Methods, media, and supplements for expanding hematopoietic cells

Abstract

The present disclosure relates to methods, media and supplements for culturing target cells such hematopoietic stem and progenitor cells (HSPCs). Methods, media and supplements of this disclosure may include a one or more epigenetic modifiers within a culture condition to culture and / or expand target cells, such as HSPCs or CD34+ cells, as may be obtained, enriched, or isolated from primary samples that are infected / diseased or normal. Output populations of HSPCs expanded in the presence of one or more epigenetic modifiers may be amenable for use in downstream applications.

Description

METHODS, MEDIA, AND SUPPLEMENTS FOR EXPANDING HEMATOPOIETIC CELLSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 586,490, filed September 29, 2023, the entire contents of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to cell culture applications, and more specifically to cell culture applications using hematopoietic stem and progenitor cells (HSPCs), and still more specifically to cell culture applications related to expansion of CD34+HSPCs and / or CD34+malignant cells.BACKGROUND

[0003] Hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPC) have been shown to have significant therapeutic potential. One limitation of clinical use of HSCs and HSPCs has been the difficulty to obtain sufficient numbers of such cells. In particular, HSCs and HSPCs are resistant to maintenance, propagation and expansion ex vivo. Another challenge relates to the loss of HSC and HSPC multi-potency, which may arise when such cells are cultured ex vivo.

[0004] Thus, research into ways to improve ex vivo maintenance, propagation, and expansion of HSCs and HSPCs that preserve their multi-potency and functionality are ongoing, and epigenetic modifiers may be promising candidates for this purpose. Particularly, use of pyrimidoindole compounds for the expansion of cord-blood derived HSCs or HSPCs is currently under investigation in the clinic.

[0005] Accordingly, there is a need for other or improved reagents and methods to expand HSCs, HSPCs, and other CD34+cells.SUMMARY

[0006] The present disclosure relates to the culture and / or expansion of target cells, which may be HSPCs, or CD34+cells, and various subsets thereof, including primitive CD34+subsets.

[0007] In one aspect of this disclosure are provided methods for culturing and / or expanding target cells (e.g. HSPC, or CD34+cells). Target cells of this disclosure may be comprised in an initial population comprising the target cells and non-target cells, or as single cells. The disclosed methods may be in vitro methods.

[0008] Methods of this disclosure may comprise contacting one or more of the target cells with a cocktail of a plurality of epigenetic modifiers in a culture medium, and culturing the target cells in the presence of the cocktail for a time sufficient to produce an expanded population of target cells, such as HSPCs or CD34+cells.

[0009] The cocktail of a plurality of epigenetic modifiers may comprise one or more of, or two or more of, or three or more of, or each of a first inhibitor of a histone deacetylase (HDACi), a second HDACi, at least one inhibitor of a histone demethylase (HDMi), and at least one inhibitor of a histone methyltransferase (HMTi).

[0010] In one embodiment, the first HDACi and the second HDACi are class I, II, or IV HDACi. In one embodiment, the first HDACi and the second HDACi are selected from: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.

[0011] In one embodiment, the at least one HDMi is a LSD1 or LSD2 inhibitor.

[0012] In one embodiment, the at least one HMTi is a G9a / GLP HMTi.

[0013] Methods of this disclosure may comprise isolating or enriching the one or more HSPC from a sample before contacting the population with the cocktail. Non-limiting examples of a sample include whole blood, cord blood, peripheral blood, or bone marrow. In addition, samples of this disclosure may be diseased or infected samples.

[0014] In one embodiment, the target cells (whether the initial one or more target cells, the initial population of target cells, or the expanded population of target cells) may comprise a primitive HSPC having a CD34+CD45RA CD90+phenotype, a CD34+CD45RA CD90+EPCR+phenotype, and / or a CD34+CD45RA CD90+EPCR+CD133+CD49f+phenotype. In one embodiment, the expanded population of target cells (e.g. HSPCs, or CD34+cells) comprises more primitive cells having a CD34+CD45RA CD90+phenotype and / or a CD34+CD45RA_CD90+EPCR+phenotype and / or a CD34+CD45RA CD90+EPCR+CD133+CD49f+phenotype compared to when the one or more target cells are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of target cells.

[0015] In one embodiment, the expanded population of target cells (e.g. HSPC, or CD34+cells) comprises more target cells compared to when the one or more target cells or the initial population of target cells are not cultured in the presence of one or more epigenetic modifiers (e.g. supplement or cocktail) for the time sufficient to produce the expanded population of target cells.

[0016] Methods of this disclosure may further comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34+cells) when the one or more target cells iscontacted with and / or cultured in the presence of one or more epigenetic modifiers (of a cocktail or supplement) comprising each of the first and second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing.

[0017] Methods of this disclosure may further comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34+cells) when the one or more target cells is contacted with and / or cultured in the presence of one or more epigenetic modifiers (of a cocktail or supplement) comprising a first HDACi, or a functional equivalent thereof, and two of the second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising two of the first and second HDACi, at least one HDMi, and at least one HMT, wherein the first HDACi is valproic acid.

[0018] Methods of this disclosure may further comprise yielding a more highly expanded population target cells (e.g. HSPCs, or CD34+cells) when the one or more target cells is contacted with and cultured in the presence of one or more epigenetic modifiers (of a cocktail or supplement) comprising a first HDACi, or a functional equivalent thereof, and one of the second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising one of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic acid.

[0019] Methods of this disclosure may further comprise yielding a more highly expanded population of HSPCs with multi-lineage differentiation capacity when the one or more HSPC is contacted with and cultured in the presence of a cocktail comprising the first HDACi and the at least one HMTi, wherein the first HDACi is valproic acid.

[0020] In one embodiment, the expanded population of target cells (e.g. HSPCs, or CD34+cells) comprises between 2-fold to more than 100-fold more target cells compared to when the target cells are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of target cells.

[0021] In one embodiment, the culture media may further comprise one or more of SCF, FLT- 3L, and / or TPO.

[0022] Different methods of this disclosure may comprise culturing or expanding a population of target cells (e.g. HSPC, or CD34+cells) by contacting the target cells with a serum-free culture media comprising at least one epigenetic modifier and one or more of SCF, FLT-3L, and / or TPO, and culturing the target cells in the culture medium for a time sufficient to produce an expanded population of target cells.

[0023] The expanded population may comprise target cells comprising primitive HSPCs or CD34+cells having a CD34+CD45RA CD90+phenotype and / or a CD34+CD45RA_CD90+EPCR+phenotype and / or a CD34+CD45RA CD90+EPCR+CD133+CD49f phenotype.

[0024] In one embodiment, the at least one epigenetic modifier is a histone deacetylase inhibitor (HDACi). In one embodiment, the HDACi is valproic acid.

[0025] In embodiments comprising more than one epigenetic modifier, the modifiers may comprise one or more of a second histone deacetylase inhibitor (HDACi), at least one histone demethylase inhibitor (HDMi), and at least one histone methyltransferase inhibitor (HMTi). Exemplary HDACi include: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin. Exemplary HDMi include a LSD1 or LSD2 inhibitor. Exemplary HMTi include a G9a / GLP HMTi.

[0026] In one embodiment, the cocktail of epigenetic modifiers comprises two or more of, three or more of, or all a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi.

[0027] In any method of this disclosure may further comprise isolating an initial population of target cells (e.g. HSPC, or CD34+cells) from a sample before contacting the population with the one or more epigenetic modifiers (or cocktail). In one embodiment, the sample is human cord blood, mobilized peripheral blood, or bone marrow. In one embodiment, the sample is a leukemia sample. In one embodiment, the initial population of CD34+cells is isolated from CML sample or AML peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs).

[0028] In one embodiment, the expanded population comprises one or more of the following primitive subsets: CD34+CD45RA CD90+; CD34+CD45RA CD90+EPCR+; and / or CD34+CD45RA CD90+EPCR+CD133+CD49f+. In one embodiment, the expanded population of target cells (e.g. HSPC, or CD34+cells) comprises more of one or more of the foregoing subsets when the target cells are contacted with and cultured in the presence of one or more epigenetic modifiers (or the cocktail) relative to when the target cells are not contacted with and / or not cultured in the presence of one or more epigenetic modifiers (or the cocktail).

[0029] In any method of this disclosure, culturing the population of target cells (e.g. HSPC, or CD34+cells) is for between about 5 to 21 days.

[0030] In another aspect of this disclosure are provided supplements (e.g. HSPC or CD34+expansion supplement) for expanding target cells (e.g. HSPC, or CD34+cells) in vitro. Supplements may comprise a cocktail of a plurality of epigenetic modifiers comprising one or more of, two or more of, three or more of, or each of the following: a first inhibitor of a histonedeacetylase (HDACi), a second HDACi, at least one inhibitor of a histone demethylase (HDMi), and at least one inhibitor of a histone methyltransferase (HMTi). In one embodiment, the cocktail may comprise a first HDACi and a second HDACi.

[0031] In one embodiment, the first HDACi and the second HDACi are class I, II, or IV HDACi. Exemplary HDACi include: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.

[0032] In one embodiment, the at least one HDMi is a LSD1 or LSD2 inhibitor.

[0033] In one embodiment, the at least one HMTi is a G9a / GLP HMTi.

[0034] In another aspect of this disclosure are provided media for culturing (e.g. expanding) a population of target cells (e.g. HSPCs, or CD34+cells). Media of this disclosure may comprise a basal medium and a supplement (comprising one or more epigenetic modifiers, as described herein). Media of this disclosure may further comprise one or more of SCF, FLT- 3L, IL-3, IL-6, and / or TPO.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0036] Figure 1 shows bar graphs quantifying expansion of human cord blood HSPC, and certain CD34+subsets thereof (A-D) and colony forming potential (E-F) after 7-day culture in the presence of CD34 Supp (StemSpan™ CD34+ Expansion Supplement) + individual or various cocktails of epigenetic modifiers, as indicated. Tested epigenetic modifiers included: a first HDACi (HDACi-1), a second HDACi (HDACi-2), a histone demethylase inhibitor (HDMi- 1), and a histone methyltransferase inhibitor (HMTi-1). Ref = a positive control comprising CD34 Supp + a published small molecule reference compound. TNC = total nucleated cells. CFU-GEMM=colony-forming unit, granulocyte, erythroid, macrophage, megakaryocyte; CFU- G / M / GM= colony-forming unit, granulocyte-macrophage; BFU-E=burst-forming unit, erythroid. Data shown are mean of two cord blood samples.

[0037] Figure 2 shows bar graphs quantifying expansion of human cord blood HSPC, and certain CD34+subsets thereof (A-D) and colony forming potential (E-F) after 7-day culture essentially as performed in Figure 1 , except the 4i cocktail of epigenetic modifiers was assessed either in the presence (4i) or absence (4i-AO) of an anti-oxidizing agent. TNC, Ref,CFU-GEMM, CFU-G / M / GM, and BFU-E are as defined in the above paragraph. Data shown are mean ± SEM of 2 cord blood samples.

[0038] Figure 3 shows bar graphs quantifying expansion of human cord blood (CB), human mobilized peripheral blood (mPB), and human bone marrow (BM) HSPC and certain CD34+subsets thereof after 7-day (A-E) and 14-day (F-J) culture, essentially as performed in Figure 1. Ref is as defined in the above paragraphs. Data shown are mean ± SEM of cell samples from 2-8 separate donors.

[0039] Figure 4 shows bar graphs quantifying expansion of CD34+cells isolated from AML samples. CD34+AML cells were cultured for 7 days in a serum-free medium comprising CD34 Supp (as described in the above paragraphs) either alone or in combination with a reference compound (Ref, as described in the paragraphs above) or a 4i cocktail of epigenetic modifiers (as described in Figure 1). Day 7 frequency (A) and cell numbers (B) of presumptive AML stem or progenitor cells are shown, including for the more primitive subsets: CD34+CD45RA’ CD90+and CD34+CD45RA 90+EPCR+. Data shown are mean ± SEM of PB samples from 5 AML patients.

[0040] Figure 5 shows bar graphs quantifying expansion of CD34+cells isolated from CML samples. CD34+CML cells were cultured for 7 days in a serum-free medium comprising CD34 Supp (as described in the above paragraphs) either alone or in combination with a reference compound (Ref, as described in the paragraphs above) or a 4i cocktail of epigenetic modifiers (as described for Figure 1). Day 7 frequency (A) and cell numbers (B) of presumptive CML stem or progenitor cells are shown, including for the more primitive subsets: CD34*CD45RA CD90+and CD34'CD45RA90'EPCRl. Data shown are mean ± SEM of PB samples from 4 CML patients.DETAILED DESCRIPTION

[0041] This disclosure relates to media compositions and / or supplements to be added into a medium, and to methods for culturing (e.g., expanding) hematopoietic stem and progenitor (HSPC) cells, specifically, CD34+HSPCs, and / or CD34+malignant cells.

[0042] Where used in this disclosure, the terms "hematopoietic stem and progenitor cell", "HSPC", “hematopoietic progenitor cell", “hematopoietic stem cell”, “hematopoietic precursor cell”, or“HPC”, used interchangeably herein, refer to a cell of the hematopoietic lineage that is capable of self-renewal and / or differentiating into a more specialized cell of the hematopoietic lineage including the myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and the lymphoid lineages (T cells, B cells, NK cells). HSPC may be obtained from bonemarrow (BM), umbilical cord blood (CB), embryonic through to adult peripheral blood (PB), thymus, peripheral lymph nodes, gastrointestinal track, tonsils, gravid uterus, liver, spleen or any other tissue having localized populations of HSPC. HSPC may also be differentiated from pluripotent stem cells such as induced pluripotent stem cells, embryonic stem cells, naive stem cells, extended stem cells, or the like. A hallmark of HSPC is the expression of the transmembrane phosphoglycoprotein CD34 on its surface, thus HSPC may be referred to as CD34+cells, though cells of the non-hematopoietic lineage, or the like, may also express CD34. A population of HSPCs may comprise a single population of CD34+cells, that is, CD34+cells of the same phenotype. Or, a population of HSPCs may comprise a plurality of CD34+cells, that is, the cells may all express CD34 but variously other surface markers. Human HSPC subsets may be further defined by expression of CD45, and may still further be defined by combinations of markers such as CD38, CD43, CD45RO, CD45RA, CD10, CD49f, CD59, CD90, CD109, CD117, CD133, CD166, HLA-DR, EPCR (CD201), and integrin-alpha3. HSPCs may lack expression or have only low expression of markers such as Glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20 and CD56; such markers may be characteristic of more mature blood cells. Examples of more common markers of HSPCs (e.g. CD34+HSPC) may include CD45RA, which is absent or only weakly expressed on primitive cells, and CD34, CD90 (Thy-1), CD201 (endothelial protein C receptor, EPCR), CD133, and CD49f, which are expressed at higher levels on primitive cells than on differentiated cells.

[0043] Where used in this disclosure, the terms “epigenetic modifier” or “epigenetic regulator”, used interchangeably herein, refer to an agent or a factor such as a protein, a small molecule, interfering RNA, messenger RNA, or other natural or synthetic compound (or catalytic domain thereof having enzymatic activity) that modify or cooperate with other factors to modify epigenetic marks of DNA and / or histones. Epigenetic modifiers may be classified depending on their mechanism of action, such as DNA methyltransferases or inhibitors thereof, histone acetyltransferases or inhibitors thereof, histone methyltransferases or inhibitors thereof, histone deacetylases or inhibitors thereof, or histone demethylases or inhibitors thereof.

[0044] Where used in this disclosure, the term “expanded population” refers to an output population of cells wherein a quantity of output cells (such as after a period of culture in the presence of one or more epigenetic modifiers) is greater than in a control population (such as one not cultured in the presence of the one or more epigenetic modifiers). In one embodiment, an expanded population of cells may specifically refer to a population of HSPC or CD34+cells expanded in the presence of one or more epigenetic modifiers. In one embodiment, an expanded population of HSPC cells or CD34+cells may more specifically refer to a progenitor or primitive population expanded in the presence of one or more epigenetic modifiers. In one embodiment, an expanded population may mean an increase of at least 10%, or at least about20%, 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% or up to and including a 100% increase, or any increase between 10-100% as compared to an input or control. In one embodiment, an expanded population may mean at least about a 2-fold, at least about a 3-fold, at least about a 5-fold, at least about a 10-fold increase, at least about a 15-fold increase, at least about a 20-fold, at least about a 25-fold increase, at least about a 30-fold increase, at least about a 35-fold increase, at least about a 40-fold increase, at least about a 45-fold increase, at least about a 50-fold increase, or any increase between 2-fold and 50- fold, or greater, as compared to an input or control. In one embodiment, the expanded population of the target cells (e.g. HSPC, or CD34+cells) may be used for gene editing applications. In one embodiment, the expanded population of the target cells (e.g. HSPC, or CD34+cells) whether gene edited or not, may be used to engraft into a subject, such as a human subject or into an animal model such as a mouse.Media and Supplements

[0045] In one aspect of this disclosure are provided supplements (which may be added to culture media) for culturing or generating or expanding a population of target cells, such as HSPC (e.g. CD34+cells) and / or progenitors or primitive subsets thereof. A composition / formulation of a supplement of this disclosure is not limited in any particular way, provided that it is suitable for contacting one or more HSPC, or a population thereof, and facilitating their culture.

[0046] Supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules. More specifically, supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that modulate epigenetic marks (e.g. epigenetic modifiers). While supplements of this disclosure may be entirely small molecule-, compound-, and / or macromolecule-based, they may comprise or be combined with one or more macromolecules (e.g. lipids, proteins, carbohydrates) that does not have a primary function as an epigenetic modifier. Thus, supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that are epigenetic modifiers, and one or more macromolecules that does not have a primary function as an epigenetic modifier, or supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that are epigenetic modifiers and be combined with one or more macromolecules that does not have a primary function as an epigenetic modifier (as may be comprised in a separate supplement or culture medium).

[0047] When combined with a basal medium, and in some cases one or more other supplements, supplements of this disclosure may support the expansion of target cells, such as HSPC cells (e.g., CD34+HSPCs and / or CD34+malignant cells).

[0048] As indicated above, supplements of this disclosure may comprise one or more epigenetic modifiers. In one embodiment, supplements of this disclosure comprise a plurality of epigenetic modifiers (e.g. a cocktail of epigenetic modifiers). Further, supplements of this disclosure may comprise either purely small molecule epigenetic modifiers, purely non-small molecule epigenetic modifiers (e.g. compounds), purely macromolecule modifiers (e.g. fatty acids), or any combination of the foregoing.

[0049] Supplements (and as applicable a cocktail) of this disclosure may comprise one or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).

[0050] Supplements (and as applicable a cocktail) of this disclosure may comprise two or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).

[0051] Supplements (and as applicable a cocktail) of this disclosure may comprise each of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).

[0052] Supplements (and as applicable a cocktail) of this disclosure may comprise one or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0053] Supplements (and as applicable a cocktail) of this disclosure may comprise two or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0054] Supplements (and as applicable a cocktail) of this disclosure may comprise three or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0055] Supplements (and as applicable a cocktail) of this disclosure may comprise each of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0056] In some embodiments, a supplement (and as applicable a cocktail) comprises at least a first HDACi (e.g. valproic acid). In some embodiments, a supplement (and as applicable a cocktail) comprises at least a first HDACi (e.g. valproic acid) and / or at least one HMTi.

[0057] HDAC inhibitors are known and widely available, and are thus not particularly limited in supplements of this disclosure. In one embodiment, one HDACi is comprised in a supplement of this disclosure. In one embodiment, two HDACi are comprised in a supplement of this disclosure. In one embodiment, more than two HDACi are comprised in a supplement of this disclosure.

[0058] The one or more HDACi comprised in a supplement may be class I, II, or IV HDACi. If only one HDACi is comprised in a supplement, then such HDACi may belong to a single class of HDACi or to multiple HDACi classes. If more than one (e.g. two or more) HDACi are comprised in a supplement, then each HDACi may belong to the same or different class(es). In one embodiment, a supplement of this disclosure comprises at least two class I HDACi.

[0059] Examples of HDACi that may be comprised in a supplement are Valproic acid (HDAC class I & II inhibitor), Trichostatin A (TSA) (HDAC class I, II & IV inhibitors), Romidepsin (HDAC class I inhibitor), Entinostat (HDAC class I inhibitor), Tacedinaline (HDAC class I inhibitor), Panobinostat (HDAC class I, II & IV inhibitors), and LMK235 (HDAC class II inhibitors). In one embodiment, the supplement comprises Valproic acid. In one embodiment, the supplement comprises Romidepsin. In one embodiment, the supplement comprises one or both of Valproic acid and Romidepsin.

[0060] A concentration of the one or more HDACi in the supplement is not particularly limited, except that the concentration should not be lethal or toxic to cells. If the HDACi is a small molecule, then its concentration may be in the nanomolar to micromolar range. For example, the concentration may range between about 0.1 nM to 100 uM, or between about 0.5 nM to 50 uM, or between about 1 nM to 10 uM, or between about 5 nM to 1 uM, or between about 10 nM to 0.5 uM, or between about 25 nM and 250 uM. If the HDACi is not a small molecule, then its concentration may be in the micromolar to millimolar range. For example, the concentration may range between about 1 uM to 1000 mM, or between about 10 uM to 500 mM, or between about 100 uM to 250 mM, or between about 250 uM to 200 mM, or between about 500 uM to 150 mM, or between about 1 mM and 100 mM.

[0061] HDM inhibitors (HDMi) are known and widely available, and are thus not particularly limited in supplements of this disclosure. In one embodiment, one HDMi is comprised in a supplement of this disclosure. In one embodiment, two HDMi are comprised in a supplement of this disclosure. In one embodiment, more than two HDMi are comprised in a supplement of this disclosure.

[0062] The one or more HDMi comprised in a supplement may be an inhibitor of lysine specific demethylases, such as LSD1 and LSD2 inhibitors. In one embodiment, the HDMi is a LSD1 inhibitor. Examples of LSD1 inhibitors that may be comprised in a supplement are 2-PCPA (tranylcypromine), RN-1 (LSD1 inhibitor IV), SP2509, ORY-100, and GSK-LSD1. In one embodiment, the LSD1 inhibitor is 2-PCPA (tranylcypromine). In one embodiment, the LSD1 inhibitor is tranylcypromine hydrochloride or a functional derivative thereof.

[0063] A concentration of the one or more HDMi in the supplement is not particularly limited, except that the concentration should not be lethal or toxic to cells. If the HDMi is a small molecule, then its concentration may be in the nanomolar to micromolar range. For example, the concentration may range between about 1 nM to 1 mM, or between about 25 nM to 750 uM, or between about 100 nM to 500 uM, or between about 250 nM to 400 uM, or between about 500 nM to 300 uM, or between about 1 uM and 250 uM. If the HDMi is not a small molecule, then its concentration may be in the micromolar to millimolar range. For example, the concentration may range between about 1 uM to 1000 mM, or between about 10 uM to 500 mM, or between about 100 uM to 250 mM, or between about 250 uM to 200 mM, or between about 500 uM to 150 mM, or between about 1 mM and 100 mM.

[0064] HMT inhibitors (HMTi) are known and widely available, and are thus not particularly limited in supplements of this disclosure. In one embodiment, one HMTi is comprised in a supplement of this disclosure. In one embodiment, two HMTi are comprised in a supplement of this disclosure. In one embodiment, more than two HMTi are comprised in a supplement of this disclosure.

[0065] The one or more HMTi comprised in a supplement may be histone methyltransferase G9a (also known as EHMT2 or KMTIC) and / or G9a-like protein (GLP, also known as EHMT1). In one embodiment, the at least one HMTi comprised in a supplement is a G9a / GLP HMTi. In one embodiment, the G9a / GLP HMTi is UNC0638.

[0066] A concentration of the one or more HMTi in the supplement is not particularly limited, except that the concentration should not be lethal or toxic to cells. If the HMTi is a small molecule, then its concentration may be in the nanomolar to micromolar range. For example, the concentration may range between about 1 nM to 1 mM, or between about 25 nM to 750 uM, or between about 100 nM to 500 uM, or between about 250 nM to 400 uM, or betweenabout 500 nM to 300 uM, or between about 1 uM and 250 uM. If the HMTi is not a small molecule, then its concentration may be in the micromolar to millimolar range. For example, the concentration may range between about 1 uM to 1000 mM, or between about 10 uM to 500 mM, or between about 100 uM to 250 mM, or between about 250 uM to 200 mM, or between about 500 uM to 150 mM, or between about 1 mM and 100 mM.

[0067] In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement of this disclosure comprises at least one HDACi, and at least one of an HDMi and HMTi. In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement of this disclosure comprises at least one HDACi, at least one HDMi, and at least one HMTi. In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement of this disclosure comprises at least two HDACi, and at least one of an HDMi and HMTi. In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement of this disclosure comprises at least two HDACi, at least one HDMi, and at least one HMTi.

[0068] In one embodiment, a supplement (and / or media to which the supplement is added) further comprises an antioxidant, such as ascorbic acid, retinoic acid, glutathione, or any derivative, analog, or functional equivalent thereof.

[0069] Supplements of this disclosure may be concentrated. In one embodiment, the supplement is at about a 2X concentration, about a 5X concentration, about a 10X concentration, about a 15X concentration, about a 20X concentration, about a 25X concentration, about a 50X concentration, about a 100X concentration, about a 200X concentration, or greater.

[0070] Supplements of this disclosure may be added to cell culture media. In formulating complete media, a supplement comprising one or more epigenetic modifier may be further combined with one or more cytokines or growth factors.

[0071] Thus, in another aspect of this disclosure are provided media for culturing / expanding target cells in vitro, such as HSPCs (e.g., CD34+cells). HSPCs expanded in media of this disclosure (and supplemented as described herein) may retain the ability to differentiate into a multitude of downstream cell types in the hematopoietic lineage. Media of this disclosure may also be used to expand CD34+malignant cells isolated from leukemias (e.g. AML and / or CML PBMCs) or other myelodysplastic syndromes.

[0072] Media of this disclosure may contain serum or may be serum-free. In one embodiment, media of this disclosure are serum-free. If serum-free, it may be necessary to include in such media a serum replacement supplement, such as BIT 9500 Serum Substitute (STEMCELL Technologies), or other commercially available serum replacement solutions. Alternatively,components ordinarily present in serum that are needed for culturing or differentiating any cells of this disclosure may be individually added at an acceptable concentration to the media.

[0073] Culture media of this disclosure will comprise a basal medium. The basal medium may be any medium that, when appropriately supplemented, supports the expansion of target cells, such as mammalian HSPC (e.g. CD34+cells, or subsets thereof). In one embodiment, the basal medium may be any medium capable of supporting the expansion of mammalian CD34+malignant cells. In one embodiment, the basal medium may be any basal medium which is supportive of culturing cells of the hematopoietic lineage. By way of non-limiting example, the basal medium may be StemSpan™ SFEM (STEMCELL Technologies), StemSpan™ SFEM II (STEMCELL Technologies), StemSpan™-XF (STEMCELL Technologies), StemSpan™ AOF (STEMCELL Technologies), or any other commercially available basal medium fit for the purpose.

[0074] Media of this disclosure (as may be combined with a supplement as described above) may comprise one or more epigenetic modifiers, or more than one epigenetic modifier. The description above with regard to the nature, combination, and concentration of epigenetic modifiers is entirely incorporated by reference into the context of cell culture media of this disclosure.

[0075] Media of this disclosure may further comprise one or more additional cytokines or growth factors, or small molecules, or other proteins that provide critical signals for HSPC expansion. By way of non-limiting example, critical signals required for HSPC expansion may include one or more of: interleukin(s) (e.g. IL-1 , IL-2, IL-3, IL-6), VEGF, G-CSF, GM-CSF, SCF, FLT3-L, TPO, and EPO.

[0076] Supplements and media of this disclosure may support fold expansion of target cells (e.g. HSPCs) on the order of at least 10%, or 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% or up to and including a 100% increase, or any increase between 10-100%, as compared to a control condition (lacking the supplement of one or more epigenetic modifier, as described above and fully incorporated herein by reference). Supplements and media of this disclosure may support fold expansion of target cells (e.g. HSPC) on the order of at least about a 2-fold, or at least about a 3-fold, or at least about a 5- fold, or at least about a 10-fold increase, or at least about a 15-fold increase, or at least about a 20-fold, or at least about a 25-fold increase, or at least about a 30-fold increase, or at least about a 35-fold increase, or at least about a 40-fold increase, or at least about a 45-fold increase, or at least about a 50-fold increase or any increase between 2-fold and 50-fold, orgreater, as compared to a control condition (lacking the supplement of one or more epigenetic modifier, as described above and fully incorporated herein by reference).

[0077] Enhanced expansion of HSPCs in the presence of supplements of one or more epigenetic modifier, and media of this disclosure comprising such supplements and one or more cytokines / growth factors beneficial to HSPC expansion, may more specifically relate to expansion of more primitive HSPC subsets (e.g. CD34+CD45RACD90+, CD34+CD45RA_CD90+EPCR+and CD34+CD45RACD90+EPCR+CD133+CD49f+), relative to a commercially available cytokine supplement alone (e.g. StemSpan™ CD34 Expansion Supplement, STEMCELL Technologies) or in combination with a reference published small molecule (e.g. a pyrimidoindole compound).

[0078] In one embodiment, higher expansion of primitive HSPC subsets (e.g. CD34+CD45RA_CD90+, CD34+CD45RA CD90+EPCR+, and CD34+CD45RACD90+EPCR+CD133+CD49r) are supported by (i) a supplement comprising each of the first and the second HDACi, the at least one HDMi, and the at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing, (ii) a supplement comprising the first HDACi, or a functional equivalent thereof, and two of the second HDACi, the at least one HDMi, and the at least one HMTi, as compared to a cocktail comprising two of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic, or (iii) a supplement comprising the first HDACi, or a functional equivalent thereof, and one of the second HDACi, the at least one HDMi, and the at least one HMTi, as compared to a cocktail comprising one of the first and the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic acid.

[0079] In some embodiments, higher expansion of primitive HSPC subsets (e.g. CD34+CD45RA CD90+, CD34+CD45RA CD90+EPCR+, and CD34+CD45RA' CD90+EPCR+CD133+CD49f) with an additional phenotype are supported by a supplement of this disclosure. In one embodiment, higher expansion of primitive HSPC subsets with in vitro multi-lineage differentiation capacity (e.g., on the colony forming potential) are supported by a supplement comprising the first HDACi and the at least one HMTi, wherein the first HDACi is valproic acid.Methods

[0080] In another aspect of this disclosure are provided methods for culturing and / or expanding target cells, such as HSPC (e.g. CD34+cells). Methods of this disclosure may encompass those steps for culturing and / or expanding target cells, such as HSPCs (e.g. CD34+cells), in a serum-free culture environment. The methods disclosed herein for culturingand / or expanding cells, such as CD34+cells - whether normal CD34+or malignant CD34+cells - are preferably in vitro methods.

[0081] The target cells are preferably HSPC, which may express at least CD34. HSPC are preferably mammalian, and more preferably human. HSPC may be directly isolated from a primary sample, such as whole blood, cord blood, (mobilized) peripheral blood, bone marrow, thymus, uterus, liver, gut or secondary lymphoid tissues. In one embodiment, the CD34+are isolated from human cord blood. In one embodiment, the CD34+are isolated from (mobilized) peripheral blood. In one embodiment, the CD34+are isolated from human bone marrow.

[0082] Target cells used in the methods of this disclosure may be CD34+cells obtained, enriched, and / or isolated from a cancer, malignant, diseased, or infected sample, such as peripheral blood mononuclear cells (PBMCs). In one embodiment, the CD34+cancer or malignant cells are obtained, enriched, and / or isolated from a chronic myeloid leukemia (CML) sample (e.g. PBMCs). In one embodiment, the CD34+cancer or malignant cells are obtained, enriched, and / or isolated from an acute myeloid leukemia (AML) sample (e.g. PBMCs).

[0083] Methods for culturing (e.g. expanding) target cells (e.g. HSPC cells) may comprise contacting one or more cells (e.g. HSPC) with one or more epigenetic modifiers in a culture medium. More specifically, methods of the disclosure may comprise contacting one or more cells (e.g. HSPC) with a cocktail of a plurality of epigenetic modifiers in a culture medium. The nature and other details, such as combinations and concentrations, of the epigenetic modifier(s) in a culture medium of this disclosure (as used in the instant methods) may be as described hereinabove.

[0084] In addition, the description above regarding culture media may also apply to the disclosed methods. Hematopoietic cells may require one or more cytokine and / or growth factors for efficient expansion; thus, culture media used in methods of this disclosure may be supplemented with one or more of SCF, FLT-3L, and / or TPO.

[0085] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise one or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).

[0086] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise two or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).

[0087] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise each of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).

[0088] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise one or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0089] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise two or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0090] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise three or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0091] Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise each of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).

[0092] In some embodiments, a supplement (and as applicable a cocktail), as may be comprised in a culture media, for use in methods of this disclosure comprises at least a first HDACi (e.g. valproic acid). In some embodiments, a supplement (and as applicable a cocktail) comprises at least a first HDACi (e.g. valproic acid) and / or at least one HMTi.

[0093] In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least one HDACi, and at least one of an HDMi and HMTi. In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least one HDACi, at least one HDMi, and at least one HMTi. In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement (as may be comprised inculture media) used in methods of this disclosure comprises at least two HDACi, and at least one of an HDMi and HMTi. In some embodiments, a HSPC (e.g. CD34+cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least two HDACi, at least one HDMi, and at least one HMTi.

[0094] The one or more target cells (e.g. HSPC, or CD34+cells) may be comprised in an initial population of cells, and the initial population of cells may either be an isolated or enriched population of target cells, or a bulk population comprising target cells and off-target cells. Thus, methods of this disclosure may comprise isolating or enriching the one or more target cells (e.g. HSPC, or CD34+cells) from a sample before contacting the population with the cocktail. Approaches to isolating or enriching target cells, as may be comprised in an initial population comprising target cells (e.g. HSPC, or CD34+cells), are known and commercially available, such as by FACS or by immunomagnetic cell separation, as commercialized under the EasySep™ brand (STEMCELL Technologies).

[0095] Following contact of target cells with a culture medium comprising one or more epigenetic modifiers, the methods may further comprise culturing the one or more target cells (e.g. HSPC, or CD34+cells) in the presence of the one or more epigenetic modifiers for a time sufficient to produce an expanded population of target cells.

[0096] Culturing duration is not particularly limited other than by build-up of metabolites, pH, etc., by nutrient availabilities, and over-crowding, all of which may be rectified by medium exchanges and / or scaling up vessel size and media volume. Notwithstanding, culturing may be for between about 1 and 35 days, between about 2 and 32 days, between about 3 and 28 days, between about 5 and 21 days, or between about 7 days to about 14 days. In some embodiments, culturing is for 7 days ± 1 day. In some embodiments, culturing is for 10 days ± 1 day. In some embodiments, culturing is for 14 days ± 1 day.

[0097] Yielding / producing an expanded population of target cells (e.g. HSPC, or CD34+cells) after culturing for a sufficient time, may result in more target cells (e.g. HSPC, or CD34+cells) compared to when the one or more target cells (as may comprised in an initial population) are not cultured in the presence of the one or more epigenetic modifiers (e.g. the cocktail, or the supplement) for the time sufficient to produce the expanded population of target cells (e.g. HSPCs or CD34+cells).

[0098] Increased expansion of target cells by practicing the methods of this disclosure may also be quantified relative to expansion of target cells (e.g. HSPC, or CD34+cells) when contacted with and / or cultured in the presence of a reference standard small molecule in the culture medium. In the field of hematopoietic cell expansion, particularly HSPC and / or CD34+cells, various such reference molecules / compounds are known including engineered cytokines (e.g. IL-6), pyrimidoindole compounds, or aryl hydrocarbon antagonists.

[0099] Increased expansion of target cells by practicing the methods of this disclosure may also be quantified relative to expansion of target cells (e.g. HSPC, or CD34+cells) when contacted with and / or cultured in the presence of fewer than the more than one epigenetic modifier comprised in a cocktail or in a culture medium. For example, higher expansion of target cells (e.g. HSPC, or CD34+cells, or primitive subsets thereof: e.g. CD34+CD45RA_CD90+; CD34+CD45RA CD90+EPCR+; or CD34+CD45RA CD90+EPCR+CD133+CD49f+) is supported (i) in the presence of four epigenetic modifiers (in an appropriate medium) relative to the presence of three, two or one epigenetic modifiers (in an appropriate medium), (ii) in the presence of three epigenetic modifiers (in an appropriate medium) relative to the presence of two or one epigenetic modifiers (in an appropriate medium), or (iii) in the presence of two epigenetic modifiers (in an appropriate medium) relative to the presence of one epigenetic modifiers (in an appropriate medium).

[0100] Thus, methods of this disclosure may comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34+cells) when one or more target cells is contacted with and / or cultured in the presence of a cocktail comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing.

[0101] Methods of this disclosure may comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34+cells) when one or more target cells is contacted with and cultured in the presence of a cocktail comprising a first HDACi, or a functional equivalent thereof, and two of a second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising two of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMT, wherein the first HDACi is valproic acid

[0102] Methods of this disclosure may comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34+cells) when one or more target cells is contacted with and / or cultured in the presence of a cocktail comprising a first HDACi, or a functional equivalent thereof, and one of either a second HDACi, at least one HDMi, or at least one HMTi, as compared to a cocktail comprising one of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic acid.

[0103] Yielding / producing an expanded population of target cells (e.g. HSPC, or CD34+cells) after culturing for a sufficient time, may result in between 2-fold to 100-fold more target cells compared to when the one or more target cells (e.g. HSPC, or CD34+cells) are not cultured in the presence of the one or more epigenetic modifies (e.g. the cocktail, or the supplement)for the time sufficient to produce the expanded population of target cells. If starting from an initial population of target cells (e.g. HSPC, or CD34+cells), culturing for a sufficient time may yield / produce between 2-fold to more than 100-fold more target cells in an expanded population compared to when the target cells (e.g. HSPC, or CD34+cells) of the initial population are not cultured in the presence of the one or more epigenetic modifies (e.g. the cocktail, or the supplement) for the time sufficient to produce the expanded population of target cells.

[0104] In some embodiments, an expanded population of target cells may result in about 2- fold more target cells, about 3-fold more target cells, about 4-fold more target cells, about 5- fold more target cells, about 10-fold more target cells, about 15-fold more target cells, about 20-fold more target cells, about 25-fold-more target cells, about 30-fold more target cells, about 40-fold more target cells, about 50-fold more target cells, about 60-fold-more target cells, about 70-fold more target cells, about 80-fold more target cells, about 90-fold more target cells, about 100-fold-more target cells, or more.

[0105] Methods of this disclosure may yield expanded populations of CD34+cells which may be further characterized as distinct subsets of CD34-expressing cells. For example, the one or more target cells (e.g. CD34+cells, or HSPC) may comprise a CD34+CD45RACD90+phenotype, a CD34+CD45RA CD90+EPCR+phenotype, and / or a CD34+CD45RA_CD90+EPCR+CD133+CD49f phenotype. Such phenotypes may correspond with more primitive subsets of CD34-expressing cells and / or HSPC. Thus, expanded populations (following contact with and culture in the presence of one or more epigenetic modifiers) may comprise expanded populations of target cells comprising a CD34+CD45RA CD90+phenotype, a CD34+CD45RA CD90+EPCR+phenotype, and / or a CD34+CD45RA_CD90+EPCR+CD133+CD49f+phenotype.

[0106] In some embodiments, an expanded population of target cells (e.g. HSPC, or CD34+cells) may comprise between about 2 to 50-fold, or between about 3 to 40-fold, or between about 4 to 30-fold, or between 5 to 25-fold more of a primitive subset of HSPC or CD34+cells (e.g CD34+CD45RA CD90+) after culturing one or more such target cells (as may be comprised in an initial population of cells, such as CD34+cells) in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium. More specifically, an expanded population of target cells (e.g. HSPC, or CD34+cells) may comprise between about 2 to 50-fold, or between about 3 to 40-fold, or between about 4 to 30-fold, or between 5 to 25-fold more of a primitive subset of HSPC or CD34+cells (e.g. CD34+CD45RA CD90+) per input CD34+cells following 7 days or 14 days of expansion culturein the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.

[0107] In some embodiments, an expanded population of target cells (e.g. HSPC, or CD34+cells) may comprise between about 2 to 25-fold, or between about 3 to 20-fold, or between about 4 to 15-fold, or between 5 to 10-fold more of a primitive subset of HSPC or CD34+cells (e.g CD34+CD45RA CD90+EPCR+) after culturing one or more such target cells (as may be comprised in an initial population of cells, such as CD34+cells) in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium. More specifically, an expanded population of target cells (e.g. HSPC, or CD34+cells) may comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more of a primitive subset of HSPC or CD34+cells (e.g. CD34+CD45RA CD90+EPCR+) per input CD34+cells following 7 days or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.

[0108] In some embodiments, an expanded population of target cells (e.g. HSPC, or CD34+cells) may comprise between about 2 to 25-fold, or between about 3 to 20-fold, or between about 4 to 15-fold, or between 5 to 10-fold more of a primitive subset of HSPC or CD34+cells (e.g. CD34+CD45RA CD90+EPCR+CD133+CD49f) after culturing one or more such target cells (as may be comprised in an initial population of cells, such as CD34+cells) in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium. More specifically, an expanded population of target cells (e.g. HSPC, or CD34+cells) may comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6- fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more of a primitive subset of HSPC or CD34+cells (e.g. CD34+CD45RA CD90+EPCR+CD133+CD49f+) per input CD34+cells following 7 days or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.

[0109] Thus, an expanded population of target cells (e.g. HSPCs, or CD34+cells) may comprise more primitive cells having a phenotype selected from one or more of CD34+CD45RA CD90+, CD34+CD45RACD90+EPCR+, and / or CD34+CD45RA' CD90+EPCR+CD133+CD49f+compared to when the one or more target cells are not culturedin the presence of the one or more epigenetic modifiers (e.g. the cocktail, or supplement) for a time sufficient to produce the expanded population of cells. Accordingly, methods (and supplements and supplemented media) of this disclosure may support the expansion of HSPC or CD34+cells. More particularly, methods (and supplements and supplemented media) of this disclosure may preferentially support the expansion of primitive subsets of HSPC or CD34+cells, such as CD34+CD45RA CD90+cells, CD34+CD45RACD90+EPCR+cells, and CD34+CD45RA CD90+EPCR+CD133+CD49f+cells.

[0110] In some embodiments, the presence of at least one HDACi (e.g. valproic acid) helps promote expansion of target cells and to yield target cells capable of downstream differentiation, such as may be measured by CFU assay. In the context of downstream differentiation potential, such as may be measured by CFU, a cocktail comprising a first HDACi, a second HDACi, and at least one HMTi (as described herein) may outperform a cocktail comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi at least in terms of CFU-GEMM, and / or CFU-G / M / GM, and / or BFU-E output. Further, in the context of downstream differentiation potential, such as may be measured by CFU, a cocktail comprising a first HDACi, a second HDACi, and at least one HMTi (as described herein) may outperform any other 3-way combination selected from a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, at least in terms of CFU-GEMM, and / or CFU-G / M / GM, and / or BFU-E output. Further, in the context of downstream differentiation potential, such as may be measured by CFU, a cocktail comprising a first HDACi, a second HDACi, and at least one HDMi (as described herein) may underperform a cocktail comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, at least in terms of CFU-GEMM, and / or CFU-G / M / GM, and / or BFU-E output. Further, in the context of downstream differentiation potential, such as may be measured by CFU, cocktails comprising a first HDACi, a second HDACi, and at least one HDMi (as described herein) may underperform any other 3-way combination selected from a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, at least in terms of CFU-GEMM, and / or CFU-G / M / GM, and / or BFU-E output.

[0111] By practicing the methods disclosed herein, it may be possible to obtain large quantities of in vitro expanded cells (e.g. HSPC, and / or CD34+cells or progenitor and / or primitive subsets thereof). Such expanded cells may be used in any relevant downstream applications, including in gene editing workflows, clinically or therapeutically, to study hematopoietic development in normal or diseased contexts, or downstream differentiation to downstream hematopoietic lineages (e.g. myeloid, erythroid, and / or lymphoid), etc.

[0112] Indeed, a functional HSPC should be capable of reconstituting the hematopoietic lineages of a sub-lethally irradiated recipient, and target cells obtained by practicing the methods disclosed herein have been shown to engraft in a subject after introduction and that they produce myeloid cells, lymphoid cells, and erythroid cells. Thus, methods of this disclosure may further comprise introducing expanded cells into a subject, wherein i) a similar or a higher level of lymphoid (CD45+CD19+B cells) engraftment occurs relative to uncultured cells in the engrafted subject, and / or ii) a similar or a higher level of myeloid (CD45+CD33+) engraftment occurs relative to uncultured cells, and / or iii) a similar or a higher level of lymphoid and / or myeloid engraftment relative to uncultured cells.

[0113] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: Procurement, preparation, and culture of cells

[0114] Cord blood units were procured from commercial suppliers. Human bone marrow samples and mobilized peripheral blood was procured from STEMCELL Technologies. AML and CML samples were procured from Discovery Life Sciences. CD34+cells were enriched from the foregoing samples using the EasySep™ Human Cord Blood CD34 Positive Selection Kit II (STEMCELL Technologies), and then either frozen in serum with 10% DMSO or used fresh.

[0115] For culture, ~4000 CD34+cells were plated per well of a 24-well plate for 7 days in serum-free StemSpan™ SFEM II (STEMCELL Technologies) supplemented with cytokines contained in StemSpan™ CD34 Expansion Supplement (STEMCELL Technologies). In addition, cells were cultured either in the presence of a reference, published small molecule pyrimidoindole compound (Ref) available commercially from STEMCELL Technologies, or one or more epigenetic modifiers.Example 2: Flow cytometry and determination of cell count and yield

[0116] During or following expansion culture as described in Example 1 , aliquots of the cultures can be harvested to assess phenotype, cell number, and frequencies, such as by flow cytometry.

[0117] Briefly, a cell sample / aliquot was harvested by centrifugation and washed. The cells were stained with fluorophore-conjugated antibodies against CD34, CD90, CD45RA, and EPCR and analyzed on the CytoFLEX S™ flow cytometer (Beckman-Coulter). Dead cells were excluded by side and forward scatter profile and 7-AAD staining.

[0118] Total viable cell counts were obtained using a Cytek® Guava® easyCyte™ flow cytometer. To calculate the yield of particular cells per input cell, total viable counts were multiplied by the % frequency of the given cell type. For example, to calculate the yield of CD34+per input CD34+cell, the viable cell count is first multiplied by the % CD34+obtained by flow cytometry. This number is then divided by the number of input cells (in the case input CD34+cells) to obtain the final value. Input CD34+cell numbers were obtained by multiplying total cells cultured in one well by frequency of CD34+cells after cell separation.Example 3: Expanding HSPCs in the presence of one or more epigenetic modifiers

[0119] Human cord blood HSPC were isolated and cultured essentially as described in Example 1 (e.g. StemSpan™ SFEM II + StemSpan™ CD34 Expansion Supplement). In certain conditions, the cells were also cultured in the presence of either a reference small molecule (Ref) or various epigenetic modifiers, whether individually (1 ; 2; 3; or 4) or in various 2-way (1-3; 1-4; 1-2; 2-3; or 2-4), 3-way (1-2-3; 2-3-4; 1-2-4; or 1-3-4), or 4-way (4i) cocktails (Figure 1).

[0120] At day 7, expansion of total nucleated cell (TNC), progenitor CD34+and primitive subsets CD34briCD45RA CD90+and CD34briCD45RA CD90+EPCR+per input CD34+were determined as described in Example 2. Each condition tested promoted the expansion of TNC and progenitor CD34+to a level comparable to the reference pyrimidoindole compound or the negative control. However, only certain 2-way (e.g. 1-3; 1-4; 1-2), 3-way (e.g. 1-2-3; 1-2-4; or 1-3-4), and the 4-way (4i) cocktails also promoted expansion of both primitive HPSC subsets (CD34briCD45RA CD90+and CD34bri45RA CD90+EPCR+) assessed, which were higher than the levels of the reference compound and control (Figure 1).

[0121] In vitro multi-lineage differentiation capacity of such expanded HSPC was measured by colony-forming unit (CFU) assay, as briefly described herein. After 7 days of expansion, 400 cells / well were cultured in MethoCult™ H4435 Enriched (STEMCELL Technologies) in a 6-well SmartDish™ plate (STEMCELL Technologies) and incubated for 14 days in a humidified incubator at 37° and 5% CO2 Day 14 colonies were counted with STEMvision™ (STEMCELL Technologies).

[0122] The results in Figure 1C and D show that expansion of primitive subsets (CD34+CD45RA CD90+and CD34+CD45RA CD90+EPCR+) of HSPC is reduced in cocktails lacking HDACi-1 (valproic acid) compared to culture conditions including HDACi-1 , suggesting that HDACi-1 is critical for expansion. However, HSPC expanded in the absence of HDACi-1 did not appear to possess a markedly impacted ability to form colonies in a CFU assay (Figure 1 E). In contrast, HSPC expanded in the absence of HMTi-1 (e.g. UNC-0638) exhibited a marked reduction in CFU output. Overall, it appears that presence of HDACi-1 and HMTi-1 ina cocktail of epigenetic modifiers helps to expand primitive subsets of HSPC, while also yielding cells having acceptable colony forming potential.Example 4: Expanding HSPC in a supplement comprising epigenetic modifiers and an antioxidant

[0123] Human cord blood HSPCs were isolated and cultured in a cytokine supplement +4i, as described in Example 1. In this experiment, impacts of the presence or absence of an antioxidant among the cocktail of epigenetic modifiers was assessed on the expansion of TNCs, HSPC and primitive HSPC subsets (CD34+CD45RA CD90+and CD34+CD45RA' CD90+EPCR+) (Figures 2A-D), and in a CFU assay (Figure 2E-F).

[0124] Figures 2E-F show that an absence of an antioxidant in the 4i cocktail (4i-AO) resulted in lower number of colonies compared to the reference compound and the condition that included the antioxidant in the 4i cocktail (4i). Thus, while presence of an anti-oxidant in the 4i cocktail of epigenetic modifiers does not appear to influence the output number of primitive HSPCs, it does appear to have a marked impact on colony forming potential.Example 5: Expanding HSPC from different sources in the presence of cocktails of epigenetic modifiers

[0125] Human cord blood, peripheral blood, and bone marrow HSPC were isolated and cultured for 7 or 14 days, essentially as described in Example 1 , except different basal media formulations were used together with a cytokine supplement and cocktail 4i: StemSpan™ SFEM (STEMCELL Technologies), StemSpan™ SFEM II (STEMCELL Technologies), StemSpan™-XF (STEMCELL Technologies) or StemSpan™ AOF (STEMCELL Technologies).

[0126] For all tested sources of HSPC, the 4i condition resulted in comparable or better expansion of day 7 (Figure 3A) and day 14 (Figure 3B) expanded HSPC and primitive HSPC subsets (e.g. CD34+CD45RA CD90+, CD34+CD45RA CD90+EPCR+, and CD34+CD45RA' CD90+EPCR+CD133+CD49f) in SFEM relative to levels using the reference pyrimidoindole compound. The same observations were made in respect of the other basal media tested (data not shown).Example 6: Expanding presumptive cancer stem or progenitor cells from leukemia samples

[0127] CD34+cells were isolated from AML and CML samples and cultured essentially as described in Example 1 . After 7 days, the cultured cells were stained with fluorescently labeled antibodies and analyzed by flow cytometry as described in Example 2.

[0128] The frequency (A) and cell numbers (B) of progenitor CD34+and primitive progenitor subsets (CD34+CD45RACD90+and CD34+CD45RA CD90+EPCR+) expanded from AMLsamples are shown in Figure 4. Higher frequencies and yields of CD34+CD45RACD90+and CD34+CD45RA CD90+EPCR+cells were measured at day 7 compared to a cytokine cocktail (CD34 supp) or the reference pyrimidoindole compound.

[0129] The frequency (A) and cell numbers (B) of progenitor CD34+and primitive progenitor subsets (CD34+CD45RA CD90+and CD34+CD45RA CD90+EPCR+) expanded from CML samples are shown in Figure 5. Higher frequencies and yields of CD34+CD45RA CD90+and CD34+CD45RA CD90+EPCR+cells were measured at day 7 compared to a cytokine cocktail (CD34 supp) or the reference pyrimidoindole compound.Example 7 Engrafting expanded HSPC into mice

[0130] Human cord blood HSPCs were isolated and cultured for 7 days in the presence of a cytokine supplement + 4i, essentially as described in Example 1. After 7 days of expansion, progeny of 2500 initial CD34+cells or 2500 uncultured CD34+cells (control) were transplanted in sub-lethally irradiated NSG mice. At week 20, long-term multilineage engraftment was measured in bone marrow of transplanted NSG mice.

[0131] Recipients of 4i expanded cells showed similar or higher frequencies of human CD45+and CD34+cells in analyzed mouse bone marrow compared to recipients of uncultured cells. Recipients of 4i expanded cells showed higher levels of HSPC (CD34), lymphoid (CD45+CD19+), and myeloid (CD45+CD33+) cell engraftment, relative to uncultured cells (see Table 1).Table 1 . Engraftment of expanded HSPC. Data in the table are mean ± SD (n = 8 mice)

Claims

CLAIMS1) A method for culturing one or more hematopoietic stem and progenitor cell (HSPC), said method comprising: contacting the one or more HSPC with a cocktail of a plurality of epigenetic modifiers in a culture medium, the cocktail comprising two or more of i) a first inhibitor of a histone deacetylase (HDACi), ii) a second HDACi, iii) at least one inhibitor of a histone demethylase (HDMi), and iv) at least one inhibitor of a histone methyltransferase (HMTi); and culturing the one or more HSPC in the presence of the cocktail for a time sufficient to produce an expanded population of HSPCs.2) The method according to claim 1 , further comprising isolating or enriching the one or more HSPC from a sample before contacting the population with the cocktail.3) The method according to claim 2, wherein the sample is whole blood, cord blood, peripheral blood, or bone marrow.4) The method according to claim 2 or 3, wherein the sample is a diseased or infected sample.5) The method according to any one of claims 1 to 4, wherein the one or more HSPC is comprised in an initial population comprising HSPCs.6) The method according to any one of claims 1 to 4, wherein the one or more HSPC and / or the expanded population of HSPCs express CD34.7) The method according to any one of claims 1 to 6, wherein the one or more HSPC is a primitive HSPC having a CD34+CD45RA CD90+phenotype, a CD34+CD45RA CD90+EPCR+phenotype, and / or a CD34+CD45RA CD90+EPCR+CD133+CD49f phenotype.8) The method according to any one of claims 1 to 7, wherein the expanded population of HSPC comprises more HSPC compared to when the one or more HSPC are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of HSPCs.9) The method according to any one of claims 1 to 8, wherein the expanded population of HSPCs comprises more primitive cells having a CD34+CD45RA CD90+phenotype and / or a CD34+CD45RA CD90+EPCR+phenotype and / or a CD34+CD45RA' CD90+EPCR+CD133+CD49f phenotype compared to when the one or more HSPC are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of HSPCs.10) The method according to any one of claims 1 to 9, wherein the first HDACi and the second HDACi are class I and / or class II and / or class IV HDACi.11) The method according to claim 10, wherein the first HDACi and the second HDACi are selected from: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.12) The method according to any one of claims 1 to 11 , wherein the at least one HDMi is a LSD1 or LSD2 inhibitor.13) The method according to any one of claims 1 to 12, wherein the at least one HMTi is a G9a / GLP HMTi.14) The method according to any one of claims 1 to 13, further comprising yielding a more highly expanded population of HSPCs when the one or more HSPC is contacted with and cultured in the presence of a cocktail comprising each of the first and second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing.15) The method according to any one of claims 1 to 13, further comprising yielding a more highly expanded population of HSPCs when the one or more HSPC is contacted with and cultured in the presence of a cocktail comprising the first HDACi, or a functional equivalent thereof, and two of the second HDACi, the at least one HDMi, and the at least one HMTi, as compared to a cocktail comprising two of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMT, wherein the first HDACi is valproic acid.16) The method according to any one of claims 1 to 13, further comprising yielding a more highly expanded population of HSPCs when the one or more HSPC is contacted with and cultured in the presence of a cocktail comprising the first HDACi, or a functional equivalent thereof, and one of the second HDACi, the at least one HDMi, and the at least one HMTi, ascompared to a cocktail comprising one of: the first HDACi; the second HDACi; the at least one HDMi; and the at least one HMTi, wherein the first HDACi is valproic acid.17) The method according to any one of claims 1 to 13, further comprising yielding a more highly expanded population of HSPCs with multi-lineage differentiation capacity when the one or more HSPC is contacted with and cultured in the presence of a cocktail comprising the first HDACi and the at least one HMTi, wherein the first HDACi is valproic acid.18) The method according to any one of claims 1 to 17, wherein the expanded population of HSPCs comprises between 2-fold to more than 100-fold more HSPCs compared to when the one or more HSPC are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of HSPCs.19) The method according to any one of claims 1 to 18, wherein the culture medium is further supplemented with one or more of IL-3, IL-6, SCF, FLT-3L, and / or TPO.20) A method for expanding hematopoietic stem and progenitor cells (HSPC), said method comprising: contacting one or more HSPC with a serum-free culture medium comprising at least a first histone deacetylase inhibitor (HDACi) and one or more of SCF, Flt3 ligand, and TPO; and culturing the one or more HSPC in the culture medium for a time sufficient to produce an expanded population of HSPCs, wherein the expanded population of HSPCs comprises primitive HSPCs having a CD34+CD45RA CD90+phenotype and / or a CD34+CD45RA_CD90+EPCR+phenotype and / or a CD34+CD45RA CD90+EPCR+CD133+CD49f+phenotype.21) The method according to claim 20, wherein at least the first HDACi is at least a class I HDACi.22) The method according to claim 20 or 21 , wherein the culture medium further comprises one or more of: a second HDACi, at least one HDMi, and at least one HMTi.23) A HSPC expansion supplement comprising a cocktail of a plurality of epigenetic modifiers, the cocktail comprising two or more of i) a first inhibitor of a histone deacetylase (HDACi), ii) a second HDACi, iii) at least one inhibitor of a histone demethylase (HDMi), andiv) at least one inhibitor of a histone methyltransferase (HMTi).24) The supplement according to claim 23, wherein the first HDACi and the second HDACi are class I and / or class II and / or class IV HDACi.25) The supplement according to claim 24, wherein the first HDACi and the second HDACi are selected from: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.26) The supplement according to claim 23, wherein the at least one HDMi is a LSD1 or LSD2 inhibitor.27) The supplement according to claim 23, wherein the at least one HMTi is a G9a / GLP HMTi.28) A culture medium comprising a basal medium and a supplement of any one of claims 23 to 27.29) The culture medium of claim 28, further comprising one or more of SCF, FLT-3L, and / or TPO.30) The culture medium of claim 28 or 29, wherein the culture medium is serum-free.

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