), and LSD1 inhibitor (tranylcypromine)), or with the addition of a p38 inhibitor (p38i, SB203580), and analyzed by flow cytometry. FIG. 40C illustrates quantification of immunophenotype HSCs (cell lineage—CD34+CD45RA−CD38−CD90+CD49F+) after 12 days of culture in the indicated conditions. [Figure 41A] Figure 41A shows that the HDAC1 / 2-specific inhibitor romidespin can replace the pan-HDAC inhibitor trichostatin A for efficient ex vivo maintenance / expansion of human HSCs. 3000 CD34+ enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated chemical combinations (Tgfβ inhibitor (A, A83-01), pan-HDAC inhibitor (TSA, trichostatin A), LSD1 inhibitor (TC, tranylcypromine), p38 inhibitor (p38i, SB203580), and HDAC1 / 2 inhibitor (Rom, romidespin)) and analyzed by flow cytometry. Figure 41A illustrates the immunophenotype of the cells. [Figure 41B]Figure 41B shows that the HDAC1 / 2-specific inhibitor romidespin can replace the pan-HDAC inhibitor trichostatin A for efficient ex vivo maintenance / expansion of human HSCs. 3000 CD34+ enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated chemical combinations (Tgfβ inhibitor (A, A83-01), pan-HDAC inhibitor (TSA, trichostatin A), LSD1 inhibitor (TC, tranylcypromine), p38 inhibitor (p38i, SB203580), and HDAC1 / 2 inhibitor (Rom, romidespin)) and analyzed by flow cytometry. Figure 41B illustrates the percentages of the indicated populations. [Figure 41C] Figure 41C shows that the HDAC1 / 2-specific inhibitor romidespin can replace the pan-HDAC inhibitor trichostatin A for efficient ex vivo maintenance / expansion of human HSCs. 3000 CD34+ enriched mobilized peripheral blood cells were cultured for 7 days in serum-free medium supplemented with cytokines (SCF, TPO, FLT3L, IL3) in the presence of the indicated chemical combinations (Tgfβ inhibitor (A, A83-01), pan-HDAC inhibitor (TSA, trichostatin A), LSD1 inhibitor (TC, tranylcypromine), p38 inhibitor (p38i, SB203580), and HDAC1 / 2 inhibitor (Rom, romidespin)) and analyzed by flow cytometry. FIG. 41C illustrates quantification of immunophenotyped HSCs (cell lineage—CD34+CD45RA−CD38−CD90+CD49F+) after 7 days of culture in the indicated conditions. [Figure 42A]Figure 42A shows the results of transplantation of human CD34+ cord blood cells cultured ex vivo for 14 days. Sublethally irradiated, immunocompromised NSG (Nod-Scid-γ) mice were transplanted with a culture of 10,000 starting CD34+ cord blood cells after 14 days of ex vivo culture in the presence of DMSO, W3 (Tgfβ inhibitor A83-01, LSD1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A), a combination of LSD1 inhibitor IV and Tgfβ inhibitor RepSox (C2), stemregenin 1 (SR1), and UM171, or with 10,000 fresh, uncultured CD34+ cord blood cells. (Figure 42A) shows peripheral blood donor chimerism. [Figure 42B] Figure 42B shows the results of transplantation of human CD34+ cord blood cells cultured ex vivo for 14 days. Sublethally irradiated immunocompromised NSG (Nod-Scid-γ) mice were transplanted with a culture of 10,000 starting CD34+ cord blood cells after 14 days of ex vivo culture in the presence of DMSO, W3 (Tgfβ inhibitor A83-01, LSD1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A), a combination of LSD1 inhibitor IV and Tgfβ inhibitor RepSox (C2), stemregenin 1 (SR1), and UM171, or with 10,000 fresh uncultured CD34+ cord blood cells. Figure 42B illustrates quantification of peripheral blood donor chimerism at 24 and 30 weeks post-transplant. [Figure 42C] Figure 42C shows the results of transplantation of human CD34+ cord blood cells cultured ex vivo for 14 days. Sublethally irradiated, immunocompromised NSG (Nod-Scid-γ) mice were transplanted with a culture of 10,000 starting CD34+ cord blood cells after 14 days of ex vivo culture in the presence of DMSO, W3 (Tgfβ inhibitor A83-01, LSD1 inhibitor tranylcypromine, HDAC inhibitor trichostatin A), a combination of LSD1 inhibitor IV and Tgfβ inhibitor RepSox (C2), stemregenin 1 (SR1), and UM171, or with 10,000 fresh, uncultured CD34+ cord blood cells. Figure 42C illustrates the lineage contribution of the transplanted cells at 30 weeks post-transplantation. [Figure 43] 4A-48C illustrate schematic diagrams of the experimental procedures for obtaining the results shown in FIGS. [Figure 44] The results of FACS analysis after 12 days of culture are shown. [Figure 45] 1 depicts a graph of total viable cell output after 12 days of culture from 300 starting cell HSC equivalents in the presence of DMSO, W7, or W3. [Figure 46] Figures 46A and 46B depict graphs of the frequency (Figure 46A) and absolute number (Figure 46B) of cell line-IL7R-ckit+Sca1+ (LSK) cells after 12 days of culture in the presence of DMSO, W7, or W3. [Figure 47] Figures 47A and 47B depict graphs of the frequency (Figure 47A) and absolute numbers (Figure 47B) of cell line-IL7R-ckit+Sca1+CD48-CD150+ HSCs after 12 days of culture in the presence of DMSO, W7, or W3. [Figure 48] Figures 48A-48C illustrate the experimental schematic (Figure 48A), a graph of donor cell engraftment 4 weeks after transplantation of 200 starter cell (HSC) equivalents cultured for 12 days in the presence of DMSO, W7, or W3 (Figure 48B), and a graph of lineage contribution (Figure 48C) of Mac1+ myeloid cells, CD3-positive T cells, and B220-positive B cells. DETAILED DESCRIPTION OF THE INVENTION

[0195] Detailed Description The present invention is based on the surprising discovery that hematopoietic stem cell populations with hematopoietic stem cell functional potential can be expanded, enriched, and maintained ex vivo by contacting hematopoietic stem cells with one or more agents that exhibit one, two, or more effects selected from the group consisting of modulating histone methylation, inhibiting TGFβ signaling, inhibiting p38 signaling, activating canonical Wnt signaling, and modulating histone acetylation. A wide variety of structurally and mechanistically distinct agents that modulate these biological events are known in the art. For example, these agents may be small molecules that can agonize or antagonize specific events in a particular pathway (e.g., small molecules that inhibit the enzymatic activity of proteins that transduce signaling cascades). These agents may also be antibodies, e.g., monoclonal antibodies or antigen-binding fragments thereof, that competitively bind to specific proteins and disrupt specific interactions (e.g., ligand-receptor interactions) by sterically hindering the association of the proteins with their cognate binding partners. Other agents, such as therapeutic proteins and structurally constrained peptides, are topologically well suited to antagonizing protein-protein interactions that occur on large molecular surfaces, and thus represent a class of inhibitors that can intervene at targets in signal transduction pathways that are difficult to disrupt with traditional small molecule therapeutics.Another class of inhibitors includes interfering RNA molecules that can attenuate target gene expression by binding to mRNA polynucleotides through complementary hydrogen bonds, for example, by inducing target mRNA degradation or sterically hindering ribosome assembly nucleation.The following section outlines examples of the types of agents that are useful in conjunction with the compositions and methods of the present invention to promote hematopoietic stem cell expansion, enrichment, and maintenance of hematopoietic stem cell functional capacity.

[0196] UM171 and its structural analogues Additional agents that can be used with the methods of the present invention include UM171, a small molecule that has been shown to induce the expansion of hematopoietic stem cells. UM171 is described, for example, in Fares et al. Science 345:1509 (2014), the disclosure of which is incorporated herein by reference. Other agents that can be used to expand, enrich, and maintain hematopoietic stem cells include UM171 analogs, such as UM171 structural analogs according to any one of formulas (I), (II), (III), (IV), (V), and (VI) of US2015 / 0011543, the disclosure of which is incorporated herein by reference. For example, UM171 analogs that can be used with the compositions and methods described herein include the compounds listed in Table 11 below.

[0197] Table 11. UM171 and its structural analogs TIFF2025116082000016.tif210165TIFF2025116082000017.tif198165TIFF2025116082000018.tif193165TIFF2025116082 000019.tif185165TIFF2025116082000020.tif189165TIFF2025116082000021.tif208165TIFF2025116082000022.tif21216 5TIFF2025116082000023.tif172165TIFF2025116082000024.tif204165TIFF2025116082000025.tif194165TIFF2025116082 000026.tif169165TIFF2025116082000027.tif164165TIFF2025116082000028.tif181165TIFF2025116082000029.tif90165

[0198] low molecule Various small molecules can be used with the methods described herein. These small molecules include modulators of enzyme-substrate interactions. Various small molecules have been developed to antagonize enzyme-substrate interactions or intervene at distinct points in signal transduction cascades. For example, tranylcypromine and its derivatives are a robust class of inhibitors that can irreversibly bind to and inhibit histone demethylases, such as LSD1, by forming covalent adducts with the isoalloxazine moiety of the FAD cofactor used by these enzymes to catalyze the oxidative demethylation of N-methylated histone tail residues. Exemplary small molecule inhibitors of histone demethylation useful with the compositions and methods of the present invention include LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I (also referred to as BHC110 inhibitor I, histone lysine demethylase inhibitor III, and / or KDM1 inhibitor I), and tranylcypromine, described above. Further examples of small molecules useful for inhibiting histone demethylases include phenelzine, propargylamine, and derivatives thereof, as described in US2013 / 0095067, the disclosure of which is incorporated herein by reference. Other tranylcypromine derivatives are described, for example, in US2014 / 0163041, the disclosure of which is incorporated herein by reference.

[0199] Further examples of small molecules that can be used to modulate histone methylation include BIX01294 (e.g., an H3K9 methylation inhibitor described in WO2014 / 057997); UNC0638 (e.g., an H3K9 methylation inhibitor described in WO2013 / 050422), the disclosures of each of which are incorporated herein by reference; and CARM1 inhibitors (PRMT inhibitor V, 3,5-bis(3-bromo-4-hydroxybenzylidene)-1-benzylpiperidin-4-one, a histone arginine methyltransferase inhibitor).

[0200] Several structurally distinct classes of small molecule inhibitors of TGFβ signaling have been reported. These agents can be classified based on the core molecular scaffold of these molecules. For example, TGFβ signaling inhibitors may contain dihydropyrrlipyrazole, imidazole, pyrazolopyridine, pyrazole, imidazopyridine, triazole, pyridopyrimidine, pyrrolopyrazole, isothiazole, or oxazole functional groups as the core structural fragment of the molecule. Some non-limiting examples of small molecule inhibitors of TGFβ signaling include the above-mentioned ALK5 inhibitor II (also known as E-616452), LY364947 (also known as ALK5 inhibitor I, TbR-I inhibitor, transforming growth factor-b type I receptor kinase inhibitor), A83-01, and DMH1.Other examples of small molecules that can be used to modulate TGFβ signaling in conjunction with the compositions and methods of the present invention include SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, Alk5 inhibitor), Galunisertib (LY2157299, Alk5 inhibitor), LY2109761 (4-[2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo [1,2-b]pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine, Alk5 / TGFβRII inhibitor), SB525334 (6-[2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl]quinoxaline, Alk5 inhibitor), GW788388 (N-(oxan-4-yl)-4-[4-(5-pyridin-2-yl-1H-pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine, Alk5 / TGFβRII inhibitor), [6-(4-(2 ... [1,2,4]triazole[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline, an Alk4 / Alk5 inhibitor), and LDN-212854 (5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline, an Alk4 / Alk5 inhibitor).

[0201] Further examples of small molecule TGFβ modulators include antagonists of the TGFβ receptor, such as 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 naphththyridine, [3-(pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole. Other small molecule inhibitors include SB-431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), described in Halder et al. Neoplasia 7:509 (2005), SM16, a small molecule inhibitor of the TGFβ receptor ALK5, the structure of which is shown below (Fu et al. Arteriosclerosis, Thrombosis and Vascular Biology 28:665 (2008)), and SB-505124 (Dacosta, Byfield et al. Molecular Pharmacology 65:744 (2004), the structure of which is shown below, and 6-bromo-indirubin-3'-oxime (described in U.S. Pat. No. 8,298,825), the disclosures of each of which are incorporated herein by reference. TIFF2025116082000030.tif94128

[0202] Further examples of TGF-β signaling inhibitors are described, for example, in Callahan et al. Journal of Medicinal Chemistry 45:999 (2002); Sawyer et al. Journal of Medicinal Chemistry 46:3953 (2003); Gellibert et al. Journal of Medicinal Chemistry 47:4494 (2004); Tojo et al. Cancer Science 96:791 (2005); Petersen et al. Kidney International 73:705 (2008); Yingling et al. Nature Reviews Drug Discovery 3:1011 (2004); Byfield et al. Molecular Pharmacology 65:744 (2004); Dumont et al. Cancer Cell 3:531 (2003); WO2002 / 094833;WO2004 / 026865;WO2004 / 067530;WO2009 / 032667;WO2004 / 013135;WO2003 / 097639;WO2007 / 048857;WO2007 / 018818;W US6,476,031 and US2009 / 0036382, the disclosures of each of which are incorporated herein by reference.

[0203] Another class of small molecules useful with the compositions and methods of the invention includes modulators of bone morphogenetic protein (BMP) signaling. BMPs are members of the TGFβ superfamily of ligands, and BMP signaling modulators, such as inhibitors of Alk2, Alk3, and Alk6, can be used with the methods of the invention, for example, to expand hematopoietic stem cells, enrich hematopoietic stem cells, and / or maintain hematopoietic stem cells in a pluripotent state. Exemplary BMP inhibitors include DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline, 4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), K02288 (3-(6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol), LDN-212854 (5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[ 1,5-a]pyrimidin-3-yl]-quinoline), LDN-193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), LDN-214117 (1-(4-(6-methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine), and ML347 (5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline).

[0204] To promote hematopoietic stem cell expansion, hematopoietic stem cell enrichment, and maintenance of hematopoietic stem cell functional capacity, receptor tyrosine kinase inhibitors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) signaling inhibitors, can also be used with the compositions and methods of the present invention. For example, an exemplary VEGF / PDGF inhibitor useful with the methods described herein is ABT-869 (Linifanib, 1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea).

[0205] Other small molecules useful with the compositions and methods of the invention include DNA methylation inhibitors, including chemical modulators of DNMT1, DNMT3a, and DNMT3B. An exemplary inhibitor of these targets that can be used with the compositions and methods of the invention to expand, enrich, and maintain the functional potential of hematopoietic stem cells is RG108 (N-phthalyl-L-tryptophan).

[0206] To date, various small molecule inhibitors of p38 MAPK have also been reported, including the pyridinylimidazole compounds SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) and SB202190 (4(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole). These compounds are a class of inhibitors that selectively antagonize p38 MAPK α- and β-isoforms without disrupting the enzymatic activity of the γ- or δ-isoforms. These compounds are described in U.S. Pat. No. 6,602,896, the disclosure of which is incorporated herein by reference. Other examples of p38 MAPK inhibitors include SB203580, BIRB796 (Doramapimod), VX702, SB202190, LY2228820, VX745, vinorelbine (navelbine), PH797804, pamapimod, CMPD-1, EO1428, JX401, ML3403, RWJ67657, SB239063, SCIO469 hydrochloride, SKF86002 dihydrochloride, SX011, and TAK715, as described, for example, in US2014 / 0127231, the disclosure of which is incorporated herein by reference. Further examples of p38 inhibitors useful with the compositions and methods of the present invention include pexmetinib (ARRY-614), PH-797804 (3-(3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N,4-dimethylbenzamide), losmapimod (GW856553X), and skepinone-L.

[0207] Small molecule agents that can inhibit proteins that promote β-catenin degradation include agents that can attenuate the activity of proteins that promote β-catenin phosphorylation. Such inhibitors serve to increase the nuclear concentration of this transcription factor, and various examples are known in the art. β-catenin phosphorylation inhibitors include compounds that inhibit glycogen synthase kinase 3 (GSK3), such as the aforementioned CHIR99021, as well as 6-bromo-indirubin-3′-oxime (Meijer et al. Chemistry and Biology 10:1255 (2003); Goessling et al. Cell 136:1136 (2009)), AR-A014418 (Bhat et al. Journal of Biological Chemistry 278:45937 (2003)), and the organometallic GSK-3 inhibitor DW21 (Williams et al. Angewandte Chemie International Edition 44:1984 (2005), the disclosures of which are incorporated herein by reference. Other small molecule modulators of Wnt signaling useful with the compositions and methods of the present invention for expanding, enriching, and maintaining the functional potential of hematopoietic stem cells include inhibitors of GSK3a and GSK3b, such as CHIR99021 and lithium chloride.

[0208] Histone deacetylase can also be targeted using small molecule therapeutic agents. Hydroxamic acids are a particularly robust class of histone deacetylase inhibitors, inhibiting histone deacetylases through a hydroxamate functional group that binds to cationic zinc within the active site of histone deacetylase. Exemplary inhibitors include the aforementioned trichostatin A, as well as vorinostat (N-hydroxy-N'-phenyl-octanediamine, described in Marks et al., Nature Biotechnology 25, 84-90 (2007); Stenger, Community Oncology 4, 384-386 (2007), the disclosure of which is incorporated herein by reference). Other histone deacetylase inhibitors include panobinostat, described in Drugs of the Future 32(4): 315-322 (2007), the disclosure of which is incorporated herein by reference. TIFF2025116082000031.tif29128

[0209] Further examples of hydroxamic acid inhibitors of histone deacetylase include the compounds shown below, as described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference. TIFF2025116082000032.tif67128TIFF2025116082000033.tif19276TIFF2025116082000034.tif71128

[0210] Other histone deacetylase inhibitors that do not contain hydroxamate substituents have also been developed, including valproic acid (Gottlicher et al. EMBO Journal 20: 6969 (2001) and mocetinostat (N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide) described in Balasubramanian et al. Cancer Letters 280: 211 (2009), the disclosures of each of which are incorporated herein by reference. Other small molecule inhibitors that utilize chemical functionality different from hydroxamate include those described by Bertrand, European Journal of Medicinal Chemistry 45:2095-2116. (2010), the disclosure of which is incorporated herein by reference. Other small molecule inhibitors that utilize chemical functionality different from hydroxamates to inhibit histone deacetylases include the small molecule inhibitors shown below. TIFF2025116082000035.tif168128

[0211] Further examples of chemical modulators of histone acetylation useful with the compositions and methods of the present invention for expanding, enriching, and maintaining the hematopoietic stem cell functional potential of hematopoietic stem cells include modulators of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, Sirt1, Sirt2, and / or HATs, e.g., butyrylhydroxamic acid, M344, LAQ824 (Dacinostat), ), AR-42, Belinostat (PXD101), CUDC-101, Scriptaid, Sodium Phenylbutyrate, Tasquinimod, Quisinostat (JNJ-26481585), Pracinostat (SB939), CUDC-907, Entinostat (MS-275), Mocetinostat (MGCD0103), Tubastatin A HCl, PCI-34051, Droxinostat, PCI-24781 (Abexinostat), RGFP966, Rocilinostat (ACY-1215), CI994 (Tacedinaline), Tubacin, RG2833 (RGFP109), Resminostat, Tubastatin A, BRD73954, BG45, 4SC-202, CAY10603, LMK-235, Nexturastat A, TMP269, HPOB, Cambinol, and Anacardic Acid.

[0212] Antibodies and other therapeutic proteins Antibodies are a chemical domain uniquely suited to targeting extracellular protein-protein interactions, such as receptor-ligand interactions. These agents possess a large molecular volume beneficial for inhibiting interactions, characterized by residues that favorably contribute to the free energy of interaction distributed over a broad surface, rather than being confined to a superficial cleft. Antibody agents possess a large molecular volume beneficial for inhibiting interactions that occur over a broad surface, rather than within a superficial cleft. Inhibitory antibodies may function by binding to extracellular receptors in a way that sterically prevents their interaction with their cognate ligand, thus maintaining the receptor in an inactive conformation. For example, inhibitory antibodies that can attenuate TGFβ receptor activity include lerdelimumab and antibodies that bind to TGFβ receptor type II. Other examples include GC-1008, an antibody that binds to and antagonizes all isoforms of human TGFβ, and ID11, an antibody that binds to all isoforms of mouse TGFβ. These antibodies are described in detail, for example, in US 8,603,818, the disclosure of which is incorporated herein by reference.

[0213] Antagonist antibodies have also been developed that inhibit β-catenin phosphorylation by transmitting Wnt signaling cascade.Such antibodies can bind to Wnt receptors, including Frizzled, and LRP family proteins, and induce simultaneous conformational changes that stimulate the propagation of the Wnt signaling pathway, including separate molecular events that inhibit β-catenin phosphorylation by GKS3.For example, antibody 1D9 has been developed as an agonist of Wnt signaling, and is described in detail in US2014 / 0044717.This disclosure is incorporated herein by reference.

[0214] Other classes of therapeutic proteins can additionally be used to expand hematopoietic stem cells, enrich hematopoietic stem cells, and inhibit biological processes to maintain the functional potential of hematopoietic stem cells. Endogenous proteins that modulate signaling events may be used ex vivo to attenuate these events and thereby enhance the natural affinity of these proteins for their cognate ligands, to antagonize or stimulate key protein-protein interactions. For example, for this purpose, various proteins that antagonize the TGFβ signaling cascade can be used, including decorin, an extracellular matrix proteoglycan that negatively regulates TGFβ activity, as well as Lefty 1, Lefty 2, follistatin, Noggin, Chordin, Cerberus, Germlin, inhibin, cystatin C, recombinant mouse Lefty 1 (ACVR2B inhibitor), and the Smad proteins Smad6 and Smad7, which help block phosphorylation of R-Smad proteins or help recruit ubiquitin ligase to TGFβ receptor type I to promote degradation of TGFβ receptor type I. These proteins are described in detail in US Pat. No. 8,298,825, the disclosure of which is incorporated herein by reference.

[0215] Another TGFβ signaling modulator that can be used with the compositions and methods of the present invention to expand hematopoietic stem cells, enrich hematopoietic stem cells, and maintain the hematopoietic stem cell functional potential of hematopoietic stem cells is recombinant amphibian TGF-β5 (ACVR2A, ACVR2B, TGFβRII activator).

[0216] In addition to the negative feedback proteins mentioned above, proteins that can induce Wnt signal transduction to inhibit β-catenin phosphorylation have also been described.For example, Rspondin (ependymal plate specific spondin) protein is also known to activate β-catenin signal transduction.Rspondin protein does not have sequence similarity with Wnt protein, and appears to enhance Wnt signal transduction through a Frizzled-independent mechanism.This protein is described in detail in Kazanskaya. O., et al., Dev. Cell 7, 525-534 (2004).This disclosure is incorporated herein by reference.

[0217] Interfering RNA RNA interference (RNAi) is an inhibitory therapy that exploits the ability of antagonist RNA (e.g., double-stranded RNA containing oligonucleotides capable of complementary base-pairing with endogenous mRNA sequences via hydrogen bonding) to attenuate intracellular gene expression. Mechanistically, this phenomenon often works by degrading complementary mRNA or by sterically inhibiting ribosome formation on mRNA transcripts. Long sequences of dsRNA are often cleaved in the cytoplasm of eukaryotic cells by the well-known ribonuclease Dicer to produce short, 21- to 25-nucleotide small interfering RNAs known as siRNAs. These siRNAs then assemble with protein components into the RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to the complementary transcript through base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, resulting in sequence-specific degradation of the mRNA, resulting in gene silencing. The molecular events underlying RISC-mediated gene silencing are described in, for example, US6,506,559; Fire et al., Nature 391(19):306-311 (1998); Timmons et al., Nature 395:854 (1998); Montgomery et al., TIG 14 (7):255-258 (1998); David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, Pa. (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003).These disclosures are incorporated herein by reference.

[0218] Importantly, siRNA molecules useful with the compositions and methods of the present invention need not be limited to siRNA molecules containing only RNA, but may also include, for example, chemically modified nucleotides and non-nucleotides that result in RNA interference, as well as molecules in which the ribose sugar is replaced with another sugar molecule or its analog. Furthermore, non-natural linkages between nucleotide residues, such as phosphorothioate linkages, which are not susceptible to degradation via phosphodiesterase, can be used. RNA oligonucleotides useful with the compositions and methods of the present invention may also be derivatized with reactive functional groups or reporter groups, such as fluorophores. Particularly useful derivatives are modified at one or more ends of the RNA strand, typically the 3' end of the sense strand. For example, the 2'-hydroxyl at the 3' end can be easily and selectively derivatized with various groups by standard nucleophilic substitution methods known in the art. Other useful RNA derivatives incorporate nucleotides with modified carbohydrate moieties, such as 2'-O-alkylated residues or 2'-O-methylribosyl and 2'-O-fluororibosyl derivatives, which confer increased structural stability to the oligonucleotide. The nucleobases of siRNA may also be chemically modified. For example, halogenated bases such as 5-bromouracil and 5-indouracil can be incorporated into siRNA molecules to adjust the strength of hydrogen bond interaction with target mRNA. Nucleic acid bases can also be strategically alkylated. For example, 7-methylguanosine can be incorporated instead of guanosine residues. Unnatural bases that promote the inhibition of target gene expression can also be incorporated into interfering RNA. Other siRNA modifications include 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides, and RNA duplexes containing phosphodiester or various numbers of phosphorothioate linkages. Such modifications are described, for example, in Braasch et al., Biochemistry 42: 7967-7975 (2003). This disclosure is incorporated herein by reference.

[0219] Synthetic siRNA molecule can be obtained by using many techniques known to those skilled in the art.For example, siRNA molecule can be chemically synthesized by using methods known in the art, for example, by using appropriately protected ribonucleoside phosphoramidite and conventional solid-phase oligonucleotide synthesis, or can be produced by recombinant (see, for example, Elbashir et al. Nature 411:494 (2001); Elbashir et al. Genes & Development 15:188 (2001); Harborth. et al. Journal of Cell Science 114:4557 (2001); Masters et al. Proceedings of the National Academy of Sciences USA 98:8012 (2001); and Tuschl et al. Genes & Development 13:3191 (1999).These disclosures are incorporated herein by reference).Additionally, dsRNA can be expressed as stem-loop structures encoded by plasmid vectors, retroviruses, and lentiviruses (e.g., Paddison et al. Genes and Development 16:948 (2002); McManus et al. RNA 8:842 (2002); Paul et al. Nature Biotechnology 20:505 (2002); Miyagishi et al. Nature Biotechnology 20:497 (2002); Sui et al. Proceedings of the National Academy of Sciences USA 99:5515 (2002); Brummelkamp et al. Cancer Cell 2:243 (2002); Lee et al. Nature Biotechnology 20:500 (2002); Yu et al. Proceedings of the National Academy of Sciences USA 99:6047 (2002); Zeng et al. Molecular Cell 9:1327 (2002); Rubinson et al. Nature See Genetics 33:401 (2003); Stewart et al. RNA 9:493 (2003), the disclosures of each of which are incorporated herein by reference.

[0220] In order to antagonize the biological process described by the method of the present invention, various inhibitors that work by RNA interference mechanism have been developed.For example, a TGFβ receptor type II siRNA polynucleotide (Genbank accession number: M85079) derived from human TGFβRII sequence has been reported.The following siRNA duplex sequences have been developed against specific target sequences in the TGFβ receptor type II gene and have been used to knock down the expression of this receptor in various whole cell models.These siRNA sequences are described in detail in US8,067,389, the disclosure of which is incorporated herein by reference.Other oligonucleotide-based TGFβ signaling modulators, such as siRNA and antisense oligonucleotides, are described in US5,731,424; US6,124,449; US2008 / 0015161; US2006 / 0229266; US2004 / 0006030; US2005 / 0227936; and US2005 / 0287128. The disclosures of each of these are incorporated herein by reference. siRNAs useful for targeting TGFβR or ALK5 expression can be readily designed and tested. A database of siRNA sequences and predictive variables for siRNA sequences has been established (Chalk et al. (Nucleic Acids Research 33: D131 (2005)). This database can be used to predict thermodynamic parameters of specific siRNA-target mRNA interactions and to characterize designed siRNA sequences for off-target interactions. This database is available as an electronic resource at www.siRNA.cgb.ki.se. TIFF2025116082000036.tif140128

[0221] Conformationally constrained peptides Peptide-based therapeutic agents are an emerging class of compounds useful for inhibiting protein-protein interactions that are often difficult to inhibit by other means. Conformationally constrained peptides offer particular advantages for therapeutic applications because these compounds often exhibit high target affinity and selectivity by presenting structurally preorganized epitopes that favor spatial interactions of specific pharmacophores with proteins of interest. Constrained peptides often feature the added benefit of increased protease resistance compared to their unconstrained (e.g., linear) counterparts by restricting protease access to internal amide bonds. The cell penetration capabilities of these compounds are also often greater than those of linear peptides because hydrogen bond donors and acceptors are sequestered from the aqueous solvent. Exemplary constrained peptide inhibitors useful with the compositions and methods of the present invention include olefin "stapled" peptides, which often feature α-helices that are structurally rigidified by the insertion of covalent bridges between residues on the same face of the α-helix. This class of constrained peptides is described, for example, in Walensky et al. Journal of Medicinal Chemistry 57:6275 (2014), the disclosure of which is incorporated herein by reference. Stapled peptide inhibitors of β-catenin phosphorylation have been developed that function by disrupting the Axin / β-catenin interaction. Axin helps anchor β-catenin to a protein complex containing GSK3, and this association has been shown to be mediated by the insertion of the α-helical region of Axin into a superficial pocket on the surface of β-catenin. The sequence α-helical structure (S) is structurally restricted to an α-helical conformation by an olefin bridge at residues R8 ((S)-α-(7-octenyl)alanine) and S5 ((S)-α-(4-pentenyl)alanine). A stapled peptide of TIFF2025116082000037.tif4128 has been reported (see, e.g., Cui et al. Cell Research 23: 581 (2013), the disclosure of which is incorporated herein by reference). This peptide competes with axin for binding to the surface of β-catenin, liberating this protein from GSK3-containing complexes and thus serving to increase the nuclear concentration of this transcription factor.

[0222] Constrained peptides have also been developed by covalently cyclizing the N- and C-termini. Exemplary inhibitors of this class include depsipeptides, which feature a lactone moiety that makes these peptides macrocyclic. Depsipeptide inhibitors useful with the compositions and methods of the present invention include histone deacetylase inhibitors, such as romidespin (also known as istodax; structure shown below). Romidespin is described, for example, in Vinodhkumar et al., Biomedicine & Pharmacotherapy 62:85-93 (2008), the disclosure of which is incorporated herein by reference. TIFF2025116082000038.tif59128

[0223] Further examples of depsipeptide inhibitors of histone deacetylase include apicidin, described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference. TIFF2025116082000039.tif54128

[0224] Combinations of Compounds Useful with the Compositions and Methods of the Invention Embodiments of the compositions and methods of the present invention may contain any combination of compounds described above that can be used, for example, to expand a hematopoietic stem cell population, enrich a hematopoietic stem cell population, and maintain the hematopoietic stem cell functional potential of a hematopoietic stem cell population. Specific combinations of compounds useful with the compositions and methods of the present invention are specified in Tables 1-10 below.

[0225] (Table 1) TIFF2025116082000040.tif227131TIFF2025116082000041.tif227108TIFF2025116082000042.tif22782

[0226] (Table 2) TIFF2025116082000043.tif184131TIFF2025116082000044.tif184154TIFF2025116082000045.tif184128

[0227] (Table 3) TIFF2025116082000046.tif227141TIFF2025116082000047.tif22756

[0228] (Table 4) TIFF2025116082000048.tif22753

[0229] (Table 5) TIFF2025116082000049.tif183128

[0230] (Table 6) TIFF2025116082000050.tif22711

[0231] (Table 7) TIFF2025116082000051.tif153140TIFF2025116082000052.tif153128TIFF2025116082000053.tif113128

[0232] (Table 8) TIFF2025116082000054.tif157128TIFF2025116082000055.tif157154TIFF20251160820 00056.tif157154TIFF2025116082000057.tif157154TIFF2025116082000058.tif157128

[0233] (Table 9) TIFF2025116082000059.tif228117TIFF2025116082000060.tif228117TIFF2025116082000061.tif22812 1TIFF2025116082000062.tif228117TIFF2025116082000063.tif228117TIFF2025116082000064.tif22871

[0234] (Table 10) TIFF2025116082000065.tif227140TIFF2025116082000066.tif22789

[0235] Additional agents that can be used to induce the expansion, enrichment, and maintenance of hematopoietic stem cells during ex vivo culture Other compounds can also be used in conjunction with the compositions and methods of the present invention to expand, enrich, and / or maintain hematopoietic stem cells during ex vivo culture.Examples of these compounds include aryl hydrocarbon receptor (AHR) antagonists, such as stemregenin 1 (SR1), a small molecule that promotes the expansion and self-renewal of human CD34+ peripheral blood and umbilical cord blood hematopoietic stem cells.SR1 is described, for example, in US2014 / 0369973; Boitano et al. Science 1345 (2010); and Smith et al. Journal of Pharmacology and Experimental Therapeutics 338:318 (2011).The disclosures of each of these are incorporated herein by reference. Other AHR inhibitors that can be used with the compositions and methods of the present invention include SR1 analogs, such as SR1 analogs containing various aryl and aliphatic substituents around the 6-aminopurine core (e.g., those described in US2014 / 0369973, the disclosure of which is incorporated herein by reference). Further examples of AHR antagonists include stilbene derivatives (E)-1-(4'-trifluoromethylphenyl)-2-(3,5-ditrifluoromethylphenyl)-ethene, (E)-1-(4'-methoxyphenyl)-2-(3,5-dichlorophenyl)-ethene, and (E)-1-(4'-chlorophenyl)-2-(3,5-dichlorophenyl)-ethene, as described in WO2004 / 041758, the disclosure of which is incorporated herein by reference.Additional stilbene derivatives useful for AHR inhibition include 3,5,4'-trihydroxystilbene (e.g., resveratrol, particularly trans-resveratrol); 3,4,3',5-tetrahydroxystilbene (also known as piceatannol); 2,3',4',5'-tetrahydrostilbene (also known as oxyresveratrol); and 4,4'-dihydroxystilbene and glycosides (e.g., galactosides, lactosides, mannosides, piceosides, and fructosides thereof), as described, for example, in WO 1999 / 056737, the disclosure of which is incorporated herein by reference. Another exemplary AHR antagonist is 2-methyl-2H-pyrazole-3-carboxylic acid-(2-methyl-4-o-tolylazophenyl)-amide (also referred to as CH-223191), which is described in detail, for example, in Kim et al. Molecular Pharmacology 69:1871 (2006) and WO2009 / 115807, the disclosures of each of which are incorporated herein by reference.

[0236] Additional agents that can be used with the methods of the present invention include UM171, another small molecule shown to induce hematopoietic stem cell expansion. UM171 is described, for example, in Fares, et al., Science, 345(6203):1509-1512 (2014), the disclosure of which is incorporated herein by reference. Other agents that can be used with the compositions and methods of the present invention include UM171 analogs, such as those described in WO2013 / 110198, the disclosure of which is incorporated herein by reference. Particularly useful UM171 analogs that can be used with the compositions and methods of the present invention include compound numbers 1-55 listed in WO2013 / 110198 and the compounds disclosed in Table 11 herein. Further examples of classes of compounds that can be further used with the compositions and methods of the present invention include prostaglandins. Particularly useful prostaglandins include, for example, the prostaglandin dmPGE2 described in US8,551,782 and US8,168,428.The disclosures of each of these are incorporated herein by reference.Additional compounds that can be used with the compositions and methods described herein include Sirtuin 1 (SIRT1) protein inhibitors, such as nicotinamide (e.g., as described in Peled et al. Experimental Hematology 40:342 (2012)) and cambinol (e.g., as described in Lugrin et al. Biochimica Biophysica Acta 1833:1498 (2013)).The disclosures of each of these are incorporated herein by reference. TIFF2025116082000067.tif106128

[0237] Additional agents that can be contacted with hematopoietic stem cells in combination with one or more agents that exhibit two or more actions selected from the group consisting of regulating histone methylation, inhibiting TGFβ signaling, inhibiting p38 signaling, activating canonical Wnt signaling, and regulating histone acetylation include activators of the Notch signaling pathway.Notch signaling agonists include, but are not limited to, proteins that contain a portion of a toporythmic protein that mediates binding to Notch and / or mediates Notch activity, such as Delta, Serrate, or Jagged (see, for example, Lindsell et al., Cell 80: 909-917 (1995) (the disclosure of which is incorporated herein by reference)), as well as nucleic acids encoding the aforementioned proteins, and proteins, nucleic acids, small molecules, or derivatives thereof that regulate the activity or gene expression of these proteins. Notch signaling agonists also include proteins or derivatives or fragments thereof, including functionally active fragments, for example, Notch ligand fragments that mediate binding to Notch protein.

[0238] Notch activity is promoted by the binding of Notch ligands (e.g., Delta and Serrate ligands) to the extracellular portion of the Notch receptor. Endogenous Notch ligands are typically membrane-bound to adjacent cells. Therefore, Notch ligands for use with the compositions and methods of the present invention can be incubated with hematopoietic stem cells in a dissolved state as a soluble protein factor or immobilized on a solid surface (e.g., tissue culture plate, bead, or nanomatrix). For example, a full-length Notch ligand expressed on the surface of a cell induces activation of the Notch signaling cascade in adjacent cells when the ligand contacts the Notch receptor. Therefore, Notch signaling agonists for use with the compositions and methods of the present invention include soluble, optionally truncated, Delta or Serrate (e.g., Jagged) molecules containing the extracellular domain or Notch-binding portion, as well as these proteins immobilized on the solid surface of a surface, e.g., a tissue culture plate, water-miscible beads, or nanomatrix. Such soluble proteins can be immobilized to a solid surface by antibodies or interacting proteins, such as antibodies directed against an epitope tag (e.g., a myc epitope tag recognized by antibody 9E10) with which Delta or Serrate are expressed as a fusion protein, or proteins that interact with an epitope tag (e.g., an immunoglobulin epitope tag to which Protein A binds) with which Delta or Serrate are expressed as a fusion protein, as described in US2014 / 0369973, the disclosure of which is incorporated herein by reference. Exemplary Notch signaling agonists include notch polypeptides, deltex polypeptides, mastermind polypeptides, split polypeptides, hairless polypeptides, RBP-Jκ polypeptides, or hesl polypeptides, as described in US2011 / 0091448, the disclosure of which is incorporated herein by reference.

[0239] The additional compound contacted with the hematopoietic stem cells contacted with one or more agents that together exhibit two or more effects selected from the group consisting of regulating histone methylation, inhibiting TGFβ signaling, inhibiting p38 signaling, activating canonical Wnt signaling, and regulating histone acetylation may include agents such as the aforementioned agents (e.g., AHR antagonists, e.g., SR1, optionally in combination with UM171, dmPGE2, Notch signaling agonists, and / or SIRT1 inhibitors, e.g., nicotinamide or cambinol). For example, the hematopoietic stem cells contacted with one or more of these agents may be further contacted with these compounds according to separate incubation regimens, such that one or more of the AHR antagonists, UM171, dmPGE2, Notch signaling agonists, and / or SIRT1 inhibitors, e.g., nicotinamide or cambinol, are introduced into the hematopoietic stem cells at different times during the culture period. Alternatively, these agents may be incubated simultaneously with the hematopoietic stem cells when desired.

[0240] Hematopoietic stem cell mobilization Hematopoietic stem cells for use with the compositions and methods of the present invention may be derived from various cell types.For example, hematopoietic cells for the methods of expanding, enriching, and maintaining the functional potential of hematopoietic stem cells listed herein may be obtained from mononuclear cells before being treated with one or more agents that together exhibit two or more effects selected from the group consisting of regulating histone methylation, inhibiting TGFβ signaling, inhibiting p38 signaling, activating canonical Wnt signaling, and regulating histone acetylation.Optionally, human hematopoietic stem cells may be CD34+ cells before being treated with one or more of these agents. For example, human hematopoietic stem cells may be within a population having a cell surface phenotype comprising CD34+ cells, CD34+CD38- cells, CD34+CD38-CD90+ cells, CD34+CD38-CD90+CD45RA- cells, or CD34+CD38-CD90+CD45RA-CD49F+ cells prior to treatment with one or more of these agents.

[0241] Hematopoietic stem cells may also be derived from human bone marrow. Alternatively, hematopoietic stem cells may be derived from human umbilical cord blood or from mobilized peripheral blood. Hematopoietic stem cells obtained from human peripheral blood can be mobilized by one of a variety of strategies. Exemplary agents that can be used to induce mobilization of hematopoietic stem cells from bone marrow to peripheral blood include chemokine (C-X-C motif) receptor 4 (CXCR4) antagonists, such as AMD3100 (also known as Plerixafor and MOZOBIL™ (Genzyme, Boston, MA)), and granulocyte colony-stimulating factor (GCSF), the combination of which has been shown to inhibit CD34 in clinical trials. +It has been shown to rapidly mobilize hematopoietic stem cells. Furthermore, chemokine (C-X-C motif) ligand 2 (CXCL2, also known as GROβ) is another agent capable of inducing hematopoietic stem cell mobilization from bone marrow to peripheral blood. Agents capable of inducing hematopoietic stem cell mobilization for use with the compositions and methods of the present invention may be used in combination with each other. For example, a CXCR4 antagonist (e.g., AMD3100), CXCL2, and / or GCSF may be administered sequentially to a subject or simultaneously in a single mixture to induce hematopoietic stem cell mobilization from bone marrow to peripheral blood. The use of these agents as inducers of hematopoietic stem cell mobilization is described, for example, in Pelus, Current Opinion in Hematology 15:285 (2008), the disclosure of which is incorporated herein by reference. TIFF2025116082000068.tif38128

[0242] Regulation of target gene expression in hematopoietic stem cells The compositions and methods of the present invention further provide strategies for regulating the expression of target genes in hematopoietic stem cell populations. For example, hematopoietic stem cell populations can be expanded, enriched, or maintained ex vivo according to the methods of the present invention, and further genetically modified to exhibit altered gene expression patterns. Alternatively, cell populations can be enriched for hematopoietic stem cells, and hematopoietic stem cell populations can be maintained in a pluripotent state, and the cells can be further modified using established genome editing methods known in the art. For example, genome editing procedures can be used to promote the expression of exogenous genes or inhibit the expression of endogenous genes in hematopoietic stem cells. Importantly, hematopoietic stem cell populations can be expanded, enriched, or maintained in a pluripotent state according to the methods of the present invention listed herein, and then genetically modified to express desired target genes. Alternatively, these cell populations can first be genetically modified, and then expanded, enriched, or maintained in a pluripotent state. A number of different methods have been established to integrate target genes into the genome of cells (eg, mammalian cells, such as mouse or human cells) to facilitate expression of such genes.

[0243] Polynucleotide encoding the target gene One example of a platform that can be used to facilitate the expression of a target gene in hematopoietic stem cells is by integrating a polynucleotide encoding the target gene into the nuclear genome of the cell. Various techniques have been developed to introduce exogenous genes into eukaryotic genomes. One such technique involves inserting the target gene into a vector, such as a viral vector. Vectors for use with the compositions and methods of the present invention can be introduced into cells by various methods, including transformation, transfection, direct uptake, projectile bombardment, and encapsulating the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are further described in, for example, Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor University Press, New York (2014); and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (2015). The disclosures of each of these are incorporated herein by reference.

[0244] Exogenous genes can also be introduced into mammalian cells by using a vector containing the gene of interest that is directed to cell membrane phospholipids.For example, vectors can be targeted to the phospholipids on the extracellular surface of cell membranes by linking vector molecules to VSV-G protein, a viral protein that has affinity for all cell membrane phospholipids.The viral vectors that contain VSV-G protein are further described in, for example, US 5,512,421; and US 5,670,354.The disclosures of each of these are incorporated herein by reference.

[0245] The recognition and binding of mammalian RNA polymerase to a polynucleotide encoding a target gene is a key molecular event for gene expression. Therefore, a polynucleotide may contain sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription initiation site. Such sequence elements include, for example, mammalian promoters, which are sequences that specific transcription initiation factors can recognize and bind, and ultimately, RNA polymerase can recognize and bind. Alternatively, promoters derived from viral genomes can be used to stably express target genes in mammalian cells. Examples of functional viral promoters that can be used to promote mammalian expression of these enzymes include the adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, and cytomegalovirus (CMV) promoter. Additional viral promoters include the SV40 late promoter from simian virus 40, the baculovirus polyhedron enhancer / promoter element, the herpes simplex virus thymidine kinase (HSV tk) promoter, and the 35S promoter from cauliflower mosaic virus. Suitable phage promoters for use with the compositions and methods of the present invention include, but are not limited to, the E. coli T7 and T3 phage promoters, the S. typhimurium phage SP6 promoter, the B. subtilis SP01 phage and B. subtilis phage phi29 promoters, and the N4 and K11 phage promoters, as described in U.S. Pat. No. 5,547,892, the disclosure of which is incorporated herein by reference.

[0246] Once a polynucleotide encoding a target gene has been integrated into the genome of a cell (e.g., the nuclear genome of a hematopoietic stem cell), transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to an external chemical reagent, such as an agent that modulates the binding of transcription factors and / or RNA polymerase to the mammalian promoter, thus regulating gene expression. The chemical reagent may serve to facilitate the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing repressor proteins bound to the mammalian promoter. Alternatively, the chemical reagent may serve to enhance the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, such that the transcription rate of a gene placed downstream of the mammalian promoter increases in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above-described mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

[0247] Other DNA sequence elements that can be included in polynucleotides for use with the compositions and methods of the present invention include enhancer sequences. Enhancers are another class of regulatory elements that induce conformational changes in polynucleotides, including genes of interest, so that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use with the compositions and methods of the present invention include polynucleotides encoding target genes and further include mammalian enhancer sequences. Many enhancer sequences derived from mammalian genes are currently known; examples include enhancers derived from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use with the compositions and methods of the present invention also include enhancers derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Further enhancer sequences that induce the activation of eukaryotic gene transcription are disclosed in Yaniv, et al., Nature, 297:17-18 (1982). This disclosure is incorporated herein by reference. The enhancer may be spliced into a vector containing a polynucleotide encoding a target gene, for example, at a position 5' or 3' from the gene. In a preferred direction, the enhancer is placed 5' from the promoter, and the promoter is then placed 5' from the polynucleotide encoding the target gene.

[0248] In addition to promoting rapid transcription and translation rates, stable expression of exogenous genes in hematopoietic stem cells can be achieved by integrating a polynucleotide containing the gene into the nuclear DNA of the cell. Various vectors have been developed for delivering and integrating polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are disclosed, for example, in WO1994 / 11026, the disclosure of which is incorporated herein by reference. Expression vectors for use with the compositions and methods of the present invention contain polynucleotide sequences encoding target genes and additional sequence elements used, for example, for the expression of these enzymes and / or the integration of these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express target genes include plasmids containing regulatory sequences that induce gene transcription, such as promoter and enhancer regions. Other useful vectors for expressing target genes contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements often encode features within the RNA transcript that enhance nuclear export, cytosolic half-life, and ribosome affinity of these molecules, such as 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites, to guide efficient transcription of the genes carried on the expression vector. Exemplary expression vectors may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Non-limiting examples of suitable markers include genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0249] Vectors for target gene expression Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes into mammalian cells.Viral genomes are particularly useful vectors for gene delivery.Because the polynucleotides contained in such genomes are typically integrated into the nuclear genome of mammalian cells by general transduction or specific transduction.These processes occur as part of the natural viral replication cycle and often do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma viruses, mammalian type C, type B, and type D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996, the disclosure of which is incorporated herein by reference).Other examples of viral vectors include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in US Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference.

[0250] Additional Transfection Methods Other techniques that can be used to introduce polynucleotides, such as DNA or RNA (e.g., mRNA, tRNA, siRNA, miRNA, shRNA, chemically modified RNA) into mammalian cells are well known in the art. For example, electroporation can be used to permeabilize mammalian cells by applying an electrostatic potential. Mammalian cells, such as hematopoietic stem cells, subjected to an external electric field in this manner subsequently become susceptible to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al. Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes the application of an electric field to stimulate the update of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al. Experimental Dermatology 14:315 (2005) and US2010 / 0317114. The disclosures of each of these are incorporated herein by reference.

[0251] Another useful technique for transfecting hematopoietic stem cells includes squeezeporation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique has the advantage that no vector is required to deliver nucleic acid to cells such as hematopoietic stem cells. Squeezeporation is described in detail in, for example, Sharei et al. Journal of Visualized Experiments 81:e50980 (2013). The disclosure of this technique is incorporated herein by reference.

[0252] Lipofection is another technique useful for transfecting hematopoietic stem cells. This method involves loading nucleic acids into liposomes. These liposomes often present cationic functional groups, such as quaternary amines or protonated amines, toward the exterior of the liposome. Because cell membranes are anionic, this promotes electrostatic interactions between the liposome and the cell, ultimately leading to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids involves contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for interaction with cell membranes include activated dendrimers (e.g., as described in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran. Their use as transfection agents is described in detail, for example, in Gulick et al. Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect hematopoietic stem cells in a gentle and efficient manner, as this method utilizes the application of a magnetic field to induce uptake of nucleic acids. This technique is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0253] Another useful tool for inducing hematopoietic stem cells to take up exogenous nucleic acid is laser infection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize cells and allow polynucleotides to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al. Methods in Cell Biology 82:309 (2007).The disclosure of this document is incorporated herein by reference.

[0254] Microvesicles are another vehicle that can potentially be used to modify the genome of hematopoietic stem cells according to the methods of the present invention described herein. For example, to prepare the cell genome for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence, microvesicles induced by co-overexpression of glycoprotein VSV-G and, for example, a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins that subsequently catalyze the site-specific cleavage of endogenous polynucleotide sequences into cells. The use of such vesicles, also called gesicles, for genetic modification of eukaryotic cells is described in detail, for example, in Quinn, TP, et al. Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0255] Targeted gene integration using gene editing In addition to viral vectors, various additional tools have been developed that can be used to integrate exogenous genes into hematopoietic stem cells. One such method involves using transposons, which can be used to integrate polynucleotides encoding target genes into hematopoietic stem cells. Transposons are polynucleotides that encode transposase enzymes and contain the polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once transposons are delivered to cells, the expression of the transposase gene begins, producing an active enzyme that excises the gene of interest from the transposon. This activity is mediated by the transposase's site-specific recognition of the transposon excision site. In certain cases, these excision sites can be terminal repeats or inverted terminal sequences. Once the gene of interest is excised from the transposon, it can be integrated into the mammalian cell genome through the transposase-catalyzed excision of similar excision sites present in the nuclear genome of mammalian cells. This allows the gene of interest to be inserted into the complementary excision site of the cut nuclear DNA, and then the integration process is completed by the covalent ligation of phosphodiester bond that connects the gene of interest to the DNA of mammalian cell genome.In some cases, transposon can be retrotransposon, so that the gene encoding target gene is first transcribed into RNA product, then reverse transcribed into DNA, and then integrated into mammalian cell genome.Exemplary transposon systems include piggyback transposon (for example, as described in WO2010 / 085699) and Sleeping Beauty transposon (for example, as described in US2005 / 0112764).The disclosures of each of these are incorporated herein by reference.

[0256] Another useful tool for integrating target genes into the hematopoietic stem cell genome is the clustered regularly interspaced short palindromic repeat (CRISPR) / Cas system, which originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR / Cas system contains palindromic repeat sequences within plasmid DNA and the associated Cas9 nuclease. This set of DNA and proteins first induces site-specific DNA cleavage of the target sequence by integrating the foreign DNA into the CRISPR locus. Next, polynucleotides containing these foreign sequences and the repeat-spacer element of the CRISPR locus are transcribed in the host cell to generate guide RNAs, which then anneal to the target sequence and localize the Cas9 nuclease to this site. In this way, highly site-specific Cas9-mediated DNA cleavage can be achieved in foreign polynucleotides. This is because the interaction that brings Cas9 into proximity with the target DNA molecule is governed by RNA:DNA hybridization. As a result, in theory, the CRISPR / Cas system can be designed to cleave any target DNA molecule of interest. This technique has been utilized to edit eukaryotic genomes (Hwang et al. Nature Biotechnology 31:227 (2013)) and can be used as an efficient means to site-specifically edit hematopoietic stem cell genomes to integrate a gene encoding a target gene after DNA cleavage. The use of CRISPR / Cas to regulate gene expression is described, for example, in US 8,697,359, the disclosure of which is incorporated herein by reference. Alternative methods for site-specific cleavage of genomic DNA in hematopoietic stem cells followed by integration of a gene of interest include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain guide polynucleotides to localize to specific target sequences.Instead, target specificity is controlled by the DNA binding domain in these enzymes.The use of ZFN and TALEN in genome editing applications is described, for example, in Urnov et al. Nature Reviews Genetics 11:636 (2010) and Joung et al. Nature Reviews Molecular Cell Biology 14:49 (2013).The disclosures of both of these are incorporated herein by reference.

[0257] Other genome editing methods that can be used to integrate polynucleotides encoding target genes into hematopoietic stem cell genome include using ARCUS™ meganuclease, which can be rationally designed to site-specifically cut genomic DNA.Considering the defined structure-activity relationship that has been demonstrated for such enzymes, it is advantageous to use these enzymes to integrate genes encoding target genes into mammalian cell genomes.Single-chain meganucleases can be modified at certain amino acid positions to create nucleases that selectively cut DNA at desired locations, allowing target genes to be site-specifically integrated into the nuclear DNA of hematopoietic stem cells.These single-chain nucleases are extensively described, for example, in US8,021,867 and US8,445,251.The disclosures of each of these are incorporated herein by reference.

[0258] Induction of hematopoietic stem cell differentiation In certain cases, it may be desirable to expand, enrich, or maintain a hematopoietic stem cell population according to the methods of the present invention, and then induce these cells to differentiate into blood cells of the hematopoietic repertoire before infusing the resulting cells into a recipient. This is a useful paradigm for administering a specific blood cell type to a recipient in need of this blood cell type. Hematopoietic stem cell populations that have been expanded, enriched, and / or maintained according to the methods of the present invention may be subjected to various conditions, such as conditions known in the art, to stimulate the differentiation of these cells into cells of the hematopoietic lineage. For example, using established protocols, hematopoietic stem cells can be induced to differentiate into one of a number of blood cell types, such as common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocytic progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, and lymphocytes, such as NK cells, B cells, and T cells.

[0259] Indications for hematopoietic stem cell therapy Hematopoietic stem cells generated (e.g., expanded, enriched, or maintained in a pluripotent state) using the compositions and methods of the present invention can be used to treat a variety of human diseases. The hematopoietic stem cells or their progeny administered to a patient can be autologous, syngeneic, or allogeneic, and can be administered with one or more agents that promote hematopoietic stem cell expansion in vivo. For example, hematopoietic stem cells or their progeny can be used to treat acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), myelodysplastic syndromes (MDS), multiple myeloma, aplastic anemia, bone marrow failure, myeloproliferative disorders such as myelofibrosis, essential thrombocytopenia, or polycythemia vera, Fanconi anemia, dyskeratosis congenita, common variable immunodeficiency (CVID, e.g., CVID1, CVID2, CVID3, CVID4, CVID5, and CVID6), human immunodeficiency virus (HIV), hemophagocytic lymphoma, and the like. It may be administered to a patient (e.g., a human patient) to treat diseases such as histiocytosis, amyloidosis, solid tumors such as neuroblastoma, germ cell tumors, breast cancer, Wilms' tumor, medulloblastoma, and neuroectodermal tumors, autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, systemic lupus erythematosus, and type I diabetes, or protein deficiencies such as adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), hemophilia A and B, Hurler syndrome, Hunter syndrome, Fabry disease, Gaucher disease, epidermolysis bullosa, globoid cell leukodystrophy, Sanfilippo syndrome, and Morquio syndrome.

[0260] Hematopoietic stem cells or their progeny may also be used to treat genetic blood disorders, such as sickle cell anemia, alpha thalassemia, beta thalassemia, delta thalassemia, hemoglobin E / thalassemia, hemoglobin S / thalassemia, hemoglobin C / thalassemia, hemoglobin D / thalassemia, chronic granulomatous disease (X-linked chronic granulomatous disease, autosomal recessive (AR) chronic granulomatous disease, chronic granulomatous disease AR I NCF1, chronic granulomatous disease AR CYBA, chronic granulomatous disease AR II NCF2, chronic granulomatous disease AR III NCF4), X-linked severe combined immunodeficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID, Rag1 / Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, congenital granulocytopenia, congenital granulocytopenia-congenital neutropenia-SCN1, congenital granulocytopenia-congenital neutropenia-SCN2, familial hemophagocytic lymphohistiocytosis (FHL), familial hemophagocytic lymphohistiocytosis type 2 (FHL2, perforin mutation), agammaglobulinemia (X-linked agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, hemolytic anemia due to erythrocyte pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, X-linked adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative disorder, unicentric Castleman disease, multicentric Castleman disease, congenital amegakaryocytic thrombocytopenia (CAMT) type I, reticular dysplasia, Fan Kohni anemia, acquired idiopathic sideroblastic anemia, systemic mastocytosis, von Willebrand disease (VWD), congenital dyserythroid anemia type 2, cartilage-hair hypoplasia syndrome, hereditary spherocytosis, Blackfan-Diamond syndrome, Shwachman-Diamond syndrome, thrombocytopenia-radial coloboma syndrome, osteopetrosis, childhood osteopetrosis, mucopolysaccharidoses, Lesch-Nyhan syndrome, glycogen storage disease, congenital mastocytosis, Omenn syndrome, X-linked immunodysregulation-polyendocrinopathy enteropathy (IPEX), IPEX characterized by FOXP3 mutations, X-linked immunodeficiency-polyendocrinopathy diarrhea syndrome (XPID), X-linked autoimmune and allergic dysregulation syndrome (XLAAD), IPEX-like syndrome, hyper-IgM type 1, hyper-IgM type 2, hyper-IgM type 3, hyper-IgM type 4, hyper-IgMThey may also be administered to human patients to treat type 5, X-linked hyperimmunoglobulin M, bare lymphocyte syndrome type I, and bare lymphocyte syndrome type II (bare lymphocyte syndrome type II, MHC class I deficiency; bare lymphocyte syndrome type II, complementation group A; bare lymphocyte syndrome type II, complementation group C; bare lymphocyte syndrome type II, complementation group D; bare lymphocyte syndrome type II, complementation group E). Hematopoietic stem cell populations expanded, enriched, or maintained by the compositions and / or methods of the invention, as well as their progeny, can also be used to treat patients suffering from hematolymphoid malignancies, non-hematolymphoid malignancies, or protein deficiencies. In other embodiments, the patient may be a tissue or cell transplant recipient, and the hematopoietic stem cells or their progeny are administered to induce tolerance to the transplanted tissue or cells.

[0261] All patents and other publications, including references, issued patents, published patent applications, and copending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methods described in such publications, which may be used in conjunction with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0262] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments and examples of the present disclosure have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as will be recognized by those skilled in the relevant art. For example, while method steps or functions are shown in a certain order, alternative embodiments may perform the functions in a different order, or functions may be accomplished substantially simultaneously. The teachings of the present disclosure provided herein may be applied to other procedures or methods, as appropriate. Various embodiments described herein may be combined to provide further embodiments. If necessary, aspects of the present disclosure may be modified to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, the concept of biological functional equivalence allows for some changes to be made to protein structure without affecting biological or chemical activity in terms of type or amount. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be encompassed within the scope of the appended claims.

[0263] Particular elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, while advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to be within the scope of the present disclosure.

[0264] Some aspects of the technology described herein can be defined by any of the following numbered items: 1. A hematopoietic stem cell population is isolated by a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation 1. A method for producing an expanded hematopoietic stem cell population ex vivo, comprising contacting a subject with one or more agents that together exhibit two or more effects selected from the group consisting of: 2. A hematopoietic cell population containing one or more hematopoietic stem cells is a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation 1. A method for enriching a cell population ex vivo for hematopoietic stem cells, comprising contacting a cell population with one or more agents that together exhibit two or more effects selected from the group consisting of: 3. The first hematopoietic stem cell population is a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and (c) contacting the first hematopoietic stem cell population with one or more agents that together exhibit two or more effects selected from the group consisting of: 4. The method according to any one of items 1 to 3, wherein the modulation of histone methylation is activation of histone methylation, maintenance of histone methylation, or inhibition of histone demethylation. 5. The method according to any one of items 1 to 4, wherein the modulation of histone acetylation is activation of histone acetylation, maintenance of histone acetylation, or inhibition of histone deacetylation. 6. The method of item 4, wherein the one or more agents comprise a compound that activates histone methylation, a compound that maintains histone methylation, or a compound that inhibits histone demethylation, and a compound that inhibits TGFβ signaling. 7. The method of item 6, wherein the compound that activates histone methylation, the compound that maintains histone methylation, or the compound that inhibits histone demethylation is a histone demethylase inhibitor, and the compound that inhibits TGFβ signaling is a TGFβ receptor inhibitor. 8. The method according to item 7, wherein the histone demethylase inhibitor is an LSD1 inhibitor. 9. The method of item 8, wherein the LSD1 inhibitor is LSD1 inhibitor IV RN-1 and the TGFβ receptor inhibitor is ALK5 inhibitor II. 10. The method of claim 8, wherein the LSD1 inhibitor is tranylcypromine and the TGFβ receptor inhibitor is ALK5 inhibitor II. 11. A hematopoietic stem cell population is a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase 1. A method for producing an expanded hematopoietic stem cell population ex vivo, comprising contacting a human hematopoietic stem cell with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 12. A hematopoietic cell population containing one or more hematopoietic stem cells, a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase 1. A method for enriching a cell population ex vivo for hematopoietic stem cells, comprising contacting a cell population with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 13. The first population of hematopoietic stem cells is isolated by: a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase and (c) contacting said first hematopoietic stem cell population with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 14. The method of any of items 11-13, wherein the one or more agents comprise a combination of agents selected from the combinations of agents in Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6. 15. The method according to any one of items 11 to 13, wherein the histone demethylase is LSD1. 16. The method of any of items 11 to 13, wherein the one or more agents comprise a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. 17. The method according to any one of items 11 to 16, wherein the protein that transmits TGFβ signaling is a TGFβ receptor. 18. The method of any of items 11 to 17, wherein the one or more agents comprise a compound that inhibits a protein that transduces TGFβ signaling, and the compound is selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1. 19. The method of any of items 11 to 18, wherein the one or more agents comprise a compound that inhibits a protein that mediates p38 signaling, and the compound is SB203580. 20. The method of any of items 11 to 19, wherein the one or more agents comprise a compound that inhibits a protein that promotes β-catenin degradation, and the compound is selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. 21. The method of any of items 11 to 20, wherein the one or more agents comprise a compound that inhibits histone deacetylase, and the compound is selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. 22. The method of any of paragraphs 11 to 21, wherein the one or more agents together inhibit the action of a histone demethylase and a protein that mediates TGFβ signaling. 23. The method of item 22, wherein the histone demethylase is LSD1. 24. The method of item 22 or 23, wherein the protein that transmits TGFβ signaling is a TGFβ receptor. 25. The method of any of items 22 to 24, wherein the one or more agents comprise LSD1 inhibitor IV RN-1 and ALK5 inhibitor II. 26. The method of any of items 22 to 25, wherein the one or more agents comprise a compound that inhibits p38 signaling. 27. The method of any of items 22 to 26, wherein the one or more agents comprise a compound that inhibits histone deacetylase. 28. The method of any one of items 22 to 27, wherein the one or more agents further comprise a compound that inhibits BMP signaling. 29. A method for generating an expanded hematopoietic stem cell population ex vivo, comprising contacting a hematopoietic stem cell population with (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax. 30. A method for enriching a cell population for hematopoietic stem cells ex vivo, comprising contacting a hematopoietic cell population containing one or more hematopoietic stem cells with (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax. 31. A method comprising contacting a first hematopoietic stem cell population with: (a) LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, or LSD1 inhibitor III. 1. A method for maintaining hematopoietic stem cell functional potential of a hematopoietic stem cell population ex vivo for at least two days, comprising contacting the first hematopoietic stem cell population with (a) a first agent selected from the group consisting of CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax, wherein after two days or later, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but not contacted with the first agent and the second agent. 32. The method of any of items 29 to 31, wherein the one or more agents comprise a combination of agents selected from the combinations of agents in Table 7, Table 8, Table 9, and Table 10. 33. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to stimulate an expansion of the hematopoietic stem cell population by 10% or more on day 7 or later of culture (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture) relative to a hematopoietic stem cell population not contacted with the one or more agents. 34. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to stimulate an expansion of the hematopoietic stem cell population by 10% or more compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or with a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture). 35. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more, relative to a hematopoietic stem cell population not contacted with the one or more agents, on day 7 or later of culture (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture). 36. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or with a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture). 37. The method of any of items 3, 13, and 31, wherein the first hematopoietic stem cell population exhibits hematopoietic stem cell functional capacity that is superior to the hematopoietic stem cell functional capacity of the control hematopoietic stem cell population on day 3 or later of culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). 38. The method according to any one of items 1 to 37, wherein the hematopoietic stem cells are mammalian cells. 39. The method of claim 38, wherein the mammalian cells are human cells. 40. The method of item 39, wherein the hematopoietic stem cells are CD34+ cells. 41. The method of item 40, wherein at least 10% of the CD34+ cells are CD34+CD38- cells, CD34+CD38-CD90+ cells, CD34+CD38-CD90+CD45RA- cells, or CD34+CD38-CD90+CD45RA-CD49F+ cells. 42. The method according to any one of items 38 to 40, wherein the hematopoietic stem cells are derived from human umbilical cord blood. 43. The method according to any one of items 38 to 40, wherein the hematopoietic stem cells are derived from human mobilized peripheral blood. 44. The method according to any one of items 38 to 40, wherein the hematopoietic stem cells are derived from human bone marrow. 45. The method according to any one of items 38 to 44, wherein the hematopoietic stem cells are freshly isolated from a human. 46. The method according to any one of items 38 to 44, wherein the hematopoietic stem cells have previously been cryopreserved. 47. The method of claim 38, wherein the mammalian cells are mouse cells. 48. The method of any of items 1 to 47, wherein the hematopoietic stem cells are cultured for 2 days or more (e.g., 3 days, 5 days, 7 days, 10 days, 12 days, 14 days, 15 days, 20 days, or more). 49. The method of any of paragraphs 1-48, wherein the hematopoietic stem cells are contacted with the one or more agents for two or more days (e.g., three days, five days, seven days, ten days, twelve days, fourteen days, fifteen days, twenty days, or more). 50. The method of any one of items 1 to 49, wherein the hematopoietic stem cells are contacted with the one or more agents simultaneously. 51. The method of any one of items 1 to 49, wherein the hematopoietic stem cells are contacted with the one or more agents at different times. 52. The method of any of items 1 to 51, wherein the hematopoietic stem cells maintain hematopoietic stem cell functional capacity on day 2 of culture (e.g., on day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). 53. The method of item 52, wherein the hematopoietic stem cells maintain hematopoietic stem cell functional capacity after transplantation on day 2 of culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). 54. The method of any of items 1 to 53, wherein the hematopoietic stem cells maintain long-term engraftment potential on day 2 of culture (e.g., day 3, day 5, day 7, day 10, day 12, day 14, day 15, day 20, or later). 55. The method of any of items 1 to 54, wherein the hematopoietic stem cells, when transplanted into a patient, elicit the recovery of a population of cells selected from the group consisting of neutrophils, platelets, erythrocytes, monocytes, macrophages, antigen-presenting cells, microglia, osteoclasts, dendritic cells, and lymphocytes. 56. The method of item 55, wherein the lymphocytes are selected from the group consisting of natural killer cells, T cells (e.g., CD4+ cells or CD8+ cells), and B cells. 57. The method of any of items 1 to 56, wherein the hematopoietic stem cells are capable of localizing to hematopoietic tissues in the transplanted recipient and re-establishing productive hematopoiesis. 58. The method according to any one of items 1 to 57, wherein the hematopoietic stem cells are cultured on a plastic surface or on a support comprising vitronectin, fibronectin, or matrigel. 59. The method according to any one of items 1 to 58, wherein the hematopoietic stem cells are cultured in the presence of 2 to 20% oxygen. 60. The method according to item 59, wherein the hematopoietic stem cells are cultured in the presence of 2 to 12% oxygen. 61. The method of item 60, wherein the hematopoietic stem cells are cultured in the presence of about 5% oxygen. 62. The method of any one of items 1 to 61, wherein the hematopoietic stem cells are originally present in a mononuclear cell fraction before being treated with the one or more agents. 63. The method of any of paragraphs 1 to 61, wherein the hematopoietic stem cells are originally present in a CD34+, CD34+CD38-, CD34+CD38-CD90+, CD34+CD38-CD90+CD45RA-, or CD34+CD38-CD90+CD45RA-CD49F+ enriched cell fraction prior to contact with the one or more agents. 64. The method of any of paragraphs 1 to 61, wherein the hematopoietic stem cells are originally present in a non-enriched cell fraction prior to contact with the one or more agents. 65. a. Inserting a polynucleotide into a hematopoietic stem cell population; and b. Expanding the hematopoietic stem cell population according to the method described in any one of items 1, 11, and 29, or maintaining the hematopoietic stem cell functional potential of the hematopoietic stem cell population according to the method described in any one of items 3, 13, and 31 to 64. a method for introducing said polynucleotide into a population of hematopoietic stem cells, comprising: 66. The method according to item 65, wherein (a) precedes (b). 67. The method according to item 65, wherein (b) precedes (a). 68. The method according to any one of items 65 to 67, comprising providing one or more reagents that cleave nucleic acids in the cell. 69. The method of item 68, wherein the one or more reagents that cleave nucleic acids in the cell comprise a zinc finger nuclease. 70. The method of item 68, wherein the one or more reagents that cleave nucleic acids in the cell comprise a transcription activator-like effector nuclease. 71. The method of item 68, wherein the one or more reagents that cleave nucleic acids in the cell comprise a CRISPR-associated protein. 72. The method of item 68, wherein the one or more agents that cleave nucleic acids in the cell comprise a meganuclease. 73. The method according to any one of items 65 to 72, comprising a step of contacting the hematopoietic stem cells with a vector selected from the group consisting of a viral vector (e.g., a retrovirus, adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, or poxvirus) and a transposable element (e.g., a piggyBac transposon or a Sleeping Beauty transposon). 74. The method according to any of items 65 to 72, comprising the step of introducing the polynucleotide into the hematopoietic stem cells by electroporation, Nucleofection™, or squeezeporation. 75. The method of any of items 65 to 72, comprising contacting the cells with a transforming agent selected from the group consisting of a cationic polymer (e.g., diethylaminoethyl-dextran), a cationic lipid, calcium phosphate, an activated dendrimer, and a magnetic bead. 76. The method according to any one of items 65 to 72, comprising the step of introducing the polynucleotide into the hematopoietic stem cells by microinjection or laserfection. 77. The method according to any one of items 65 to 76, wherein the polynucleotide comprises a regulatory sequence selected from the group consisting of a promoter sequence, an enhancer sequence, or a silencer sequence. 78. The method according to any one of items 65 to 76, wherein the polynucleotide encodes a molecule selected from the group consisting of a protein and RNA (mRNA, tRNA, siRNA, miRNA, shRNA). 79. The method according to any one of items 65 to 76, wherein the polynucleotide is chemically modified RNA. 80. The method of any of items 65 to 79, further comprising the step of introducing the expanded hematopoietic stem cell population or its progeny into a recipient. 81. a. Providing a population of hematopoietic stem cells; b. Expanding the hematopoietic stem cell population according to the method described in any of items 1, 11, 29, and 32-64; c. Optionally, differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocyte progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, and lymphocytes, NK cells, B cells, and / or T cells; and d. Introducing the expanded hematopoietic stem cell population or its progeny into a recipient. a method of treating said recipient with hematopoietic stem cells or progeny thereof, comprising: 82. a. Providing a hematopoietic stem cell population; b. Enriching the hematopoietic stem cell population according to the method described in any of items 2, 12, 30, and 32-64; c. Optionally, differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocyte progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, and lymphocytes, NK cells, B cells, and / or T cells; and d. Introducing the cell population enriched for hematopoietic stem cells, or their progeny, into a recipient. a method of treating said recipient with hematopoietic stem cells or progeny thereof, comprising: 83. a. Providing a population of hematopoietic stem cells; b. Maintaining the hematopoietic stem cell functional potential of the hematopoietic stem cell population according to the method described in any one of items 3, 13, and 31-64; c. Optionally, differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocyte progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, and lymphocytes, NK cells, B cells, and / or T cells; and d. Introducing the hematopoietic stem cell population or its progeny into a recipient. a method of treating said recipient with hematopoietic stem cells or progeny thereof, comprising: 84. a. Providing a hematopoietic stem cell population generated by the method according to any one of items 1 to 64; b. optionally differentiating the hematopoietic stem cells into common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, granulocyte-megakaryocyte progenitors, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, and lymphocytes, NK cells, B cells, and / or T cells; and c. introducing said hematopoietic stem cell population or its progeny into a recipient. a method of treating said recipient with hematopoietic stem cells or progeny thereof, comprising: 85. The method according to any one of items 80 to 84, wherein the recipient is a human. 86. The method of item 85, wherein the hematopoietic stem cells are derived from one or more hematopoietic stem cells isolated from a human donor. 87. The method of item 86, wherein the hematopoietic stem cells are derived from mobilized peripheral blood of the donor. 88. The method of item 87, wherein the donor has previously been administered one or more mobilizing agents selected from the group consisting of a CXCR4 antagonist (e.g., AMD3100), GCSF, and GROβ. 89. The method of any one of items 1 to 88, wherein the hematopoietic stem cells are further contacted with a substance that inhibits aryl hydrocarbon receptor signaling. 90. The method of any one of items 34, 36, and 89, wherein the substance that inhibits aryl hydrocarbon receptor signaling is SR1 or an analog thereof. 91. The method of any one of items 1 to 90, wherein the hematopoietic stem cells are further contacted with UM171 or an analog thereof. 92. The method of any one of items 1 to 91, wherein the hematopoietic stem cells are further contacted with a prostaglandin. 93. The method of item 92, wherein the prostaglandin is dmPGE2 or an analog thereof. 94. The method of any one of items 1 to 93, wherein the hematopoietic stem cells are further contacted with a Notch signaling agonist. 95. The method of any one of items 1 to 94, wherein the hematopoietic stem cells are further contacted with a SIRT1 inhibitor. 96. The method of item 95, wherein the inhibitor or SIRT1 is selected from the group consisting of nicotinamide, cambinol, and analogs thereof. 97. The recipient has acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), myelodysplastic syndrome (MDS), multiple myeloma, aplastic anemia, bone marrow failure, myeloproliferative disorders such as myelofibrosis, essential thrombocytopenia, or polycythemia vera, Fanconi anemia, dyskeratosis congenita, common variable immunodeficiency 97. The method of any of items 80 to 96, wherein the human patient is suffering from a disease selected from the group consisting of HIV (CVID, e.g., CVID1, CVID2, CVID3, CVID4, CVID5, and CVID6), human immunodeficiency virus (HIV), hemophagocytic lymphohistiocytosis, amyloidosis, solid tumors such as neuroblastoma, germ cell tumors, breast cancer, Wilms' tumor, medulloblastoma, and neuroectodermal tumors, autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, systemic lupus erythematosus, and type 1 diabetes, or protein deficiency diseases such as adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), hemophilia A and B, Hurler syndrome, Hunter syndrome, Fabry disease, Gaucher disease, epidermolysis bullosa, globoid cell leukodystrophy, Sanfilippo syndrome, and Morquio syndrome. 98. The recipient is a patient with sickle cell anemia, alpha thalassemia, beta thalassemia, delta thalassemia, hemoglobin E / thalassemia, hemoglobin S / thalassemia, hemoglobin C / thalassemia, hemoglobin D / thalassemia, chronic granulomatous disease (X-linked chronic granulomatous disease, autosomal recessive (AR) chronic granulomatous disease, chronic granulomatous disease AR I NCF1, chronic granulomatous disease AR CYBA, chronic granulomatous disease AR II NCF2, chronic granulomatous disease AR III NCF4), X-linked severe combined immunodeficiency (SCID), ADA SCID, IL7-RA SCID, CD3 SCID, Rag1 / Rag2 SCID, Artemis SCID, CD45 SCID, Jak3 SCID, congenital granulocytopenia, congenital granulocytopenia-congenital neutropenia-SCN1, congenital granulocytopenia-congenital neutropenia-SCN2, familial hemophagocytic lymphohistiocytosis (FHL), familial hemophagocytic lymphohistiocytosis type 2 (FHL2, perforin mutation), agammaglobulinemia (X-linked agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, hemolytic anemia due to erythrocyte pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, X-linked adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative disorder, unicentric Castleman syndrome disease, multicentric Castleman disease, congenital amegakaryocytic thrombocytopenia (CAMT) type I, reticular dysplasia, Fanconi anemia, acquired idiopathic sideroblastic anemia, systemic mastocytosis, von Willebrand disease (VWD), congenital dyserythroid anemia type 2, cartilage-hair hypoplasia syndrome, hereditary spherocytosis, Blackvan-Diamond syndrome, Shwachman-Diamond syndrome, thrombocytopenia-radial coloboma syndrome, osteopetrosis, childhood osteopetrosis, mucopolysaccharidoses, Lesch-Nyhan syndrome, glycogen storage disease, congenital mastocytosis, Omenn syndrome, X-linked immunodysregulation-polyendocrinopathy enteropathy (IPEX), IPEX characterized by FOXP3 mutations, X-linked immune deficiency and polyendocrinopathy diarrhea syndrome (XPID), X-linked autoimmune and allergic dysregulation syndrome (XLAAD), IPEX-like syndrome, hyper-IgM type 1, hyper-IgM type 2, hyper-IgM type 3, hyper-IgM type 4, hyper-IgM97. The method of any of items 80 to 96, wherein the human patient is suffering from a disease selected from the group consisting of type 5, X-linked hyperimmunoglobulin M, bare lymphocyte syndrome type I, and bare lymphocyte syndrome type II (bare lymphocyte syndrome type II, MHC class I deficiency; bare lymphocyte syndrome type II, complementation group A; bare lymphocyte syndrome type II, complementation group C; bare lymphocyte syndrome type II, complementation group D; bare lymphocyte syndrome type II, complementation group E). 99. The method of any of items 80 to 96, wherein the recipient is a human patient suffering from a hematolymphoid malignancy, a non-hematolymphoid malignancy, or a protein deficiency, or is a tissue or cell transplant recipient (e.g., to induce tolerance to the transplanted tissue or cells). 100. The method according to any one of items 80 to 99, wherein the hematopoietic stem cells are autologous or syngeneic. 101. The method according to any one of items 80 to 99, wherein the hematopoietic stem cells are allogeneic. 102. a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation A composition comprising one or more active substances which together exhibit two or more effects selected from the group consisting of: 103. The composition described in item 102, wherein the modulation of histone methylation is activation of histone methylation, maintenance of histone methylation, or inhibition of histone demethylation. 104. The composition described in item 102 or 103, wherein the modulation of histone acetylation is activation of histone acetylation, maintenance of histone acetylation, or inhibition of histone deacetylation. 105. The composition described in item 103, wherein the one or more agents include a compound that activates histone methylation, a compound that maintains histone methylation, or a compound that inhibits histone demethylation, and a compound that inhibits TGFβ signaling. 106. The composition described in item 105, wherein the compound that activates histone methylation, the compound that maintains histone methylation, or the compound that inhibits histone demethylation is a histone demethylase inhibitor, and the compound that inhibits TGFβ signaling is a TGFβ receptor inhibitor. 107. The composition described in item 106, wherein the histone demethylase inhibitor is an LSD1 inhibitor. 108. The composition of item 107, wherein the LSD1 inhibitor is LSD1 inhibitor IV RN-1 and the TGFβ receptor inhibitor is ALK5 inhibitor II. 109. The composition of item 107, wherein the LSD1 inhibitor is tranylcypromine and the TGFβ receptor inhibitor is ALK5 inhibitor II. 110. a.Histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase A composition comprising one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 111. The composition described in item 110, wherein the histone demethylase is LSD1. 112. The composition of item 110, wherein the one or more agents comprise a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. 113. The composition according to any one of items 110 to 112, wherein the protein that transmits TGFβ signaling is a TGFβ receptor. 114. The composition of any of items 110 to 113, wherein the one or more agents comprise a compound that inhibits a protein that transduces TGFβ signaling, and the compound is selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1. 115. The composition of any of items 110-114, wherein the one or more agents comprise a compound that inhibits a protein that mediates p38 signaling, and the compound is SB203580. 116. The composition of any of items 110-115, wherein the one or more agents comprise a compound that inhibits a protein that promotes β-catenin degradation, and the compound is selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. 117. The composition of any of items 110-116, wherein the one or more agents comprise a compound that inhibits histone deacetylase, and the compound is selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. 118. The composition of any of paragraphs 110-117, wherein the one or more agents together inhibit the action of a histone demethylase and a protein that transduces TGFβ signaling. 119. The composition of item 118, wherein the histone demethylase is LSD1. 120. The composition described in item 118 or 119, wherein the protein that transmits TGFβ signaling is a TGFβ receptor. 121. The composition of any of items 118-120, wherein the one or more agents include LSD1 inhibitor IV RN-1 and ALK5 inhibitor II. 122. The composition of any of items 118-121, wherein the one or more agents comprise a compound that inhibits p38 signaling. 123. The composition of any of items 118-122, wherein the one or more agents include a compound that inhibits histone deacetylase. 124. The composition of any of items 118-123, wherein the one or more agents include a compound that inhibits BMP signaling. 125. A composition comprising: (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine; and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax. 126. The composition of any of paragraphs 102-125, wherein the one or more agents are present in an amount sufficient to generate an expanded hematopoietic stem cell population. 127. The composition of any of items 102-125, wherein the one or more agents are present in an amount sufficient to produce a cell population enriched for hematopoietic stem cells. 128. The composition of any of paragraphs 102-125, wherein the one or more agents are present in an amount sufficient to maintain hematopoietic stem cell functional capacity of the hematopoietic stem cell population for at least 2 days. 129. The composition of any of items 126-128, wherein the one or more active substances are present in an aqueous solution. 130. The composition of any of items 126-128, wherein the one or more active substances are present as a lyophilized solid. 131. The composition of any of paragraphs 126-130, wherein the one or more agents are present in an amount sufficient to stimulate an expansion of the hematopoietic stem cell population by 10% or more on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture) compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or with a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof. 132. The composition of any of paragraphs 126-130, wherein the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or with a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture). 133. The composition of any of paragraphs 126-130, wherein the one or more agents are present in an amount sufficient to maintain the long-term viability of the hematopoietic stem cells after transplantation following contact with the hematopoietic stem cells in culture for two or more days (e.g., three days, five days, seven days, ten days, twelve days, fourteen days, fifteen days, twenty days, or more). 134. A composition comprising a combination of agents selected from the combinations of agents in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, and Table 10. 135. A cell culture medium comprising the composition according to any one of items 102 to 134. 136. The cell culture medium according to item 135, which is substantially free of serum. 137. The composition of any of items 102-134, further comprising a hematopoietic stem cell population in contact with the one or more agents. 138. The composition of item 137, wherein the hematopoietic stem cells have been cultured in the presence of the one or more agents for two or more days (e.g., three days, five days, seven days, ten days, twelve days, fourteen days, fifteen days, twenty days, or more). 139. A hematopoietic stem cell population is (1) a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation a first agent exhibiting one or more effects selected from the group consisting of: (2) a second agent selected from the group consisting of SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor; 1. A method for producing an expanded hematopoietic stem cell population ex vivo, comprising the step of contacting, wherein the first agent and the second agent are present in amounts together sufficient to produce an expanded hematopoietic stem cell population. 140. A hematopoietic cell population containing one or more hematopoietic stem cells, (1) a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation a first agent exhibiting one or more effects selected from the group consisting of: (2) a second agent selected from the group consisting of SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor; A method for enriching a cell population ex vivo for hematopoietic stem cells, comprising the step of contacting, wherein the first agent and the second agent are present together in amounts sufficient to produce a cell population enriched for hematopoietic stem cells. 141. A first population of hematopoietic stem cells is isolated from: (1) a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation a first agent exhibiting one or more effects selected from the group consisting of: (2) a second agent selected from the group consisting of SR1 or an analog thereof, UM171 or an analog thereof, a prostaglandin, a Notch signaling agonist, or a SIRT1 inhibitor; 1. A method for maintaining the hematopoietic stem cell functional potential of a hematopoietic stem cell population ex vivo for at least two days, comprising contacting the hematopoietic stem cell population with one or more agents and one or more substances, wherein after two days or more, the hematopoietic stem cell population exhibits a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the hematopoietic stem cell population but not contacted with the one or more agents and one or more substances. 142. A hematopoietic stem cell population produced by the method described in any one of items 1 to 101 and 139 to 141. 143. A kit comprising the composition according to any one of items 102 to 134, 137, and 138, and further comprising an attached document. 144. The kit of item 143, wherein the package insert instructs a user of the kit to expand a hematopoietic stem cell population ex vivo, enrich a hematopoietic stem cell population, or maintain the hematopoietic stem cell functional potential of a hematopoietic stem cell population. 145. The kit of item 143, wherein the package insert instructs the user to express a polynucleotide in the hematopoietic stem cells. 146. The kit of item 143, wherein the package insert instructs the user to administer the hematopoietic stem cells to a patient.

[0265] Some aspects of the technology described herein can be defined by any of the following numbered items: 1. A hematopoietic stem cell population is isolated by a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation 1. A method for producing an expanded hematopoietic stem cell population ex vivo, comprising contacting a subject with one or more agents that together exhibit two or more effects selected from the group consisting of: 2. A hematopoietic cell population containing one or more hematopoietic stem cells is a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation 1. A method for enriching a cell population ex vivo for hematopoietic stem cells, comprising contacting a cell population with one or more agents that together exhibit two or more effects selected from the group consisting of: 3. The first hematopoietic stem cell population is a. Regulation of histone methylation; b.Inhibition of TGFβ signaling; c.Inhibition of p38 signaling; d. Activation of canonical Wnt signaling; and e. Regulation of histone acetylation and (c) contacting the first hematopoietic stem cell population with one or more agents that together exhibit two or more effects selected from the group consisting of: 4. The method according to any one of items 1 to 3, wherein the modulation of histone methylation is activation of histone methylation, maintenance of histone methylation, or inhibition of histone demethylation. 5. The method according to any one of items 1 to 4, wherein the modulation of histone acetylation is activation of histone acetylation, maintenance of histone acetylation, or inhibition of histone deacetylation. 6. The method of item 4, wherein the one or more agents comprise a compound that activates histone methylation, a compound that maintains histone methylation, or a compound that inhibits histone demethylation, and a compound that inhibits TGFβ signaling. 7. The method of item 6, wherein the compound that activates histone methylation, the compound that maintains histone methylation, or the compound that inhibits histone demethylation is a histone demethylase inhibitor, and the compound that inhibits TGFβ signaling is a TGFβ receptor inhibitor. 8. The method according to item 7, wherein the histone demethylase inhibitor is an LSD1 inhibitor. 9. The method of item 8, wherein the LSD1 inhibitor is LSD1 inhibitor IV RN-1 and the TGFβ receptor inhibitor is ALK5 inhibitor II. 10. The method of claim 8, wherein the LSD1 inhibitor is tranylcypromine and the TGFβ receptor inhibitor is ALK5 inhibitor II. 11. A hematopoietic stem cell population is a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase 1. A method for producing an expanded hematopoietic stem cell population ex vivo, comprising contacting a human hematopoietic stem cell with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 12. A hematopoietic cell population containing one or more hematopoietic stem cells, a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase 1. A method for enriching a cell population ex vivo for hematopoietic stem cells, comprising contacting a cell population with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 13. The first population of hematopoietic stem cells is isolated by: a. histone demethylase; b. Proteins that transmit TGFβ signaling; c. p38 signal transduction protein; d. proteins that promote β-catenin degradation; and e. histone deacetylase and (c) contacting said first hematopoietic stem cell population with one or more agents that together inhibit the action of two or more proteins selected from the group consisting of: 14. The method of any of items 11-13, wherein the one or more agents comprise a combination of agents selected from the combinations of agents in Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6. 15. The method according to any one of items 11 to 13, wherein the histone demethylase is LSD1. 16. The method of any of items 11 to 13, wherein the one or more agents comprise a histone demethylase inhibitor selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine. 17. The method according to any one of items 11 to 16, wherein the protein that transmits TGFβ signaling is a TGFβ receptor. 18. The method of any of items 11 to 17, wherein the one or more agents comprise a compound that inhibits a protein that transduces TGFβ signaling, and the compound is selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1. 19. The method of any of items 11 to 18, wherein the one or more agents comprise a compound that inhibits a protein that mediates p38 signaling, and the compound is SB203580. 20. The method of any of items 11 to 19, wherein the one or more agents comprise a compound that inhibits a protein that promotes β-catenin degradation, and the compound is selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. 21. The method of any of items 11 to 20, wherein the one or more agents comprise a compound that inhibits histone deacetylase, and the compound is selected from the group consisting of trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. 22. The method of any of paragraphs 11 to 21, wherein the one or more agents together inhibit the action of a histone demethylase and a protein that mediates TGFβ signaling. 23. The method of item 22, wherein the histone demethylase is LSD1. 24. The method of item 22 or 23, wherein the protein that transmits TGFβ signaling is a TGFβ receptor. 25. The method of any of items 22 to 24, wherein the one or more agents comprise LSD1 inhibitor IV RN-1 and ALK5 inhibitor II. 26. The method of any of items 22 to 25, wherein the one or more agents comprise a compound that inhibits p38 signaling. 27. The method of any of items 22 to 26, wherein the one or more agents comprise a compound that inhibits histone deacetylase. 28. The method of any one of items 22 to 27, wherein the one or more agents further comprise a compound that inhibits BMP signaling. 29. A method for generating an expanded hematopoietic stem cell population ex vivo, comprising contacting a hematopoietic stem cell population with (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax. 30. A method for enriching a cell population for hematopoietic stem cells ex vivo, comprising contacting a hematopoietic cell population containing one or more hematopoietic stem cells with (a) a first agent selected from the group consisting of LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax. 31. A method comprising contacting a first hematopoietic stem cell population with: (a) LSD1 inhibitor IV RN-1, LSD1 inhibitor II S2101, LSD1 inhibitor LSD1-C76, or LSD1 inhibitor III. 1. A method for maintaining hematopoietic stem cell functional potential of a hematopoietic stem cell population ex vivo for at least two days, comprising contacting the first hematopoietic stem cell population with (a) a first agent selected from the group consisting of CBB1007, LSD1 inhibitor I, and tranylcypromine, and (b) a second agent selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, trichostatin A, SB203580, CHIR99021, DMH1, sodium acetate, and istodax, wherein after two days or later, the first hematopoietic stem cell population exhibits a hematopoietic stem cell functional potential that is superior to the hematopoietic stem cell functional potential of a control hematopoietic stem cell population cultured under the same conditions and for the same period of time as the first hematopoietic stem cell population but not contacted with the first agent and the second agent. 32. The method of any of items 29 to 31, wherein the one or more agents comprise a combination of agents selected from the combinations of agents in Table 7, Table 8, Table 9, and Table 10. 33. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to stimulate an expansion of the hematopoietic stem cell population by 10% or more on day 7 or later of culture (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture) relative to a hematopoietic stem cell population not contacted with the one or more agents. 34. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to stimulate an expansion of the hematopoietic stem cell population by 10% or more compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a UM171 analog selected from Table 11, a prostaglandin, a Notch signaling agonist, or with a SIRT1 inhibitor, e.g., nicotinamide, cambinol, or an analog thereof, on day 7 or later of culture (e.g., on day 7, 10, 12, 14, 15, 20, or later of culture). 35. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more, relative to a hematopoietic stem cell population not contacted with the one or more agents, on day 7 or later of culture (e.g., on day 7, day 10, day 12, day 14, day 15, day 20, or later of culture). 36. The method of any of paragraphs 1-32, wherein the one or more agents are present in an amount sufficient to enrich the cell population for hematopoietic stem cells by 10% or more compared to a hematopoietic stem cell population contacted with an agent that inhibits aryl hydrocarbon receptor signaling, e.g., SR1 or an analog thereof, UM171 or an analog thereof, a UM171 analog selected from Table 11, a prostaglandin, a Notch signaling agonist, or with a...

Claims

[Claim 1] The invention described herein.

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