Methods, media and supplements for expanding hematopoietic cells

Abstract

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

Description

Cross-reference of related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 586,490, filed September 29, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to cell culture applications, and more specifically to cell culture applications using hematopoietic stem cells and progenitor cells (HSPCs), and still more specifically to applications related to CD34. + HSPC and / or CD34 + Cell culture applications related to the amplification of malignant cells. Background Technology

[0003] Hematopoietic stem cells (HSCs) or hematopoietic stem cells and progenitor cells (HSPCs) have shown significant therapeutic potential. One limitation to the clinical use of HSCs and HSPCs is the difficulty in obtaining sufficient quantities of these cells. Specifically, HSCs and HSPCs are resistant to in vitro maintenance, proliferation, and expansion. Another challenge involves the loss of pluripotency in HSCs and HSPCs, which can occur during in vitro culture of these cells.

[0004] Therefore, research continues on improving the methods for maintaining, proliferating, and amplifying HSCs and HSPCs in vitro, in a way that preserves their pluripotency and functionality, and epigenetic modifiers are promising candidates for this purpose. Specifically, the use of pyrimidine indole compounds for the amplification of umbilical cord blood-derived HSCs or HSPCs is currently under clinical investigation.

[0005] Therefore, for the amplification of HSC, HSPC and other CD34 + There is a need for other or improved reagents and methods for cells. Summary of the Invention

[0006] This disclosure relates to the culture and / or expansion of target cells, which may be HSPCs or CD34 cells. + Cells and their various subsets, including primitive CD34 + Subset.

[0007] In one aspect of this disclosure, methods for culturing and / or expanding target cells (e.g., HSPCs or CD34 cells) are provided. + The method disclosed herein can be a cell-based approach. The target cells can be contained in an initial population containing both target and non-target cells, or as a single cell. The disclosed method can be an in vitro method.

[0008] The method disclosed herein may include contacting one or more target cells with a mixture of multiple epigenetic modifiers in a culture medium, and culturing the target cells in the presence of the mixture to produce target cells (such as HSPCs or CD34). + The time of expansion of the cell population.

[0009] The mixture of multiple epigenetic modifiers may include one or more, two or more, three or more, or each of the following: a first histone deacetylase inhibitor (HDACi), a second HDACi, at least one histone demethylase inhibitor (HDMi), and at least one histone methyltransferase inhibitor (HMTi).

[0010] In one embodiment, the first HDACi and the second HDACi are Class I, Class II, or Class 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 an LSD1 or LSD2 inhibitor.

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

[0013] The methods disclosed herein may include isolating or enriching one or more HSPCs from a sample prior to contacting the population with the mixture. Non-limiting examples of samples include whole blood, umbilical cord blood, peripheral blood, or bone marrow. Additionally, the samples disclosed herein may be diseased or infected samples.

[0014] In one implementation, the target cells (one or more initial target cells, the initial population of target cells, or the expanded population of target cells) may include primitive HSPCs having the following phenotype: CD34 + CD45RA - CD90 + Phenotype, CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR+ CD133 + CD49f + Phenotype. In one embodiment, compared to the time required to culture the one or more target cells sufficiently to generate the expanded population of target cells without the presence of the mixture, the target cells (e.g., HSPCs or CD34) show a higher phenotype. + The expanded population of cells contained more original cells with the following phenotype: CD34 + CD45RA - CD90 + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Phenotype.

[0015] In one implementation, compared to the time required to culture the initial population of one or more target cells or target cells in the absence of one or more epigenetic modifiers (e.g., supplements or mixtures) to generate the expanded population of target cells, the target cells (e.g., HSPCs or CD34s) are cultured in a manner sufficient to produce the target cell expansion population. + The expanded population of cells contains more target cells.

[0016] The method disclosed herein may further include, when contacting the one or more target cells with one or more epigenetic modifiers (mixtures or supplements) (which include each of the first and second HDACi, at least one HDMi, and at least one HMTi) and / or culturing the one or more target cells in the presence of the one or more epigenetic modifiers, producing target cells (e.g., HSPCs or CD34) compared to a mixture containing only one, two, or three of the foregoing. + A higher degree of expansion of the population (cells).

[0017] The method disclosed herein may further include, when contacting the one or more target cells with one or more epigenetic modifiers (mixtures or supplements) (which include a first HDACi or a functional equivalent thereof, and two of a second HDACi, at least one HDMi, and at least one HMTi) and / or culturing the one or more target cells in the presence of the one or more epigenetic modifiers, producing target cells (e.g., HSPCs or CD34) as compared to a mixture containing two of the first and second HDACi, at least one HDMi, and at least one HMTi. + A higher degree of expansion of the population of cells, wherein the first HDACi is valproic acid.

[0018] The method disclosed herein may further include, when contacting the one or more target cells with one or more epigenetic modifiers (mixtures or supplements) (which include a first HDACi or a functional equivalent thereof, and a second HDACi, at least one HDMi, and at least one HMTi) and culturing the one or more target cells in the presence of the one or more epigenetic modifiers, producing target cells (e.g., HSPCs or CD34) as compared to a mixture containing one of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi. + A higher degree of expansion of the population of cells, wherein the first HDACi is valproic acid.

[0019] The method disclosed herein may further include producing a more extensive population of HSPCs with multi-lineage differentiation capacity by contacting the one or more HSPCs with a mixture comprising the first HDACi and the at least one HMTi and culturing the one or more HSPCs in the presence of the mixture, wherein the first HDACi is valproic acid.

[0020] In one embodiment, compared to the time required to culture the target cells sufficiently to generate the expanded population of target cells without the presence of the mixture, the target cells (e.g., HSPCs or CD34) [are more readily cultured]. + The expanded population of cells contains between 2 and more than 100 times the number of target cells.

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

[0022] Different methods disclosed herein may include culturing or expanding target cells (e.g., HSPCs or CD34 cells) in the following ways. +Population of cells: The target cells are contacted with a serum-free culture medium containing at least one epigenetic modifier and one or more of SCF, FLT-3L and / or TPO, and the target cells are cultured in the culture medium for a time sufficient to generate an expanded population of target cells.

[0023] The expanded population may contain target cells, including naïve HSPCs or CD34 cells with the following phenotypes. + Cells: CD34 + CD45RA - CD90 + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + Phenotype and / or 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 including more than one epigenetic modifier, the modifier may include one or more of the following: 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), entenostatin, acetyldinaline, pabisostat, LMK235, and romidesin. Exemplary HDMi includes LSD1 or LSD2 inhibitors. Exemplary HMTi includes G9a / GLP HMTi.

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

[0027] In any method disclosed herein, it may further include making target cells (e.g., HSPCs or CD34 cells) +The initial population of cells is isolated from the sample before being contacted with one or more of the epigenetic modifiers (or mixtures). In one embodiment, the sample is human umbilical cord blood, mobilized peripheral blood, or bone marrow. In one embodiment, the sample is a leukemia sample. In one embodiment, CD34... + The initial population of cells was isolated from CML samples or peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs).

[0028] In one implementation, the amplified population comprises one or more of the following original subsets: CD34 + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + ; and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + In one embodiment, when the target cells are contacted with one or more epigenetic modifiers (or the mixture) and cultured in the presence of the one or more epigenetic modifiers, the target cells (e.g., HSPCs or CD34) are more likely to develop into more efficient cells compared to when the target cells are not contacted with one or more epigenetic modifiers (or the mixture) and / or cultured in the absence of the one or more epigenetic modifiers. + The expanded population of cells contains one or more of the aforementioned subsets.

[0029] In any of the methods disclosed herein, target cells (e.g., HSPCs or CD34 cells) are cultured. + The population of cells lasted for approximately 5 to 21 days.

[0030] In another aspect of this disclosure, methods for expanding target cells (e.g., HSPCs or CD34 cells) in vitro are provided. + Cellular supplements (such as HSPC or CD34) +(Amplification supplement). The supplement may include a mixture of various epigenetic modifiers, said mixture comprising one, two or more, three or more, or each of the following: a first histone deacetylase inhibitor (HDACi), a second HDACi, at least one histone demethylase inhibitor (HDMi), and at least one histone methyltransferase inhibitor (HMTi). In one embodiment, the mixture may comprise a first HDACi and a second HDACi.

[0031] In one implementation, the first HDACi and the second HDACi are Class I, Class II, or Class IV HDACi. Exemplary HDACi include: valproic acid, trichostatin A (TSA), entenostatin, acetyldinarin, pabistamine, LMK235, and romidesin.

[0032] In one embodiment, the at least one HDMi is an 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, methods for culturing (e.g., expanding) target cells (e.g., HSPCs or CD34 cells) are provided. + The culture medium for a population of cells. The culture medium of this disclosure may contain a basal medium and supplements (including one or more epigenetic modifiers, as described herein). The culture medium of this disclosure may further contain one or more of SCF, FLT-3L, IL-3, IL-6 and / or TPO. Attached Figure Description

[0035] To better understand the various embodiments described herein, and to more clearly show how these various embodiments can be implemented, reference will be made to the accompanying drawings by way of example, which illustrate at least one exemplary embodiment and are now described. The drawings are not intended to limit the scope of the teachings herein.

[0036] Figure 1 The bar graph shows the effects of human umbilical cord blood HSPCs and certain CD34 amplification factors on their growth after 7 days of culture in the presence of CD34 supplement (StemSpan™ CD34+ amplification supplement) + one or more of the indicated epigenetic modifiers. +Subset amplification (AD) and colony-forming potential (EF) were quantified. Epigenetic modifiers tested included: first HDACi (HDACi-1), second HDACi (HDACi-2), histone demethylase inhibitor (HDMi-1), and histone methyltransferase inhibitor (HMTi-1). Ref = positive control, containing CD34 supplement plus the published small molecule reference compound. TNC = total nucleated cells. CFU-GEMM = colony-forming units, granulocytes, erythroids, macrophages, megakaryocytes; CFU-G / M / GM = colony-forming units, granulocyte-macrophage; BFU-E = burst-forming units, erythroids. Data shown are averages from two umbilical cord blood samples.

[0037] Figure 2 Displaying a bar chart, which is essentially as follows: Figure 1 After 7 days of culture, human umbilical cord blood HSPCs and certain CD34 were obtained. + Amplification (AD) and colony-forming potential (EF) of subsets were quantified, but 4i mixtures of epigenetic modifiers were evaluated in the presence (4i) or absence (4i-AO) of antioxidants. TNC, Ref, CFU-GEMM, CFU-G / M / GM, and BFU-E were as defined in the paragraphs above. Data shown are mean ± SEM values ​​from two umbilical cord blood samples.

[0038] Figure 3 Displaying a bar chart, which is essentially as follows: Figure 1 After 7 days (AE) and 14 days (FJ) of culture, human umbilical cord blood (CB), human mobilized peripheral blood (mPB), and human bone marrow (BM) HSPCs and certain CD34 were cultured. + The amplification of subsets was quantified. Ref is as defined in the paragraph above. The data shown are the mean ± SEM values ​​from cell samples from 2–8 individual donors.

[0039] Figure 4 The bar graph shows the effect of CD34 isolated from the AML sample. + Quantification of cell amplification was performed. CD34 was... + AML cells were cultured for 7 days in a serum-free medium containing either a single compound or a mixture of a reference compound (Ref, as described in the preceding paragraph) or an epigenetic modifier (e.g., 4i). Figure 1 The combination of CD34 supplements (as described in the paragraph above) is shown. Day 7 frequency (A) and cell number (B) of presumed AML stem cells or progenitor cells are shown, including for a more primitive subset: CD34 + CD45RA - CD90 + and CD34+ CD45RA - 90 + EPCR + The data shown are the mean ± SEM values ​​of PB samples from 5 AML patients.

[0040] Figure 5 The bar graph shows the effect of separating CD34 from CML samples. + Quantification of cell amplification was performed. CD34 was... + CML cells were cultured for 7 days in a serum-free medium containing either a single compound or a 4i mixture with a reference compound (Ref, as described in the preceding paragraph) or an epigenetic modifier (e.g., ...). Figure 1 The combination of CD34 supplements (as described in the paragraph above) is shown. Day 7 frequency (A) and cell number (B) of presumed CML stem cells or progenitor cells are shown, including for a more primitive subset: CD34. + CD45RA - CD90 + and CD34 + CD45RA - 90 + EPCR + The data shown are the mean ± SEM values ​​of PB samples from 4 CML patients. Detailed Implementation

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

[0042] As used herein, the terms "hematopoietic stem cells and progenitor cells," "HSPC," "hematopoietic progenitor cells," "hematopoietic stem cells," "hematopoietic precursor cells," or "HPC" are used interchangeably and refer to cells of a hematopoietic lineage capable of self-renewal and / or differentiation into more specialized cells of the hematopoietic lineage, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). HSPCs can be obtained from bone marrow (BM), umbilical cord blood (CB), embryonic to adult peripheral blood (PB), thymus, peripheral lymph nodes, gastrointestinal tract, tonsils, pregnant uterus, liver, spleen, or any other tissue with a localized population of HSPCs. HSPCs can also differentiate from pluripotent stem cells (such as induced pluripotent stem cells), embryonic stem cells, juvenile stem cells, extended stem cells, etc. The hallmark of HSPC is the expression of the transmembrane phosphoglycoprotein CD34 on its surface; therefore, HSPC can be referred to as CD34. + Cells, including those not of the hematopoietic lineage, can express CD34. The HSPC population may include CD34-expressing cells. + A single population of cells, that is, CD34 cells with the same phenotype. + Cells. Alternatively, the HSPC population may contain multiple CD34 cells. + Cells, that is, all of these cells can express CD34, but express other surface markers differently. A subset of human HSPCs can be further defined by CD45 expression, and even further by combinations of markers such as CD38, CD43, CD45RO, CD45RA, CD10, CD49f, CD59, CD90, CD109, CD117, CD133, CD166, HLA-DR, EPCR (CD201), and integrin-α3. HSPCs may lack expression of markers such as blood group glycoprotein A, CD3, CD4, CD8, CD14, CD15, CD19, CD20, and CD56; such markers may be characteristic of more mature blood cells. HSPCs (e.g., CD34) + More common markers for HSPC may include CD45RA, which is absent or 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] As used herein, the terms “epigenetic modifier” or “epigenetic regulator” are used interchangeably and refer to agents or factors that modify or cooperate with other factors to modify epigenetic markers of DNA and / or histones, such as proteins, small molecules, interfering RNA, messenger RNA, or other natural or synthetic compounds (or their enzymatically active catalytic domains). Epigenetic modifiers can be classified according to their mechanism of action, such as DNA methyltransferases or their inhibitors, histone acetyltransferases or their inhibitors, histone methyltransferases or their inhibitors, histone deacetylases or their inhibitors, or histone demethylases or their inhibitors.

[0044] As used in this disclosure, the term "amplified population" refers to an output population of cells in which the quantity of output cells (e.g., after culturing for a period of time in the presence of one or more epigenetic modifiers) is greater than that of a control population (e.g., a population not cultured in the presence of one or more epigenetic modifiers). In one embodiment, the expanded population of cells may specifically refer to HSPCs or CD34 cells amplified in the presence of one or more epigenetic modifiers. + A population of cells. In one implementation, HSPC cells or CD34 cells. + The expanded population of cells can more specifically refer to an ancestral or original population expanded in the presence of one or more epigenetic modifiers. In one embodiment, the expanded population can mean an increase 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 100%, or any increase between 10% and 100%, compared to input or control. In one embodiment, the expanded population can mean an increase of at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, or any increase between 2-fold and 50-fold, compared to input or control. In one embodiment, target cells (e.g., HSPCs or CD34 cells) + An expanded population of target cells (e.g., HSPCs or CD34 cells) can be used for gene editing applications. In one implementation, target cells (e.g., HSPCs or CD34 cells) + Whether or not gene-edited, expanded populations of cells can be implanted into subjects (such as human subjects) or into animal models (such as mice).

[0045] Culture media and supplements In one aspect of this disclosure, a supplement (which may be added to a culture medium) is provided for culturing or generating or expanding a population of target cells such as HSPCs (e.g., CD34). + (cells) and / or their ancestors or original subsets. The compositions / formulations of supplements disclosed herein are not limited in any particular way, provided they are suitable for contact with one or more HSPCs or populations thereof and conducive to their culture.

[0046] The supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules. More specifically, the supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that regulate epigenetic markers (e.g., epigenetic modifiers). Although the supplements of this disclosure may be entirely based on small molecules, compounds, and / or macromolecules, they may comprise one or more macromolecules (e.g., lipids, proteins, carbohydrates) that do not have a primary function as epigenetic modifiers, or be in combination with said one or more macromolecules. Therefore, the supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that act as epigenetic modifiers, and one or more macromolecules that do not have a primary function as epigenetic modifiers, or the supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that act as epigenetic modifiers and be in combination with one or more macromolecules that do not have a primary function as epigenetic modifiers (e.g., may be included in a separate supplement or culture medium).

[0047] When combined with a basal culture medium and (in some cases) one or more other supplements, the supplements disclosed herein can support the expansion of target cells, such as HSPC cells (e.g., CD34 cells). + HSPC and / or CD34 + Malignant cells).

[0048] As described above, the supplements of this disclosure may contain one or more epigenetic modifiers. In one embodiment, the supplements of this disclosure contain multiple epigenetic modifiers (e.g., a mixture of epigenetic modifiers). Furthermore, the supplements of this disclosure may contain only small molecule epigenetic modifiers, only non-small molecule epigenetic modifiers (e.g., compounds), only macromolecule modifiers (e.g., fatty acids), or any combination thereof.

[0049] The supplements (and mixtures where applicable) disclosed herein may comprise one or more of the following: (i) at least one histone deacetylase inhibitor (HDACi); (ii) at least one histone demethylase inhibitor (HDMi); and (iii) at least one histone methyltransferase inhibitor (HMTi).

[0050] The supplements (and mixtures where applicable) disclosed herein may contain two or more of the following: (i) at least one histone deacetylase inhibitor (HDACi); (ii) at least one histone demethylase inhibitor (HDMi); and (iii) at least one histone methyltransferase inhibitor (HMTi).

[0051] The supplements (and mixtures where applicable) disclosed herein may comprise each of: (i) at least one histone deacetylase inhibitor (HDACi); (ii) at least one histone demethylase inhibitor (HDMi); and (iii) at least one histone methyltransferase inhibitor (HMTi).

[0052] The supplements (and mixtures where applicable) disclosed herein may comprise one or more of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0053] The supplements (and mixtures where applicable) disclosed herein may contain two or more of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0054] The supplements (and mixtures where applicable) disclosed herein may contain three or more of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0055] The supplements (and mixtures where applicable) disclosed herein may comprise each of: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

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

[0057] HDAC inhibitors are known and widely available, therefore there are no particular limitations in the supplements disclosed herein. In one embodiment, the supplement of this disclosure contains one HDACi. In one embodiment, the supplement of this disclosure contains two HDACi. In one embodiment, the supplement of this disclosure contains more than two HDACi.

[0058] The supplement may contain one or more HDACi, which can be Class I, Class II, or Class IV HDACi. If the supplement contains only one HDACi, that HDACi may belong to a single HDACi class or multiple HDACi classes. If the supplement contains more than one (e.g., two or more) HDACi, each HDACi may belong to the same or different classes. In one embodiment, the supplement of this disclosure contains at least two Class I HDACi.

[0059] Examples of HDAC inhibitors that may be included in supplements are valproic acid (HDAC class I and II inhibitors), trichostatin A (TSA) (HDAC class I, II, and IV inhibitors), romidesin (HDAC class I inhibitor), entenoxetine (HDAC class I inhibitor), acetyldenalin (HDAC class I inhibitor), pabisostat (HDAC class I, II, and IV inhibitors), and LMK235 (HDAC class II inhibitor). In one embodiment, the supplement contains valproic acid. In one embodiment, the supplement contains romidesin. In one embodiment, the supplement contains one or both of valproic acid and romidesin.

[0060] There are no particular restrictions on the concentration of one or more HDACi in the supplement, but the concentration should not be lethal or toxic to cells. If the HDACi is a small molecule, its concentration can be in the nanomolar to micromolar range. For example, the concentration range can be between about 0.1 nM and 100 μM, or between about 0.5 nM and 50 μM, or between about 1 nM and 10 μM, or between about 5 nM and 1 μM, or between about 10 nM and 0.5 μM, or between about 25 nM and 250 μM. If the HDACi is not a small molecule, its concentration can be in the micromolar to millimolar range. For example, the concentration range can be between about 1 μM and 1000 mM, or between about 10 μM and 500 mM, or between about 100 μM and 250 mM, or between about 250 μM and 200 mM, or between about 500 μM and 150 mM, or between about 1 mM and 100 mM.

[0061] HDM inhibitors (HDMi) are known and widely available, therefore there are no particular limitations in the supplements disclosed herein. In one embodiment, the supplement of this disclosure contains one HDMi. In one embodiment, the supplement of this disclosure contains two HDMis. In one embodiment, the supplement of this disclosure contains more than two HDMis.

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

[0063] There are no particular restrictions on the concentration of one or more HDMis in the supplement, but the concentration should not be lethal or toxic to cells. If the HDMi is a small molecule, its concentration can be in the nanomolar to micromolar range. For example, the concentration range can be between about 1 nM and 1 mM, or between about 25 nM and 750 μM, or between about 100 nM and 500 μM, or between about 250 nM and 400 μM, or between about 500 nM and 300 μM, or between about 1 μM and 250 μM. If the HDMi is not a small molecule, its concentration can be in the micromolar to millimolar range. For example, the concentration range can be between about 1 μM and 1000 mM, or between about 10 μM and 500 mM, or between about 100 μM and 250 mM, or between about 250 μM and 200 mM, or between about 500 μM and 150 mM, or between about 1 mM and 100 mM.

[0064] HMT inhibitors (HMTi) are known and widely available, therefore there are no particular limitations in the supplements disclosed herein. In one embodiment, the supplement of this disclosure contains one HMTi. In one embodiment, the supplement of this disclosure contains two HMTi. In one embodiment, the supplement of this disclosure contains more than two HMTi.

[0065] One or more HMTi contained in the supplement may be histone methyltransferase G9a (also known as EHMT2 or KMT1C) and / or G9a-like protein (GLP, also known as EHMT1). In one embodiment, at least one HMTi contained in the supplement is a G9a / GLP HMTi. In one embodiment, the G9a / GLP HMTi is UNC0638.

[0066] There are no particular restrictions on the concentration of one or more HMTi in the supplement, but the concentration should not be lethal or toxic to cells. If the HMTi is a small molecule, its concentration can be in the nanomolar to micromolar range. For example, the concentration range can be between about 1 nM and 1 mM, or between about 25 nM and 750 μM, or between about 100 nM and 500 μM, or between about 250 nM and 400 μM, or between about 500 nM and 300 μM, or between about 1 μM and 250 μM. If the HMTi is not a small molecule, its concentration can be in the micromolar to millimolar range. For example, the concentration range can be between about 1 μM and 1000 mM, or between about 10 μM and 500 mM, or between about 100 μM and 250 mM, or between about 250 μM and 200 mM, or between about 500 μM and 150 mM, or between about 1 mM and 100 mM.

[0067] In some implementations, the HSPC of this disclosure (e.g., CD34) + The cell expansion supplement contains at least one HDACi, and at least one of HDMi and HMTi. In some embodiments, the HSPC (e.g., CD34) disclosed herein... + The cell expansion supplement contains at least one HDACi, at least one HDMi, and at least one HMTi. In some embodiments, the HSPC (e.g., CD34) disclosed herein... + The cell expansion supplement contains at least two HDACi, and at least one of HDMi and HMTi. In some embodiments, the HSPC (e.g., CD34) disclosed herein... + Cellular expansion supplements contain at least two HDACi, at least one HDMi, and at least one HMTi.

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

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

[0070] The supplements disclosed herein can be added to cell culture media. When preparing complete culture media, the supplements containing one or more epigenetic modifiers can be further combined with one or more cytokines or growth factors.

[0071] Therefore, in another aspect of this disclosure, a culture medium is provided for culturing / expanding target cells in vitro, such as HSPCs (e.g., CD34). + HSPCs expanded in the media disclosed herein (and supplemented as described herein) retain the ability to differentiate into many downstream cell types in the hematopoietic lineage. The media disclosed herein can also be used to expand CD34 cells isolated from leukemia (e.g., AML and / or CML PBMCs) or other myelodysplastic syndromes. + Malignant cells.

[0072] The culture medium of this disclosure may contain serum or may be serum-free. In one embodiment, the culture medium of this disclosure is serum-free. If serum-free, it may be necessary to include a serum replacement supplement, such as BIT9500 serum substitute (STEMCELL Technologies) or other commercially available serum replacement solutions, in such a culture medium. Alternatively, components typically present in serum required for culturing or differentiating any of the cells of this disclosure may be added separately to the culture medium at acceptable concentrations.

[0073] The culture medium disclosed herein will include a basal medium. The basal medium can be any medium that, when appropriately supplemented, supports the expansion of target cells, such as mammalian HSPCs (e.g., CD34). + (cells or a subset thereof). In one implementation, the basal culture medium may be capable of supporting mammalian CD34. + Any culture medium for the expansion of malignant cells. In one embodiment, the basal medium can be any basal medium that supports the culture of cells of hematopoietic lineage. By way of non-limiting example, the basal medium can 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 suitable for the purposes described herein.

[0074] The culture medium of this disclosure (which may be combined with supplements as described above) may contain one or more epigenetic modifiers. The foregoing description of the properties, combinations and concentrations of epigenetic modifiers is incorporated herein by reference entirely within the context of the cell culture medium of this disclosure.

[0075] The culture medium disclosed herein may further contain one or more additional cytokines or growth factors, or small molecules, or other proteins that provide key signals for HSPC amplification. By way of non-limiting examples, the key signals required for HSPC amplification may include one or more of the following: one or more interleukins (e.g., IL-1, IL-2, IL-3, IL-6), VEGF, G-CSF, GM-CSF, SCF, FLT3-L, TPO, and EPO.

[0076] The supplements and culture media disclosed herein can support the following fold increases in target cells (e.g., HSPCs) compared to control conditions (supplements lacking one or more epigenetic modifiers, as described above and fully incorporated herein by reference): approximately 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 100%, or any increase between 10% and 100%. The supplements and culture media disclosed herein can support the following fold increases in target cells (e.g., HSPCs) compared to control conditions (supplements lacking one or more epigenetic modifiers, as described above and fully incorporated herein by reference): approximately at least about 2-fold, or at least about 3-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold, or at least about 20-fold, or at least about 25-fold, or at least about 30-fold, or at least about 35-fold, or at least about 40-fold, or at least about 45-fold, or at least about 50-fold, or any increase between 2-fold and 50-fold or more.

[0077] Enhanced HSPC amplification in the presence of supplements to one or more epigenetic modifiers and in the presence of a culture medium comprising such supplements and one or more cytokines / growth factors beneficial to HSPC amplification, as disclosed herein, can more specifically refer to the amplification of a larger subset of the original HSPCs (e.g., CD34) relative to either a commercially available cytokine supplement alone (e.g., StemSpan™ CD34 amplification supplement, STEMCELL Technologies) or a commercially available cytokine supplement in combination with a disclosed reference small molecule (e.g., a pyrimidine-indole compound). + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + and CD34 + CD45RA - CD90 + EPCR+ CD133 + CD49f + ).

[0078] In one implementation, the original HSPC subset (e.g., CD34) + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + and CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + The higher amplification is supported by: (i) a supplement containing each of the first and second HDACi, at least one HDMi, and at least one HMTi, as compared to a mixture containing only one, two, or three of the foregoing; (ii) a supplement containing the 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 mixture containing two of the first HDACi, the second HDACi, at least one HDMi, and at least one HMTi, wherein the first HDACi is valproic acid; or (iii) a supplement containing the 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 mixture containing one of the first and second HDACi, at least one HDMi, and at least one HMTi, wherein the first HDACi is valproic acid.

[0079] In some implementations, a subset of the original HSPC with a different phenotype (e.g., CD34) + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + and CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f +Further amplification of the original HSPC subset with in vitro multilineage differentiation capacity (e.g., based on colony formation potential) is supported by a supplement comprising a first HDACi and at least one HMTi, wherein the first HDACi is valproic acid.

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

[0081] The target cells are preferably HSPCs that express at least CD34. The HSPCs are preferably mammalian, and more preferably human. The HSPCs can be directly isolated from primary samples such as whole blood, umbilical cord blood, (mobilized) peripheral blood, bone marrow, thymus, uterus, liver, intestine, or secondary lymphoid tissue. In one embodiment, CD34... + It is isolated from human umbilical cord blood. In one implementation, CD34 + It is isolated from (mobilized) peripheral blood. In one implementation, CD34 + It was separated from human bone marrow.

[0082] The target cells used in the methods disclosed herein may be CD34 cells obtained, enriched, and / or isolated from cancerous, malignant, diseased, or infected samples (such as peripheral blood mononuclear cells (PBMCs)). + Cells. In one implementation, CD34 + Cancerous or malignant cells are obtained, enriched, and / or isolated from chronic myeloid leukemia (CML) samples (e.g., PBMCs). In one embodiment, CD34... + Cancer or malignant cells are obtained, enriched and / or isolated from acute myeloid leukemia (AML) samples (e.g., PBMCs).

[0083] Methods for culturing (e.g., expanding) target cells (e.g., HSPC cells) may include contacting one or more cells (e.g., HSPCs) with one or more epigenetic modifiers in a culture medium. More specifically, methods of this disclosure may include contacting one or more cells (e.g., HSPCs) with a mixture of multiple epigenetic modifiers in a culture medium. The properties and other details (e.g., combinations and concentrations) of one or more epigenetic modifiers in the culture medium of this disclosure (such as those used in the methods of this invention) may be as described above.

[0084] Furthermore, the above description of the culture medium can also be applied to the disclosed methods. Effective expansion of hematopoietic cells may require one or more cytokines and / or growth factors; therefore, the culture medium used in the methods of this disclosure may be supplemented with one or more of SCF, FLT-3L, and / or TPO.

[0085] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include one or more of the following: (i) at least one histone deacetylase inhibitor (HDACi); (ii) at least one histone demethylase inhibitor (HDMi); and (iii) at least one histone methyltransferase inhibitor (HMTi).

[0086] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include two or more of the following: (i) at least one histone deacetylase inhibitor (HDACi); (ii) at least one histone demethylase inhibitor (HDMi); and (iii) at least one histone methyltransferase inhibitor (HMTi).

[0087] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include each of the following: (i) at least one histone deacetylase inhibitor (HDACi); (ii) at least one histone demethylase inhibitor (HDMi); and (iii) at least one histone methyltransferase inhibitor (HMTi).

[0088] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include one or more of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0089] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include two or more of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0090] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include three or more of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0091] The supplements (and, where applicable, mixtures) in the culture medium used in the methods of this disclosure may include each of the following: (i) at least a first histone deacetylase inhibitor (HDACi); (ii) at least a second histone deacetylase inhibitor (HDACi); (iii) at least one histone demethylase inhibitor (HDMi); and (iv) at least one histone methyltransferase inhibitor (HMTi).

[0092] In some embodiments, the supplement (and, where applicable, the mixture) used in the methods of this disclosure (e.g., may be contained in a culture medium) contains at least a first HDACi (e.g., valproic acid). In some embodiments, the supplement (and, where applicable, the mixture) contains at least a first HDACi (e.g., valproic acid) and / or at least one HMTi.

[0093] In some embodiments, the HSPC (e.g., CD34) used in the methods of this disclosure + The cell expansion supplement (e.g., may be included in the culture medium) contains at least one HDACi, and at least one of HDMi and HMTi. In some embodiments, the HSPC (e.g., CD34) used in the methods of this disclosure... + The cell expansion supplement (which may be included in the culture medium) contains at least one HDACi, at least one HDMi, and at least one HMTi. In some embodiments, the HSPC (e.g., CD34) used in the methods of this disclosure... + The cell expansion supplement (e.g., which may be included in the culture medium) contains at least two HDACi, and at least one of HDMi and HMTi. In some embodiments, the HSPC (e.g., CD34) used in the methods of this disclosure...+ Cell expansion supplements (which may be included in the culture medium) contain at least two HDACi, at least one HDMi, and at least one HMTi.

[0094] One or more target cells (e.g., HSPCs or CD34) + Cells can be included in an initial population of cells, and the initial population of cells can be an isolated or enriched population of target cells, or a bulk population containing both target cells and off-target cells. Therefore, the methods of this disclosure can include isolating or enriching one or more target cells (e.g., HSPCs or CD34 cells) from a sample before contacting the population with the mixture. + Cells). Used for isolating or enriching target cells (such as those that may be included in an initial population containing target cells (e.g., HSPCs or CD34). + Cellular methods are known and commercially available, such as those by FACS or by immunomagnetic cell separation, as commercially available under the EasySep™ brand (STEMCELL Technologies).

[0095] After contacting the target cells with a culture medium containing one or more epigenetic modifiers, the method may further include introducing one or more target cells (e.g., HSPCs or CD34 cells) into the culture medium. + The time during which cells are cultured in the presence of one or more epigenetic modifiers to produce an expanded population of target cells.

[0096] Aside from being affected by metabolite accumulation, pH, nutrient availability, and overcrowding, there is no particular limitation on the culture duration; these factors can be corrected by changing the culture medium and / or scaling up the container size and culture medium volume. Nevertheless, cultures can last between approximately 1 to 35 days, approximately 2 to 32 days, approximately 3 to 28 days, approximately 5 to 21 days, or approximately 7 to approximately 14 days. In some embodiments, the culture lasts 7 days ± 1 day. In some embodiments, the culture lasts 10 days ± 1 day. In some embodiments, the culture lasts 14 days ± 1 day.

[0097] Culture one or more target cells (which may be included in the initial population) in the absence of one or more epigenetic modifiers (e.g., mixtures or supplements) to produce target cells (e.g., HSPCs or CD34 cells). + Compared to the time required to expand a population of cells, the production / generation of target cells (e.g., HSPCs or CD34) after sufficient culture time is significantly higher. + Expanding the population of cells can yield more target cells (e.g., HSPCs or CD34 cells). + cell).

[0098] The increased amplification of target cells by practicing the methods of this disclosure can also be achieved relative to the contact with and / or culture of reference standard small molecules in the culture medium and in the presence of said reference standard small molecules. + Quantification is based on the expansion of hematopoietic cells (especially HSPCs and / or CD34 cells). + In the field of cellular biology, a variety of such reference molecules / compounds are known, including engineered cytokines (e.g., IL-6), pyrimidine indole compounds, or aryl hydrocarbon antagonists.

[0099] The increased expansion of target cells by practicing the methods of this disclosure can also be achieved relative to the contact with and / or culture of target cells (e.g., HSPCs or CD34) in the presence of less than one epigenetic modifier contained in the mixture or culture medium and / or in the presence of said epigenetic modifier. + Quantification is achieved through the expansion of target cells (e.g., HSPCs or CD34 cells). + Cells or their primitive subsets: for example, CD34 + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + ; or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Higher amplification is supported in the following cases: (i) in the presence of four epigenetic modifiers (in appropriate culture medium), relative to the presence of three, two, or one epigenetic modifier (in appropriate culture medium); (ii) in the presence of three epigenetic modifiers (in appropriate culture medium), relative to the presence of two or one epigenetic modifier (in appropriate culture medium); or (iii) in the presence of two epigenetic modifiers (in appropriate culture medium), relative to the presence of one epigenetic modifier (in appropriate culture medium).

[0100] Therefore, the method of this disclosure may include, when contacting one or more target cells with a mixture comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi and / or culturing the one or more target cells in the presence of said mixture, producing target cells (e.g., HSPCs or CD34) compared to a mixture comprising only one, two, or three of the foregoing. + A higher degree of expansion of the population (cells).

[0101] The method disclosed herein may include, when contacting one or more target cells with a mixture comprising two of a first HDACi or a functional equivalent thereof, a second HDACi, at least one HDMi, and at least one HMTi, and culturing the one or more target cells in the presence of said mixture, producing target cells (e.g., HSPCs or CD34 cells) compared to a mixture comprising two of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi. + A higher degree of expansion of the population of cells, wherein the first HDACi is valproic acid.

[0102] The method disclosed herein may include, when contacting one or more target cells with a mixture comprising a first HDACi or a functional equivalent thereof and a second HDACi, at least one HDMi, or at least one HMTi and / or culturing the one or more target cells in the presence of said mixture, producing target cells (e.g., HSPCs or CD34 cells) compared to a mixture comprising a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi. + A higher degree of expansion of the population of cells, wherein the first HDACi is valproic acid.

[0103] With the absence of one or more epigenetic modifiers (e.g., mixtures or supplements), one or more target cells (e.g., HSPCs or CD34 cells) + Compared to culturing cells for a sufficient time to produce an expanded population of target cells, producing / generating target cells (e.g., HSPCs or CD34) after a sufficient culture time is significantly different. + Expanding the initial population of target cells (e.g., HSPCs or CD34 cells) can yield target cells that are 2 to 100 times larger. This is achieved by expanding the initial population of target cells (e.g., HSPCs or CD34 cells) without the presence of one or more epigenetic modifiers (e.g., mixtures or supplements). + Compared to the time required to culture target cells (e.g., HSPCs or CD34 cells) to generate an expanded population of target cells, this time is significantly longer. + Starting with an initial population of cells, culturing them for a sufficient time can produce / generate target cells that are 2 to more than 100 times larger in the expanded population.

[0104] In some implementations, the expanded population of target cells can yield approximately 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more target cells.

[0105] The method disclosed herein can produce CD34 + The expanded population of cells can be further characterized as different subsets of CD34-expressing cells. For example, one or more target cells (e.g., CD34-expressing cells). + Cells or HSPCs may include CD34 + CD45RA - CD90 + Phenotype, CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Phenotype. Such phenotypes may correspond to a further original subset of CD34-expressing cells and / or HSPCs. Therefore, the expanded population (after exposure to one or more epigenetic modifiers and culture in the presence of said one or more epigenetic modifiers) may include an expanded population of target cells containing the following phenotypes: CD34 + CD45RA - CD90 + Phenotype, CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Phenotype.

[0106] In some embodiments, one or more such target cells (e.g., which may be included in the initial population of cells, such as CD34) are cultured in the presence of one or more epigenetic modifiers (e.g., mixtures or supplements) in the culture medium. +After culturing such cells in the absence of one or more of the aforementioned epigenetic modifiers in the culture medium, the target cells (e.g., HSPCs or CD34 cells) show improved performance compared to culturing such cells in the absence of these epigenetic modifiers in the culture medium. + The expanded population of cells can include HSPCs or CD34 cells at approximately 2 to 50 times the original size, or approximately 3 to 40 times the original size, or approximately 4 to 30 times the original size, or approximately 5 to 25 times the original size. + A primitive subset of cells (e.g., CD34) + CD45RA - CD90 + More specifically, after 7 or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g., mixtures or supplements) in the culture medium, compared to culturing such cells in the absence of said one or more epigenetic modifiers in the culture medium, the target cells (e.g., HSPCs or CD34 cells) showed increased activity compared to culturing such cells in the absence of said one or more epigenetic modifiers in the culture medium. + The expanded population of cells can contain each input CD34 + Cells were HSPCs or CD34 cells at approximately 2 to 50 times, or approximately 3 to 40 times, or approximately 4 to 30 times, or approximately 5 to 25 times. + A primitive subset of cells (e.g., CD34) + CD45RA - CD90 + ).

[0107] In some embodiments, one or more such target cells (e.g., which may be included in the initial population of cells, such as CD34) are cultured in the presence of one or more epigenetic modifiers (e.g., mixtures or supplements) in the culture medium. + After culturing such cells in the absence of one or more of the aforementioned epigenetic modifiers in the culture medium, the target cells (e.g., HSPCs or CD34 cells) show improved performance compared to culturing such cells in the absence of these epigenetic modifiers in the culture medium. + The expanded population of cells can include HSPCs or CD34 cells at approximately 2 to 25-fold, or approximately 3 to 20-fold, or approximately 4 to 15-fold, or approximately 5 to 10-fold. + A primitive subset of cells (e.g., CD34) + CD45RA - CD90 + EPCR + More specifically, after 7 or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g., mixtures or supplements) in the culture medium, compared to culturing such cells in the absence of said one or more epigenetic modifiers in the culture medium, the target cells (e.g., HSPCs or CD34 cells) showed increased activity compared to culturing such cells in the absence of said one or more epigenetic modifiers in the culture medium. + The expanded population of cells can contain each input CD34 +Cells approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times greater than HSPCs or CD34. + A primitive subset of cells (e.g., CD34) + CD45RA - CD90 + EPCR + ).

[0108] In some embodiments, one or more such target cells (e.g., which may be included in the initial population of cells, such as CD34) are cultured in the presence of one or more epigenetic modifiers (e.g., mixtures or supplements) in the culture medium. + After culturing such cells in the absence of one or more of the aforementioned epigenetic modifiers in the culture medium, the target cells (e.g., HSPCs or CD34 cells) show improved performance compared to culturing such cells in the absence of these epigenetic modifiers in the culture medium. + The expanded population of cells can include HSPCs or CD34 cells at approximately 2 to 25-fold, or approximately 3 to 20-fold, or approximately 4 to 15-fold, or approximately 5 to 10-fold. + A primitive subset of cells (e.g., CD34) + CD45RA - CD90 + EPCR + CD133 + CD49f + More specifically, after 7 or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g., mixtures or supplements) in the culture medium, compared to culturing such cells in the absence of said one or more epigenetic modifiers in the culture medium, the target cells (e.g., HSPCs or CD34 cells) showed increased activity compared to culturing such cells in the absence of said one or more epigenetic modifiers in the culture medium. + The expanded population of cells can contain each input CD34 + Cells approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times greater than HSPCs or CD34. + A primitive subset of cells (e.g., CD34) + CD45RA - CD90 + EPCR + CD133 + CD49f + ).

[0109] Therefore, compared to the time required to culture one or more target cells in the absence of one or more epigenetic modifiers (e.g., mixtures or supplements) to generate an expanded population of cells, target cells (e.g., HSPCs or CD34s) require significantly more time to produce an expanded population of cells. +An expanded population of cells can contain more original cells with one or more phenotypes selected from the following: CD34 + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Therefore, the methods disclosed herein (and supplements and supplemented culture media) can support HSPC or CD34. + Cell expansion. More specifically, the methods disclosed herein (and supplements and supplemental culture media) can preferentially support HSPCs or CD34. + Amplification of a primitive subset of cells, such as CD34 + CD45RA - CD90 + Cells, CD34 + CD45RA - CD90 + EPCR + Cells and CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + cell.

[0110] In some embodiments, the presence of at least one HDACi (e.g., valproic acid) helps promote the proliferation of target cells and the production of target cells capable of downstream differentiation (e.g., measurable by CFU assay). In the context of downstream differentiation potential (e.g., measurable by CFU), a mixture comprising a first HDACi, a second HDACi, and at least one HMTi (as described herein) may outperform a mixture comprising each of the first HDACi, the 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. Furthermore, in the context of downstream differentiation potential (e.g., measurable by CFU), a mixture comprising the first HDACi, the second HDACi, and at least one HMTi (as described herein) may outperform any other 3-combination selected from the first HDACi, the 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. Furthermore, in scenarios involving downstream differentiation potential (e.g., measurable via CFU), mixtures comprising a first HDACi, a second HDACi, and at least one HDMi (as described herein) may underperform, at least in terms of CFU-GEMM and / or CFU-G / M / GM and / or BFU-E output, compared to mixtures comprising each of the first HDACi, the second HDACi, at least one HDMi, and at least one HMTi. Additionally, in scenarios involving downstream differentiation potential (e.g., measurable via CFU), mixtures comprising a first HDACi, a second HDACi, and at least one HDMi (as described herein) may underperform, at least in terms of CFU-GEMM and / or CFU-G / M / GM and / or BFU-E output, compared to any other 3-combination selected from the first HDACi, the second HDACi, at least one HDMi, and at least one HMTi.

[0111] By practicing the methods disclosed in this paper, it may be possible to obtain large quantities of in vitro expanded cells (e.g., HSPCs and / or CD34 cells). + Cells or their ancestors and / or original subsets). Such expanded cells can be used in any relevant downstream applications, including in gene editing workflows, clinically or therapeutically, to study hematopoietic development in normal or diseased settings, or to differentiate downstream into hematopoietic lineages (e.g., myeloid, erythroid, and / or lymphoid).

[0112] In practice, functional HSPCs should be able to reconstruct the hematopoietic lineage of recipients subjected to sublethal doses of irradiation, and it has been shown that target cells obtained by practicing the methods disclosed herein engraft into subjects after introduction, and that they generate myeloid, lymphoid, and erythroid cells. Therefore, the methods of this disclosure may further include introducing expanded cells into a subject, wherein i) similar or higher levels of lymphoid (CD45) cells occur relative to the uncultured cells of the engrafted subject. + CD19 + B cell engraftment, and / or ii) similar or higher levels of myeloid (CD45) cells compared to uncultured cells. + CD33 + (iii) Implantation, and / or (iv) similar or higher levels of lymphoid and / or myeloid implantation relative to uncultured cells.

[0113] The following non-limiting examples illustrate this disclosure.

[0114] Example Example 1: Cell Procurement, Preparation and Culture Umbilical cord blood units were purchased from commercial suppliers. Human bone marrow samples and mobilized peripheral blood were purchased from STEMCELL Technologies. AML and CML samples were purchased from Discovery Life Sciences. CD34 + Cells were enriched from the aforementioned samples using the EasySep™ Human Umbilical Cord Blood CD34 Positive Selection Kit II (STEMCELL Technologies) and then frozen in serum containing 10% DMSO or used fresh.

[0115] For cultivation, approximately 4000 CD34 + Cells were plated in 24-well plates in serum-free StemSpan™ SFEM II (STEMCELL Technologies) medium supplemented with cytokines contained in StemSpan™ CD34 amplification supplement (STEMCELL Technologies). Additionally, cells were cultured in the presence of a published reference small molecule pyrimidine indole compound (Ref) or one or more epigenetic modifiers commercially available from STEMCELL Technologies.

[0116] Example 2: Flow cytometry and determination of cell counting and yield During or after the amplification culture as described in Example 1, aliquots of the culture can be harvested to assess phenotype, cell number, and frequency, such as by flow cytometry.

[0117] In short, cell samples / amplified samples are harvested by centrifugation and washed. Cells are stained with fluorophore-conjugated antibodies against CD34, CD90, CD45RA, and EPCR and analyzed on a CytoFLEX S™ flow cytometer (Beckman-Coulter). Dead cells are excluded by lateral and forward scattering profiles and 7-AAD staining.

[0118] Total viable cell counts are obtained using the Cytek® Guava® easyCyte™ flow cytometer. To calculate the output of a specific cell for each input cell, the total viable cell count is multiplied by the frequency % of the given cell type. For example, to calculate the output of CD34 for each input cell type... + CD34 of cells + The output is first calculated by multiplying the live cell count by the CD34 count obtained by flow cytometry. + %. Then divide that number by the input cell (in this case, CD34). + The final value is obtained by multiplying the total number of cells cultured in a well by the number of CD34 cells after cell separation. + The frequency of cell input to obtain CD34 + Cell count.

[0119] Example 3: Amplification of HSPC in the presence of one or more epigenetic modifiers Human umbilical cord blood HSPCs were essentially isolated and cultured as described in Example 1 (e.g., StemSpan™ SFEM II + StemSpan™ CD34 amplification supplement). Under certain conditions, cells were also cultured in the presence of a reference small molecule (Ref) or various epigenetic modifiers, either alone (1; 2; 3; or 4) or in various combinations of 2 (1-3; 1-4; 1-2; 2-3; or 2-4), 3 (1-2-3; 2-3-4; 1-2-4; or 1-3-4), or 4 (4i) mixtures. Figure 1 ).

[0120] On day 7, each input CD34 is determined as described in Example 2. + Total nucleated cells (TNC), ancestral CD34 + and the original subset CD34 bri CD45RA - CD90 + and CD34 bri CD45RA - CD90 + EPCR + Amplification. Each condition tested promoted TNC and ancestral CD34. +The amplification reached levels comparable to reference pyrimidine and indole compounds or negative controls. However, only certain 2-drug combinations (e.g., 1-3; 1-4; 1-2), 3-drug combinations (e.g., 1-2-3; 1-2-4; or 1-3-4), and 4-drug (4i) mixtures also promoted the amplification of the two original HPSC subsets (CD34) evaluated. bri CD45RA - CD90 + and CD34 bri 45RA - CD90 + EPCR + The amplification was higher than that of the reference compound and the control. Figure 1 ).

[0121] The in vitro multilineage differentiation capacity of these expanded HSPCs was measured using colony-forming unit (CFU) assays, as briefly described in this article. After 7 days of expansion, 400 cells / well were cultured in 6-well SmartDish™ plates (STEMCELL Technologies) in MethoCult™ H4435 enrichment medium (STEMCELL Technologies) and incubated for 14 days in a humidified incubator at 37°C and 5% CO2. Colonies were counted on day 14 using STEMvision™ (STEMCELL Technologies).

[0122] Figure 1 C and Figure 1 The results in D show that, compared with culture conditions including HDACi-1, the original subset of HSPC (CD34) + CD45RA - CD90 + and CD34 + CD45RA - CD90 + EPCR + The amplification of HSPCs was reduced in the mixture lacking HDACi-1 (valproic acid), indicating that HDACi-1 is crucial for amplification. However, HSPCs amplified in the absence of HDACi-1 did not show a significantly affected ability to form colonies in CFU assays. Figure 1 E). Conversely, HSPCs amplified in the absence of HMTi-1 (e.g., UNC-0638) exhibited a significant reduction in CFU output. In summary, it appears that the presence of HDACi-1 and HMTi-1 in a mixture of epigenetic modifiers facilitates the amplification of a primitive subset of HSPCs while also producing cells with acceptable colony-forming potential.

[0123] Example 4: Amplification of HSPC in a supplement containing epigenetic modifiers and antioxidants Human umbilical cord blood HSPCs were isolated and cultured in cytokine supplement +4i, as described in Example 1. In this experiment, TNCs, HSPCs, and a subset of the original HSPCs (CD34) were targeted. + CD45RA - CD90 + and CD34 + CD45RA - CD90 + EPCR + amplification of ) Figure 2 A- Figure 2 D) and in CFU assay ( Figure 2 E- Figure 2 F) Assess the effect of the presence or absence of antioxidants in a mixture of epigenetic modifiers.

[0124] Figure 2 E- Figure 2 F shows that the absence of an antioxidant in the 4i mixture (4i-AO) resulted in a lower colony number compared to the reference compound and conditions (4i) that included an antioxidant in the 4i mixture. Therefore, although the presence of an antioxidant in the 4i mixture of epigenetic modifiers did not appear to affect the output number of the original HSPC, it did appear to have a significant impact on the colony formation potential.

[0125] Example 5: Amplification of HSPCs from different sources in the presence of a mixture of epigenetic modifiers Human umbilical cord blood, peripheral blood, and bone marrow HSPCs were basically isolated and cultured for 7 or 14 days as described in Example 1, but different basal culture medium formulations were used with cytokine supplements and mixture 4i: StemSpan™ SFEM (STEMCELL Technologies), StemSpan™ SFEM II (STEMCELL Technologies), StemSpan™-XF (STEMCELL Technologies), or StemSpan™ AOF (STEMCELL Technologies).

[0126] For all tested HSPC sources, the 4i conditions yielded amplified HSPCs and subsets of the original HSPCs (e.g., CD34) in SFEM relative to the levels using reference pyrimidine and indole compounds. + CD45RA - CD90 + CD34 + CD45RA - CD90 + EPCR + and CD34 + CD45RA- CD90 + EPCR + CD133 + CD49f + The comparable or better 7th day () Figure 3 A) and day 14 ( Figure 3 B) Amplification. The same observations were obtained with other basal media tested (data not shown).

[0127] Example 6: Amplification of putative cancer stem cells or progenitor cells from leukemia samples CD34 was basically isolated from AML and CML samples as described in Example 1. + Cells were cultured. After 7 days, the cultured cells were stained with fluorescently labeled antibodies and analyzed by flow cytometry as described in Example 2.

[0128] Ancestor CD34 amplified from AML samples + and primitive ancestor subset (CD34) + CD45RA - CD90 + and CD34 + CD45RA - CD90 + EPCR + The frequency (A) and cell number (B) are shown in Figure 4 In comparison, CD34 was measured on day 7 with a cytokine mixture (CD34 supplement) or a reference pyrimidine indole compound. + CD45RA - CD90 + and CD34 + CD45RA - CD90 + EPCR + Higher frequency and output of cells.

[0129] Ancestor CD34 amplified from CML samples + and primitive ancestor subset (CD34) + CD45RA - CD90 + and CD34 + CD45RA - CD90 + EPCR + The frequency (A) and cell number (B) are shown in Figure 5 In comparison, CD34 was measured on day 7 with a cytokine mixture (CD34 supplement) or a reference pyrimidine indole compound. + CD45RA - CD90+ and CD34 + CD45RA - CD90 + EPCR + Higher frequency and output of cells.

[0130] Example 7: Implantation of amplified HSPCs into mice Human umbilical cord blood HSPCs were isolated and cultured for 7 days in the presence of cytokine supplement +4i, essentially as described in Example 1. After 7 days of expansion, 2500 initial CD34 cells were transplanted into sublethal irradiated NSG mice. + Cells or 2500 uncultured CD34 + Cells (control). Long-term multilineage engraftment was measured in the bone marrow of transplanted NSG mice at week 20.

[0131] Compared to recipients of uncultured cells, recipients of 4i-expanded cells showed similar or higher levels of human CD45 in the bone marrow of the analyzed mice. + and CD34 + Cell frequency. Compared to uncultured cells, recipients of 4i-expanded cells showed higher levels of HSPCs (CD34) and lymphocytes (CD45). + CD19 + ) and myeloid (CD45) + CD33 + Cell implantation (see Table 1).

[0132] Table 1. Implantation of expanded HSPCs. Data in the table are mean ± SD (n = 8 mice).

Claims

1. A method for culturing one or more hematopoietic stem cells and progenitor cells (HSPCs), the method comprising: The one or more HSPCs are contacted with a mixture of multiple epigenetic modifiers in a culture medium, the mixture comprising two or more of the following: i) Group 1 protein deacetylase inhibitors (HDACi), ii) Second HDACi, iii) At least one histone demethylase inhibitor (HDMi), and iv) at least one histone methyltransferase inhibitor (HMTi); and The one or more HSPCs are cultured in the presence of the mixture for a sufficient time to generate an amplified population of HSPCs.

2. The method of claim 1, further comprising separating or enriching the one or more HSPCs from the sample prior to contacting the population with the mixture.

3. The method according to claim 2, wherein the sample is whole blood, umbilical 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 HSPCs are included in an initial population containing HSPCs.

6. The method according to any one of claims 1 to 4, wherein the one or more HSPCs and / or the amplified population of HSPCs express CD34.

7. The method according to any one of claims 1 to 6, wherein the one or more HSPCs are primitive HSPCs having the following phenotype: CD34 + CD45RA - CD90 + Phenotype, CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Phenotype.

8. The method according to any one of claims 1 to 7, wherein the amplified population of HSPCs contains more HSPCs than when the one or more HSPCs are cultured in the absence of the mixture for a time sufficient to generate the amplified population of HSPCs.

9. The method according to any one of claims 1 to 8, wherein the expanded population of HSPCs contains more primitive cells having the following phenotype compared to the time required to culture the one or more HSPCs sufficiently to generate the expanded population of HSPCs without the presence of the mixture: CD34 + CD45RA - CD90 + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Phenotype.

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 of claim 10, wherein the first HDACi and the second HDACi are selected from: valproic acid, trichostatin A (TSA), entinolide, acetyldinarin, pabistamine, LMK235, and romidesin.

12. The method according to any one of claims 1 to 11, wherein the at least one HDMi is an LSD1 or LSD2 inhibitor.

13. The method according to any one of claims 1 to 12, wherein the at least one HMTi is G9a / GLPHMTi.

14. The method according to any one of claims 1 to 13, the method further comprising, when contacting the one or more HSPCs with a mixture comprising each of the first and second HDACi, at least one HDMi and at least one HMTi and culturing the one or more HSPCs in the presence of the mixture, producing a higher degree of amplification of the HSPCs compared to a mixture comprising only one, two or three of the foregoing.

15. The method according to any one of claims 1 to 13, the method further comprising, when contacting the one or more HSPCs with a mixture comprising two of the first HDACi or a functional equivalent thereof and the second HDACi, the at least one HDMi and the at least one HMTi, and culturing the one or more HSPCs in the presence of the mixture, producing a higher degree of amplification of the HSPCs compared to a mixture 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 acid.

16. The method according to any one of claims 1 to 13, the method further comprising, when contacting the one or more HSPCs with a mixture comprising the first HDACi or a functional equivalent thereof and the second HDACi, the at least one HDMi and the at least one HMTi, and culturing the one or more HSPCs in the presence of the mixture, producing a higher degree of amplification of the HSPCs compared to a mixture comprising 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, the method further comprising, when contacting the one or more HSPCs with a mixture comprising the first HDACi and the at least one HMTi and culturing the one or more HSPCs in the presence of the mixture, producing a higher-degree amplified population of HSPCs with multi-lineage differentiation capacity, wherein the first HDACi is valproic acid.

18. The method according to any one of claims 1 to 17, wherein the amplified population of HSPCs contains 2 to more than 100 times more HSPCs than when the one or more HSPCs are cultured in the absence of the mixture to generate the amplified 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 cells and progenitor cells (HSPCs), the method comprising: Contact one or more HSPCs with a serum-free culture medium containing at least a first-group protein deacetylase inhibitor (HDACi) and one or more of SCF, Flt3 ligand, and TPO; and The one or more HSPCs are cultured in the culture medium for a time sufficient to generate an amplified population of HSPCs, wherein the amplified population of HSPCs comprises primitive HSPCs having the following phenotype: CD34. + CD45RA - CD90 + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + Phenotype and / or CD34 + CD45RA - CD90 + EPCR + CD133 + CD49f + Phenotype.

21. The method of 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 the following: a second HDACi, at least one HDMi, and at least one HMTi.

23. An HSPC amplification supplement comprising a mixture of multiple epigenetic modifiers, said mixture comprising two or more of the following: i) Group 1 protein deacetylase inhibitors (HDACi), ii) Second HDACi, iii) At least one histone demethylase inhibitor (HDMi), and iv) At least one histone methyltransferase inhibitor (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), entinolide, acetyldinarin, pabistat, LMK235 and romidesin.

26. The supplement according to claim 23, wherein the at least one HDMi is an LSD1 or LSD2 inhibitor.

27. The supplement according to claim 23, wherein the at least one HMTi is G9a / GLP HMTi.

28. A culture medium comprising a basal culture medium and a supplement according to any one of claims 23 to 27.

29. The culture medium according to claim 28, wherein the culture medium further comprises one or more of SCF, FLT-3L and / or TPO.

30. The culture medium according to claim 28 or 29, wherein the culture medium is serum-free.

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