Diarylheptanoid-based compounds for ex VIVO expansion of hematopoietic stem cell populations and a method thereof

Abstract

The present invention relates to diarylheptanoid-based compounds and a method for inducing ex vivo expansion of hematopoietic stem cell populations. This method comprises incubating HSCs in a culture medium comprising diarylheptanoid-based compounds, leading to improved ex vivo expansion of HSCs and preservation of their major specific properties. This includes self-renewal and multilineage differentiation. These resultant enhancements demonstrate the potential of HSCs for use in treating hematological diseases and blood cancers prior to HSCT.

Description

DIARYLHEPTANOID-BASED COMPOUNDS FOR EX VIVO EXPANSION OF HEMATOPOIETIC STEM CELL POPULATIONS AND A METHOD THEREOF

[0001] This invention relates to biotechnology, more particularly, diarylheptanoid-based compounds forex vivoexpansion of hematopoietic stem cell populations and a method thereof.

[0002] Hematopoietic stem cells (HSCs) are crucial for treatment of blood disorders due to their ability to differentiate into red blood cells, white blood cells, and platelets. HSCs play a role in hematopoietic stem cell transplantation (HSCT), a highly effective therapy for hematological diseases, bone marrow failure conditions, and various congenital diseases such as leukemia and hemoglobinopathies. The primary sources of HSCs include bone marrow, umbilical cord blood, and peripheral blood. Peripheral blood hematopoietic stem cells (PB-HSCs) are a major source of HSCT, obtained after treatment with granulocyte stimulating factor (G-CSF) to mobilize HSCs from the bone marrow, which are then harvested via peripheral blood apheresis, known as mPB-HSCs.

[0003] However, theex vivoexpansion of mPB-HSC populations is limited in both quantity and quality, making the expanded HSCs insufficient for HSCT in some hematological diseases. To address these limitations, many developments inex vivoexpansion techniques aim to maintain essential properties of HSCs for HSCT. These involve expanding the HSC populations while preserving their major specific properties, including self-renewal and multilineage differentiation. Consequently, expanded mPB-HSCs hold potential for treating hematological diseases and various blood cancers. Nevertheless, HSCs expanded throughex vivoexhibit heterogeneity, requiring fluorescence staining techniques specific to HSC surface proteins to confirm their identity as HSCs. While CD34+ protein is a common marker, it is not specific to HSCs, requiring additional markers specific to adult blood cells and those clearly expressed in HSCs (e.g., CD38-, CD45RA-, CD90+, and CD49f+ proteins) to ensure the identification of HSCs that retain their properties.

[0004] Recent advances in hematological disorder treatment, particularly theex vivoexpansion using chemical small molecules, have shown promise in increasing HSC populations while preserving their essential properties. These small molecules show potential in enhancing self-renewal, delaying differentiation, improving homing, and inhibiting apoptosis of HSCs. Despite these advantages, the clinical application of these small molecules is impeded by concerns over their ability to stimulate unwanted erythroid cell types, thereby increasing a risk of cancer development. This requires further exploration to identify and develop safer small molecules with minimal risk.

[0005] European Journal of Medicinal Chemistry, 2010, 45 (10), 4446 disclosed that curcuminoids, the major constituents ofCurcuma longaL., were structurally modified into 55 curcuminoid analogs and evaluated for antimycobacterial activity againstMycobacterium tuberculosis. Among the active curcuminoids, isoxazole analogs were the most active group, with mono-O-methylcurcumin isoxazole being the most active compound. It exhibited higher antimycobacterial activity against multidrug-resistant M.tuberculosisclinical isolates than curcumin (parent compound). However, the study suggests the use of curcumin and curcuminoid analogs as antimycobacterial agents and does not reveal their use in the treatment of hematological diseases for the proliferation of HSCs that maintain specific functional activity.

[0006] Advancing the properties of curcuminoids, diarylheptanoid-based compounds are derived from curcumin through the addition of at least one isoxazole ring. These compounds have shown effects on blood and immune systems, exhibiting anti-inflammatory properties and stimulating immune erythroid cells. This leads to increased proliferation in various white blood cell types and an increase in the number of human T-cells and B-cells. This suggests that diarylheptanoid-based compounds could be beneficial in enhancing immune responses and possibly in the expansion of HSCs. The compounds can be used either alone, exhibiting outstanding properties, or in combination with other small molecules to achieve synergistic effects, enhancing the expansion of HSCs and their functional preservation.

[0007] Furthermore, Biomedicine and Pharmacotherapy, 2021, 143, 112102 studied theex vivoexpansion and functional preservation of HSCs using a small molecule treatment, particularly a diarylheptanoid compound isolated fromCurcumacomosa(ASPP049). The study demonstrated that culturing HSCs with this compound in an expansion medium led to improved HSC populations and functional preservation. These HSCs did not express cancer-related markers in immune-free mice for up to seven months, indicating the safety of the diarylheptanoid compound. Additionally, the diarylheptanoid compound treatment resulted in a substantial fold expansion of CD34+CD38-CD90+ cells compared to those cultured without the compound. However, the efficacy of the disclosed diarylheptanoid compound was limited as the fold-expansion level of CD34+CD38-CD90+ cells did not reach that of the positive control. Therefore, there remains a need for more efficient small molecules to improveex vivoexpansion methods of HSCs to enhance the quality of HSCs for the treatment of hematological diseases.

[0008] Based on the above disclosures ofex vivoexpansion and small molecules utilized in the hematological disorder treatment, as well as awareness of the potential drawbacks of existing compounds, the present invention develops diarylheptanoid-based compounds and a method for inducingex vivoexpansion of HSC populations. Specifically, the method comprises incubating HSCs in a culture medium comprising diarylheptanoid-based compounds, leading to improvedex vivoexpansion of HSCs and preservation of their major specific properties. This includes self-renewal and multilineage differentiation, which are crucial for effective HSC treatments. The resultant enhancements demonstrate the potential of these HSCs for use in treating hematological diseases and blood cancers prior to HSCT.

[0009] In one embodiment of the invention, the present invention relates to a diarylheptanoid-based compound for inducing anex vivoexpansion of hematopoietic stem cell populations having Formula (I) ……(I)

[0010] wherein

[0011] R1and R2are independently selected from hydrogen (H) or C1to C2alkane alkoxy group;

[0012] R3and R4are independently selected from hydrogen, hydroxy group (OH), C1to C12alkane alkoxy group, C7to C12aryl alkoxy group, C1to C4carboxyl group, C1to C6alkyl alkanoate group, or C1to C6alkenyl alkanoate group;

[0013] R5and R6are independently selected from hydrogen, hydroxy group, carbonyl group, oxime group, or R5and R6are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time.

[0014] In another embodiment of the invention, the present invention also relates to a diarylheptanoid-based compound for inducing anex vivoexpansion of hematopoietic stem cell populations having Formula (II) ……(II)

[0015] wherein

[0016] R7and R8are independently selected from hydrogen (H), hydroxy group (OH), or C1to C2alkane alkoxy group;

[0017] R9and R10are independently selected from hydrogen, hydroxy group, C1to C12alkane alkoxy group, or C2to C5alkene alkoxy group;

[0018] R11and R12are independently selected from hydrogen, hydroxy group, carbonyl group, methoxy group, or R11and R12are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time.

[0019] In another embodiment of the invention, the present invention also relates to a method for inducing anex vivoexpansion of hematopoietic stem cell populations comprising a step of incubating hematopoietic stem cells (HSCs) in a culture medium comprising a diarylheptanoid-based compound of Formula (I) ……(I)

[0020] wherein

[0021] R1and R2are independently selected from hydrogen (H) or C1to C2alkane alkoxy group;

[0022] R3and R4are independently selected from hydrogen, hydroxy group (OH), C1to C12alkane alkoxy group, C7to C12aryl alkoxy group, C1to C4carboxyl group, C1to C6alkyl alkanoate group, or C1to C6alkenyl alkanoate group;

[0023] R5and R6are independently selected from hydrogen, hydroxy group, carbonyl group, oxime group, or R5and R6are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time; and

[0024] wherein the method does not comprise a method of modifying the germline genetic identity of human beings.

[0025] In another embodiment of the invention, the present invention also relates to a method for inducing anex vivoexpansion of hematopoietic stem cell populations comprising a step of incubating hematopoietic stem cells (HSCs) in a culture medium comprising a diarylheptanoid-based compound of Formula (II) ……(II)

[0026] wherein

[0027] R7and R8are independently selected from hydrogen (H), hydroxy group (OH), or C1to C2alkane alkoxy group;

[0028] R9and R10are independently selected from hydrogen, hydroxy group, C1to C12alkane alkoxy group, or C2to C5alkene alkoxy group;

[0029] R11and R12are independently selected from hydrogen, hydroxy group, carbonyl group, methoxy group, or R11and R12are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time; and

[0030] wherein the method does not comprise a method of modifying the germline genetic identity of human beings.Fig.1

[0031] shows (A) cell viability and HSC markers from screening with diarylheptanoid-based compounds analyzed using flow cytometry, (B) number of live populations and CD34+CD90+ cell populations analyzed using flow cytometry.Fig.2

[0032] [Rectified under Rule 91, 18.11.2024]shows (A) an effect of three diarylheptanoid-based compounds on increasing CD34+CD90+ cells counts at various concentrations, and (B) proportion of CD34+CD90+ cells to total cells in response to the three diarylheptanoid-based compounds at various concentrations. The data are represented as mean ± standard error of mean (SEM).Fig.3

[0033] shows (A) an absolute cell numbers of each condition including negative control (DMSO), test groups (IDAH 107), and positive controls (UM171 and SR1) at various time points and (B) HSC populations analyzed using flow cytometry showing the effect of IDAH 107 on increasing the proportion of HSCs stained with protein-specific antibodies compared to DMSO.Fig.4

[0034] shows (A) a fold expansion of CD34+ cells, and (B) a fold expansion of CD34+CD38-CD45RA-CD90+CD49f+ cells at various time points. The data are presented as mean ± SEM from three independent experiments with three subjects. The difference was calculated by t-test in comparison to the negative control group (DMSO) and IDAH 107 (*p<0.05, **p<0.01, ***p<0.01), DMSO and UM171 (##p <0.01,###p<0.001), DMSO and SR1 ($p<0.05).Fig.5

[0035] shows (A) a diagram of HSC populations specific to various cell surface proteins analyzed using flow cytometry, showing attenuation of cytopainter fluorescence intensity at various time points (one peak of graph per one cycle of cell division) and (B) mean cytopainter fluorescence intensity in HSC populations specific to various cell surface proteins. The difference was calculated by t-test in comparison to negative control group (DMSO) and IDAH 107 (*p<0.05, **p<0.01, ***p<0.01), DMSO and UM171 (##p <0.01,###p<0.001), DMSO and SR1 ($p<0.05).Fig.6

[0036] [Rectified under Rule 91, 18.11.2024]shows (A) an analysis of CD34+ protein-specific HSC populations analyzed using flow cytometry showing amounts of DNA and RNA for cell cycle identification, and (B) proportion of cell cycle phases at various time points. The data are presented as mean ± SEM from three independent experiments with three subjects. The difference was calculated by t-test in comparison to negative control group (DMSO) and IDAH 107 (*p<0.05, **p<0.01, ***p<0.01), DMSO and UM171 (##p <0.01,###p<0.001), DMSO and SR1 ($p<0.05).Fig.7

[0037] shows aneffect of IDAH 107 on morphology of various blood colony types under a 100x magnification microscope.Fig.8

[0038] shows an ability of CD34+ cells cultured with IDAH 107 at different time points to differentiate into various blood cells types, including the number of blood colonies (A) and percentage distribution (B). The data are presented as mean ± SEM from three independent experiments with three subjects. The difference was calculated by t-test in comparison to negative control group and IDAH 107 of total CFUs (*p<0.05, **p<0.01), CFU-GM (##p <0.01,###p<0.001), BFU-E ($p<0.05,$$p<0.05), and CFU-GEMM (%p<0.05,%%p<0.01,%%%p<0.001,%%%%p<0.0001), for 3 or 9 days before being cultured without IDAH 107 for 11 days.Fig.9

[0039] shows fold expansion of CD34+ cells (A) and CD34+CD38-CD45RA-CD90+CD49f+ cells (B) compared to IDAH 107-cultured CD34+ cells for 3 or 9 days before being cultured without IDAH 107 for 11 days. The data are presented as mean ± SEM from three independent experiments with three subjects. The difference was calculated by t-test in comparison to negative control group (DMSO) and IDAH 107 (*p<0.05, ***p<0.001).Fig.10

[0040] shows a volcano diagram of gene expression levels induced by IDAH 107.Fig.11

[0041] shows a heatmap diagram comparing all gene patterns between pre-conditioning and ex vivo expansion with IDAH 107.Fig.12

[0042] shows (A) a gene enrichment ratio analysis and (B) a heatmap diagram comparing gene patterns in the MAPK pathway between pre-conditioning andex vivoexpansion with IDAH 107.Fig.13

[0043] shows an expression level evaluation diagram and relationships of biomolecules related to MAPK pathway via KEGG pathway analysis.Fig.14

[0044] shows (A) a diagram of blood cell population specific to human CD45+ (hCD45+) protein analyzed using flow cytometry, showing the proportion of human blood cells to mice blood cells after HSCT for 12 weeks and (B) HSCT of hCD45+ cells stimulated with IDAH 107 in various organs of the blood system after HSCT for 12 weeks. The data are presented as mean ± SEM from five mice per group (*p<0.05, **p<0.01).Fig.15

[0045] shows (A) a diagram of blood cell population specific to human CD45+ (hCD45+) protein analyzed using flow cytometry exhibited the proportion of human blood cells to mice blood cells after HSCT for 20 weeks and (B) HSCT of hCD45+ cells stimulated with IDAH 107 in various organs of the blood system after HSCT for 20 weeks. The data are presented as mean ± SEM from five mice per group (*p<0.05, **p<0.01).Fig.16

[0046] shows (A) an effect of IDAH 107 on the persistence of human blood cells lines after HSCT for 12 weeks and (B) an effect of IDAH 107 on the persistence of human blood cells lines after HSCT for 20 weeks. The data are presented as mean ± SEM from five mice per group (*p<0.05, **p<0.01).

[0047] The present invention relates to diarylheptanoid-based compounds and a method for inducingex vivoexpansion of HSC populations. The compounds and method enhance theex vivoexpansion of HSCs while preserving their major specific properties, including self-renewal and multilineage differentiation. These enhancements demonstrate the potential of HSCs for use in treating hematological diseases and blood cancers prior to HSCT.

[0048] Any embodiments depicted herein shall encompass modification to other aspects of this invention, unless stated otherwise.

[0049] Definition

[0050] Technical terms or scientific terms used herein have definitions as understood by those having ordinary skills in the art unless stated otherwise.

[0051] Equipment, apparatus, methods, or chemicals mentioned here refer to those commonly operated or used by those skilled in the art, unless explicitly stated otherwise, that they are equipment, apparatus, methods, or chemicals specifically used in this invention.

[0052] The terms “a,” “an,” and “the” in the claims or the specification should be interpreted as “one” as well as “one or more,” “at least one,” and “one or more than one,” unless the context clearly dictates otherwise, and so forth.

[0053] The use of singular or plural nouns with the term “comprising” in the claims or the specification should be interpreted as “one” as well as “one or more,” “at least one,” and “one or more than one.”

[0054] All compositions and / or processes disclosed and claimed are intended to encompass aspects of the invention that involve actions, operation, modifications, or changes of any parameters without deviating from experiments performed, examples described, or data shown in this invention, and obtaining similar objects with the same utilities and results as those described in the present invention by persons skilled in the art, even without specific mention in the claims. Therefore, substitutions or similar objects to the present invention, including minor modifications or changes that are apparent to persons skilled in the art, should be considered within the scope, and concept of the invention as defined by the appended claims.

[0055] Throughout this application, the term “about” is used to indicate that any value presented herein may potentially vary or deviate due to variety of factors, such as calculation errors, discrepancies in apparatus or methods, or differences between individual operators implementing the apparatus or methods.

[0056] The following specification is not intended to limit the scope of the invention in any manner.

[0057] In one embodiment of the invention, the present invention relates to a diarylheptanoid-based compound for inducing anex vivoexpansion of hematopoietic stem cell populations having Formula (I) ……(I)

[0058] wherein

[0059] R1and R2are independently selected from hydrogen (H) or C1to C2alkane alkoxy group;

[0060] R3and R4are independently selected from hydrogen, hydroxy group (OH), C1to C12alkane alkoxy group, C7to C12aryl alkoxy group, C1to C4carboxyl group, C1to C6alkyl alkanoate group, or C1to C6alkenyl alkanoate group;

[0061] R5and R6are independently selected from hydrogen, hydroxy group, carbonyl group, oxime group, or R5and R6are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time.

[0062] In a preferred exemplary embodiment of the invention, R1and R2are methoxy group.

[0063] In a preferred exemplary embodiment of the invention, R3and R4are independently selected from hydroxy group, methoxy group, phenylmethoxy group, acetate group, or methacrylate group.

[0064] In a preferred exemplary embodiment of the invention, R5and R6are independently selected from hydrogen, carbonyl group, oxime group, or R5and R6are taken together form a heterocyclic ring having structure .

[0065] In an exemplary embodiment of the invention, the diarylheptanoid-based compound is select from , , , , or .

[0066] In a preferred exemplary embodiment of the invention, the diarylheptanoid-based compound is .

[0067] In another embodiment of the invention, the present invention also relates to a diarylheptanoid-based compound for inducing anex vivoexpansion of hematopoietic stem cell populations having Formula (II) ……(II)

[0068] wherein

[0069] R7and R8are independently selected from hydrogen (H), hydroxy group (OH), or C1to C2alkane alkoxy group;

[0070] R9and R10are independently selected from hydrogen, hydroxy group, C1to C12alkane alkoxy group, or C2to C5alkene alkoxy group;

[0071] R11and R12are independently selected from hydrogen, hydroxy group, carbonyl group, methoxy group, or R11and R12are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time.

[0072] In a preferred exemplary embodiment of the invention, R7and R8are independently selected from hydrogen or methoxy group.

[0073] In a preferred exemplary embodiment of the invention, R9and R10are independently selected from hydrogen, hydroxy group, or penthoxy group.

[0074] In a preferred exemplary embodiment of the invention, R11and R12are independently selected from hydrogen, methoxy group, or R11and R12are taken together to form a heterocyclic ring having structures , or .

[0075] In an exemplary embodiment of the invention, the diarylheptanoid-based compound is select from , , or .

[0076] In a preferred exemplary embodiment of the invention, the diarylheptanoid-based compound is .

[0077] In another embodiment of the invention, the present invention also relates to a method for inducing anex vivoexpansion of hematopoietic stem cell populations comprising a step of incubating hematopoietic stem cells (HSCs) in a culture medium comprising a diarylheptanoid-based compound of Formula (I) ……(I)

[0078] wherein

[0079] R1and R2are independently selected from hydrogen (H) or C1to C2alkane alkoxy group;

[0080] R3and R4are independently selected from hydrogen, hydroxy group (OH), C1to C12alkane alkoxy group, C7to C12aryl alkoxy group, C1to C4carboxyl group, C1to C6alkyl alkanoate group, or C1to C6alkenyl alkanoate group;

[0081] R5and R6are independently selected from hydrogen, hydroxy group, carbonyl group, oxime group, or R5and R6are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time; and

[0082] wherein the method does not comprise a method of modifying the germline genetic identity of human beings.

[0083] In a preferred exemplary embodiment of the invention, R1and R2are methoxy group.

[0084] In a preferred exemplary embodiment of the invention, R3and R4are independently selected from hydroxy group, methoxy group, phenylmethoxy group, acetate group, or methacrylate group.

[0085] In a preferred exemplary embodiment of the invention, R5and R6are independently selected from hydrogen, carbonyl group, oxime group, or R5and R6are taken together form a heterocyclic ring having structure .

[0086] In an exemplary embodiment of the invention, the diarylheptanoid-based compound is select from , , , , or .

[0087] In a preferred exemplary embodiment of the invention, the diarylheptanoid-based compound is .

[0088] In an exemplary embodiment of the invention, an amount of the diarylheptanoid-based compound ranges from 1 to 10 µM per HSC density of 1x106cells / mL.

[0089] In an exemplary embodiment of the invention, the HSCs are obtained from mammalian cells.

[0090] In a preferred exemplary embodiment of the invention, the said mammalian cells are human cells.

[0091] In a particularly preferred exemplary embodiment of the invention, the said HSCs are selected from a group comprising human bone marrow, human peripheral blood, or human cord blood.

[0092] In an exemplary embodiment of the invention, the HSCs are differentiated into erythrocytes, granulocytes, macrophages, megakaryocytes, or multipotent mixed lineage colony type cells.

[0093] In another embodiment of the invention, a method for inducing anex vivoexpansion of hematopoietic stem cell populations comprising a step of incubating hematopoietic stem cells (HSCs) in a culture medium comprising a diarylheptanoid-based compound of Formula (II) ……(II)

[0094] wherein

[0095] R7and R8are independently selected from hydrogen (H), hydroxy group (OH), or C1to C2alkane alkoxy group;

[0096] R9and R10are independently selected from hydrogen, hydroxy group, C1to C12alkane alkoxy group, or C2to C5alkene alkoxy group;

[0097] R11and R12are independently selected from hydrogen, hydroxy group, carbonyl group, methoxy group, or R11and R12are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N);  represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time; and

[0098] wherein the method does not comprise a method of modifying the germline genetic identity of human beings.

[0099] In a preferred exemplary embodiment of the invention, R7and R8are independently selected from hydrogen or methoxy group.

[0100] In a preferred exemplary embodiment of the invention, R9and R10are independently selected from hydrogen, hydroxy group, or penthoxy group.

[0101] In a preferred exemplary embodiment of the invention, R11and R12are independently selected from hydrogen, methoxy group, or R11and R12are taken together to form a heterocyclic ring having structures , or .

[0102] In an exemplary embodiment of the invention, the diarylheptanoid-based compound is select from , , or .

[0103] In a preferred exemplary embodiment of the invention, the diarylheptanoid-based compound is .

[0104] In an exemplary embodiment of the invention, an amount of the diarylheptanoid-based compound ranges from 1 to 10 µM per HSC density of 1x106cells / mL.

[0105] In an exemplary embodiment of the invention, the HSCs are obtained from mammalian cells.

[0106] In a preferred exemplary embodiment of the invention, the said mammalian cells are human cells.

[0107] In a particularly preferred exemplary embodiment of the invention, the said HSCs are selected from a group comprising human bone marrow, human peripheral blood, or human cord blood.

[0108] In an exemplary embodiment of the invention, the HSCs are differentiated into erythrocytes, granulocytes, macrophages, megakaryocytes, or multipotent mixed lineage colony type cells.

[0109] It should be understood that examples of the invention described hereinafter have been presented by way of explanatory and exemplary embodiments of the present invention only. Thus, the breadth and scope of the present invention should be defined only in accordance with the claims and their equivalents issuing from this disclosure.Examples

[0110] Example 1 Preparation of diarylheptanoid-based compounds

[0111] The preparation method is disclosed in European Journal of Medicinal Chemistry, 2010, 45(3), 941-956 and European Journal of Medicinal Chemistry, 2010, 45(10), 4446-4457, which are hereby incorporated by reference in their entirety. It is further understood that the diarylheptanoid-based compounds of the present invention can be prepared by any methods known to one of ordinary skill in the art. The diarylheptanoid-based compounds are delineated in Table 1.

[0112] [Table 1] shows examples of diarylheptanoid-based compounds used for inducingex vivoexpansion of HSCsCode NameStructureIDAH025 IDAH026 IDAH027 IDAH028 IDAH079 IDAH089 IDAH092 IDAH093 IDAH095 IDAH097 IDAH103 IDAH105 IDAH106 IDAH107 IDAH108 IDAH110 IDAH111 IDAH112 IDAH113 IDAH114 IDAH115 IDAH116 IDAH117 IDAH119 IDAH120 IDAH122 IDAH123 IDAH124 IDAH125 IDAH126

[0113] Example 2 Diarylheptanoid-based compounds inducedex vivoexpansion of hematopoietic stem cells (HSCs)

[0114] Isolation and culture of CD34+hematopoietic stem cells (CD34+ cells)

[0115] Adult CD34+ cells were isolated from mobilized peripheral blood mononuclear cells (mPBMCs) obtained from leftover specimens from three subjects. These CD34+ cells were collected from the Stem Cell Transplant Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University. The protocol for CD34+ cell collection was approved by the Ethical Committee on Human Rights Related to Research Involving Human Subjects at Ramathibodi Hospital (COA.MURA2019 / 916). The CD34+ cells were then separated from the mPBMC fraction using an EasySep human CD34 Positive Selection Kit II following a manufacturer’s protocol.

[0116] [Rectified under Rule 91, 18.11.2024]The isolated adult CD34+ cells were cultured in a Stemspan-AOF cGMP expansion medium (Stem cell technology, Vancouver, Canada) supplemented with approximately 1 U / mL of penicillin, approximately 1 U / mL of streptomycin, approximately 50 ng / mL of stem cell factor (SCF), approximately 50 ng / mL of thrombopoietin (TPO), approximately 20 ng / mL of FMS-like tyrosine kinase 3 ligands (Flt-3L), and approximately 20 ng / mL of Interleukin 6 (IL-6). Initially, the CD34+ cells were seeded at a density of 1x106cells / mL. The cultures were incubated at about 37°C, with about 5% CO2in an about 80% humidified atmosphere. The CD34+ cells were cultured for at least 24 h and then subjected to further experiments with changing the medium every 2 days.

[0117] Cell number scoring

[0118] The CD34+ cells in the culture medium were harvested at days 1, 3, 6, and 9 in approximately 10 µL of volume. The harvested CD34+ cells were mixed with about 0.4%w / v Trypan blue in a ratio of about 1:1, resulting in a dilution factor of about 2. These mixtures were used to calculate concentration and population of CD34+ cells using dye exclusion assay with a hemocytometer under a microscope. The following formulas were used for these calculations:

[0119] 1) Cell concentration (cells / mL) = cell number x dilution factor x 104

[0120] 2) Total cell number = cell concentration x volume of measured cell culture medium.

[0121] Flow cytometry analysis

[0122] [Rectified under Rule 91, 18.11.2024]About 5x104cells of CD34+ cells were harvested from the culture medium in a sterile test tube based on the volume calculated from the above formula. Subsequently, PBE buffer (PBS, bovine serum albumin, and EDTA) was added in an equal volume to wash the cells, removing culture medium and excess protein. The cell suspension was then subjected to centrifugation at about 500x g at room temperature for about 5 min, and the supernatant was removed to obtain cell pellets. The obtained cell pellets were stained with a fluorescent dye or antibodies at about 4°C for about 20 min. After that, the cells were washed with PBE buffer and centrifuged again at about 500x g for about 5 min. The cell pellets were resuspended in about 200 to 500 µL of PBE buffer for protein signal detection using flow cytometers of BD Accuri C6 Plus Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and Attune Nxt Flow Cytometer (ThermoFisher scientific, Waltham, MA, USA). The CD34+ cells were analyzed using FlowJo VX (BD Biosciences, Franklin Lakes, NJ, USA) which exhibited both the percentage and number of fluorescently stained cells compared to unstained or antibody-stained control cells.

[0123] Diarylheptanoid-based compounds inducedex vivoexpansion of HSCs

[0124] The CD34+ cells were cultured for 9 days with medium replacement every 2 days, as described in the “Isolation and culture of CD34+ cells” section. The CD34+ cells were cultured with different groups of compounds: (1) negative controls (without diarylheptanoid-based compounds), (2) examples of diarylheptanoid-based compounds as listed in the Table 1, and (3) positive controls (commercial small molecules of a pyrimido-[4,5-b]-indole derivative (UM171) or StemRegenin1 (SR1)). The experimental screening concentration was set at about 5 µM for all compounds. The CD34+ cells were then harvested for flow cytometry analysis using anti-CD34+CD90+ fluorescence-conjugated antibodies to determine the biological activity of each diarylheptanoid-based compound in increasing the percentage of CD34+CD90+ cells and CD34+CD90+ cell populations.

[0125] As shown in Figure 1A, the flow cytometry results revealed that five diarylheptanoid-based compounds, i.e., IDAH 103, IDAH 106, IDAH 107, IDAH 114, and IDAH 115 could increase percentage of CD34+CD90+ cells with cell viability ranging from about 80% to 100%. Additionally, IDAH 107, IDAH 114, and IDAH 115 could increase the number of CD34+CD90+ cell populations as shown in Figure 1B. Although IDAH 026, IDAH 028, and IDAH 092 increased the number of CD34+CD90+ cell populations more than IDAH 103, IDAH 106, IDAH 107, IDAH 114, and IDAH 115, their cell viability was lower. Therefore, they were not chosen for further investigation.

[0126] The CD34+ cells from three subjects were cultured with each diarylheptanoid-based compound (IDAH 103, IDAH 106, IDAH 107, IDAH 114, or IDAH 115) at various concentrations ranging from about 1 to about 10 µM to determine the optimal condition for expanding CD34+CD90+ cell populations. The results demonstrated that the IDAH 107 at the concentration of about 10 µM could increase the cell population 3-fold compared to the negative control and 1.5-fold compared to the positive control as shown in Figure 2A. In addition, the proportion of CD34+CD90+ cells (black bars) to total cells (gray bars) according to five diarylheptanoid-based compounds at various concentrations demonstrated that IDAH 107 enhanced the CD34+CD90+ cell populations as shown in Figure 2B. Based on these findings, the IDAH 107 was chosen for use in further experiments due to its efficacy.

[0127] Example 3 Diarylheptanoid-based compounds induced primaryex vivoexpansion of hematopoietic stem cells (HSCs)

[0128] To investigate efficacy of IDAH 107 on the primaryex vivoexpansion of HSCs that express specific HSC markers (CD34+CD38-CD45RA-CD90+CD49f+), CD34+ cells from three subjects were cultured with different groups of compounds: (1) negative controls (without IDAH 107), (2) IDAH 107 at a concentration of about 10 µM, and (3) positive controls (with commercial small molecules; UM171 or SR1). The CD34+ cells were cultured for about 9 days, and the live cell population was evaluated using the “Cell number scoring” section and the percentage of CD34+CD38-CD45RA-CD90+CD49f+ cells was determined using the method described in “Flow cytometry analysis” section.

[0129] The absolute cell number under all conditions exhibited an increasing trend in CD34+ cell populations throughout the culture period. Notably, an increase in the cell population was observed on day 3 of CD34+ cells cultured with IDAH 107, as shown in Figure 3A. Moreover, IDAH 107 increased the percentage of CD34+, CD90+, CD49f+, CD38-, and CD45RA- protein expression higher than the negative control as shown in Figure 3B. The fold expansion of CD34+ cells at different times under all conditions showed that IDAH 107 increased CD34+ cell populations from day 3 to day 9, which was similar to the activity of UM171 (Figure 4A). Moreover, IDAH 107 also had an effect on the expansion of CD34+, CD90+, CD49f+, CD38-, and CD45RA- cells, stimulating the expansion of these cells more than the negative and positive controls (Figure 4B).

[0130] Example 4 Effect of diarylheptanoid-based compounds on cell division and cell cycle

[0131] Cell division measurement

[0132] CD34+ cells from three subjects were stained with a cytopainter fluorescent dye, to mark proteins within living cells. These stained cells were then cultured with different groups of compounds as described in Example 3 for about 9 days. The evaluation of live HSC populations was conducted according to the “Cell number scoring” section and the cell division of HSCs was determined using the method described in “Flow cytometry analysis” section.

[0133] The standard cell proliferation staining assay analysis was performed to study the decrease in cytopainter fluorescence over time. The principle behind cytopainter staining analysis is that if cell populations increase their rate of cell division, the fluorescence intensity decreases upon the increasing time.

[0134] The cell division pattern throughout the 9-day culture period, with or without IDAH 107, showed no different pattern in the total cells and the specific cell population as shown in Figure 5A. Mean fluorescence intensity analysis demonstrated that cell division rates in various cell populations with IDAH 107 remained stable and were not different from those in the control group (Figure 5B).

[0135] CD34+ cell cycle measurements

[0136] The CD34+ cells from three subjects were cultured with different groups of compounds as described in Example 3 for about 9 days. The live HSC populations were evaluated according to the “Cell number scoring” section. The CD34+ cells were harvested, stained with anti-CD34+ fluorescence-conjugated antibodies and subsequently stained with Hoechst 34580 / DNA-staining buffer and Pyronin Y / RNA-staining buffer according to the “Flow cytometry analysis” section.

[0137] The measurements were in the following order:

[0138] (a) measuring expression of anti-CD34+ fluorescence-conjugated antibodies that proliferated in the culture to study the cell cycle of specific CD34+ cell populations;

[0139] (b) measuring the expression of the stained cell cycle fluorescent dye in the HSC populations specific to only CD34+ cells using Hoechst’s principle of DNA distribution. This method indicates phases of the cell cycle: prophases (G1, S, and G2) and M phase in CD34+ cells. Additionally, Pyronin Y fluorescence intensity was used to determine RNA expression in CD34+ cells across all phases, except for quiescence (G0) phase, where no RNA was found, and G0 / G1 phase, where RNA expression was rarely found. This approach allowed for the identification of all phases of CD34+ cell cycle.

[0140] The effect of IDAH 107 on the CD34+ cell cycle exhibited variations in DNA and RNA amounts in different phases as shown in Figure 6A. IDAH 107 affected changes in the G0 / G1 (dark gray bar) and the G1 (light gray bar) phases at days 6 and 9 of the culture (Figure 6B) which corresponded to a steady state of HSCs undergoing metabolic processes in preparation for entering cell division to increase cell populations.

[0141] The results indicated that IDAH 107 did not affect the expansion of HSCs through stimulating cell division or altering cell cycle. Instead, it might increase the population through preserving HSCs properties. Moreover, the results were consistent with the results in Figure 2B, where IDAH 107 did not stimulate overall cell growth but rather specifically increased the specific proportion of HSCs.

[0142] Example 5 Effect of diarylheptanoid-based compounds on multilineage differentiation analysis

[0143] CD34+ cells from three subjects were cultured with different groups of compounds as described in Example 3 for about 3 and about 9 days. After that, CD34+ cells were purified using the method detailed in the “Isolation and culture of CD34+ cells” section. The isolated CD34+ cells were then cultured in Methocult H4434 Classic medium (Stem cell technology, Vancouver, Canada), which induces multilineage differentiation within about 14 days without additional stimulation of the cells. This aimed to simulate transplantation conditions in patients. The effect of IDAH 107 on preserving multilineage differentiation properties was observed when stimulated for about 3 or about 9 days with and without IDAH 107. Then, the CD34+ cells were cultured in Methocult medium without IDAH 107 for about 14 days.

[0144] After that, the induced CD34+ cells formed blood colonies in a hematopoietic lineage based on each of the lineage-committed HSCs. Each colony clearly had different characteristics. The colony-forming cell (CFU) assay was performed to count and separate the colonies based on size, color, and specific cell characteristics. They were divided into red blood cell colonies (burst-forming unit of erythrocytes; BFU-E), leukocyte colonies (CFU of granulocytes and macrophages; CFU-GM), and mixed colonies (CFU of granulocytes, erythrocytes, macrophages, and megakaryocytes; CFU-GEMM).

[0145] shows the differentiated CD34+ cells formed colonies comprised red blood cells (BFU-E), white blood cells (CFU-GM), and mixed blood cells (CFU-GEMM). Statistical analysis of the number of blood colonies showed that CD34+ cells cultured with IDAH 107 increased the total number of colonies and the number of colonies and the number of each type of blood cell compared to the control group at both culture times (Figure 8A).

[0146] Moreover, when comparing the percentage of lineage distribution, it was found that the proportion of blood lineage from CD34+ cells cultured with IDAH 107 was not different from the control group at the same time point. However, when comparing the proportions of blood lineage from CD34+ cells cultured at different times, it was found that the proportion of white blood cells increased, whereas the proportion of mixed blood cells decreased. The proportion of red blood cells did not show a difference (Figure 8B).

[0147] Example 6 Diarylheptanoid-based compound-expanded CD34+ cells preserve HSC biological functions

[0148] CD34+ cells in the culture medium were cultured for about 3 or about 9 days with or without IDAH 107 (both control groups and experimental group) from Example 3. These cells underwent CD34+ cell purification as described in the “Isolation and culture of CD34+ cells” section. The purified cells were then cultured again without further stimulation of CD34+ cells. This was done to study the effect of preserving specific biological function of HSCs when stimulated for only about 3 or about 9 days in the culture medium, followed by 11 days without IDAH 107 compared to CD34+ cells without stimulation (control group).

[0149] The culture medium was divided as follows:

[0150] (a) culture medium 1 (3+11) with IDAH 107 for 3 days, followed by 11 days without IDAH 107;

[0151] (b) culture medium 2 (9+11) with IDAH 107 for 9 days, followed by 11 days without IDAH 107.

[0152] After that, the measurements were performed according to the “Cell number scoring” and “Flow cytometry analysis” sections as well as the method described in Example 3.

[0153] The results exhibited that the conditions stimulated with IDAH 107 for 3 days, followed by 11 days without IDAH 107 (a) showed increase in fold expansion of CD34+ cells by 1.4-fold (Figure 9A). Moreover, both the (a) and (b) conditions showed increase in the fold expansion of CD34+CD38-CD45RA-CD90+CD49f+ cells by 1.4-fold and 1.6-fold, respectively, compared to the control group (Figure 9B).

[0154] Example 7 Effect of diarylheptanoid-based compounds on transcriptome

[0155] CD34+ cells from three subjects were cultured with different groups of compounds as described in Example 3 for about 24 h and about 9 days to simulate pre-conditioning andex vivoexpansion. After that, the CD34+ cells cultured both in the presence and absence of IDAH 107 underwent CD34+ cell purification and RNA extraction from nuclei using RNeasy Mini Kit (Qiagen, MD, USA) following a manufacturer’s protocol.

[0156] The obtained RNA was adjusted to a concentration of about 20 ng / μL before being employed to measure stem cell biology using 770 mRNA probes in the nCounter Human Stem Cell Characterization Panel (NanoString Tech). Also, the RNA was hybridized with these mRNA probes and then incubated at about 65℃ for about 18 h using a PCR machine. Subsequently, the samples were loaded onto nCounter Sprint Cartridge for gene expression evaluation with nCounter Digital Analyzer. Data were exported and analyzed using nSlover software v4.0.

[0157] The analyzed data were compared between pre-conditioning andex vivoexpansion, including comparisons of conditions with and without IDAH 107 using VolcaNoseR program. Biomolecular mechanisms related to gene enrichment analysis were selected through WEB-based GEne SeT analysis Toolkit using a false discovery rate (FDR) as a criterion. Relationships and functions of expressed genes in related mechanisms were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0158] The results demonstrated that IDAH 107 increased transcriptome gene expression patterns in both pre-conditioning andex vivoexpansion. A volcano diagram showed the biological mechanisms induced by IDAH 107 duringex vivoexpansion compared to pre-conditioning as shown in. However, there was variation in the response of relevant genes in CD34+ cells from three subjects to IDAH 107, indicated individual differences in gene expression patterns (). The variation was related to the differential expression of 56 genes commonly involved in signaling functions in the MAPK pathway (Figure 12A and 12B). In addition, gene enrichment ratio analysis revealed the biological mechanisms related to the effects of IDAH 107 from a group of expressed genes and KEGG pathway analysis provided that the expression levels of all genes played a signaling role in the process due to the effect of IDAH 107 ().

[0159] Example 8 Effect of diarylheptanoid-based compounds on hematopoietic stem cell transplantation (HSCT) in immunodeficiency mice

[0160] Animal studies were conducted under protocol no. MUSC65-032-625, approved by the Institutional Animal Care and Use Committee (MUSC–IACUC), Faculty of Science, Mahidol University. Female NOD / Shi-scid IL2rgamma (null) (NOG) mice, approximately 6 weeks old, were acclimatized before the experiment for about 5 to about 7 days, in a sterile closed system with 12-h light / dark cycles, at about 22°C to about 24°C, with a humidity of about 50% to about 60%, The mice had free access to food and water.

[0161] Before HSCT, all mice were pre-conditioned with Busulfan at a dose of about65 mg of drug / kg of body weight, administered in four equal doses (about 15 mg / kg) every 24 h. The experimental group was divided into three groups: (1) non-HSCT group, (2) HSCT-control group, and (3) HSCT-IDAH 107 group.

[0162] For HSCT procedure, the mice were anesthetized with isoflurane. CD34+ cells were injected intravenously at about 1x106cells / mouse. The CD34+ cells mobilized themselves to the mice’s bone marrow. After 12 weeks, all mice were euthanized to collect peripheral blood, bone marrow, and spleen.

[0163] HSC expression was detected using a CD45 blood cell-specific antibody. In addition, staining was performed with antibodies against the surface markers of other types of blood cells, including CD3 (T-cell lineage), CD20 (B-cell lineage), CD33 / 15 (myeloid lineage), and CD14 (monocytic lineage). The staining was done to study the changes that occur after transplantation.

[0164] The results revealed that the HSCT-IDAH 107 mice exhibited higher human CD45+ (hCD45+) expression than the HSCT-control group in the bone marrow and spleen of mice after 12 weeks of HSCT as shown in Figures 14A and 14B. Moreover, hCD45+ expression was higher than the negative control group in the bone marrow and bloodstream after 20 weeks of HSCT as shown in Figures 15A and 15B. Additionally, when the CD34+ cells in the bone marrow from HSCT-IDAH 107 mice were cultured and stimulated to differentiate into different types of hematopoietic cells for about 14 days, it was found that the number of all types of blood colonies was higher at both HSCT time points compared to the HSCT-control group as shown in Figures 16A and 16B.

[0165] To enhance HSC expansion and functional preservation while minimizing side effects, the diarylheptanoid-based compounds described in this invention can be used either alone or in combination with other compounds. This combination approach may increase HSC populations and cell viability, potentially achieving synergistic effects.

[0166] The subject matter described above is provided by way of illustration only and should not be construed as limiting. Any of the various features and elements disclosed herein may be combined with one or more other disclosed features and elements, unless indicated otherwise. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from scope of the present invention, which is set forth in the following claims.BEST MODE OF THE INVENTION

[0167] Best mode of the invention is as provided in the description of the invention.

Claims

[Rectified under Rule 91, 18.11.2024]A diarylheptanoid-based compound for inducing an ex vivo expansion of hematopoietic stem cell populations having Formula (I)……(I)whereinR1and R2are independently selected from hydrogen (H) or C1to C2alkane alkoxy group;R3and R4are independently selected from hydrogen, hydroxy group (OH), C1to C12alkane alkoxy group, C7to C12aryl alkoxy group, C1to C4carboxyl group, C1to C6alkyl alkanoate group, or C1to C6alkenyl alkanoate group;R5and R6are independently selected from hydrogen, hydroxy group, carbonyl group, oxime group, or R5and R6are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N); represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time.The diarylheptanoid-based compound according to claim 1, wherein R1and R2are methoxy group.The diarylheptanoid-based compound according to claim 1, wherein R3and R4are independently selected from hydroxy group, methoxy group, phenylmethoxy group, acetate group, or methacrylate group.The diarylheptanoid-based compound according to claim 1, wherein R5and R6are independently selected from hydrogen, carbonyl group, oxime group, or R5and R6are taken together form a heterocyclic ring having structure.The diarylheptanoid-based compound according to claim 1, wherein the diarylheptanoid-based compound is select from,,,, or.The diarylheptanoid-based compound according to claim 5, wherein the diarylheptanoid-based compound is.[Rectified under Rule 91, 18.11.2024]A diarylheptanoid-based compound for inducing an ex vivo expansion of hematopoietic stem cell populations having Formula (II)……(II)whereinR7and R8are independently selected from hydrogen (H), hydroxy group (OH), or C1to C2alkane alkoxy group;R9and R10are independently selected from hydrogen, hydroxy group, C1to C12alkane alkoxy group, or C2to C5alkene alkoxy group;R11and R12are independently selected from hydrogen, hydroxy group, carbonyl group, methoxy group, or R11and R12are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N); represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time.The diarylheptanoid-based compound according to claim 7, wherein R7and R8are independently selected from hydrogen or methoxy group.The diarylheptanoid-based compound according to claim 7, wherein R9and R10are independently selected from hydrogen, hydroxy group, or penthoxy group.The diarylheptanoid-based compound according to claim 7, wherein R11and R12are independently selected from hydrogen, methoxy group, or R11and R12are taken together to form a heterocyclic ring having structures, or.The diarylheptanoid-based compound according to claim 7, wherein the diarylheptanoid-based compound is select from,, or.The diarylheptanoid-based compound according to claim 11, wherein the diarylheptanoid-based compound is.[Rectified under Rule 91, 18.11.2024]A method for inducing an ex vivo expansion of hematopoietic stem cell populations comprising a step of incubating hematopoietic stem cells (HSCs) in a culture medium comprising a diarylheptanoid-based compound of Formula (I)……(I)whereinR1and R2are independently selected from hydrogen (H) or C1to C2alkane alkoxy group;R3and R4are independently selected from hydrogen, hydroxy group (OH), C1to C12alkane alkoxy group, C7to C12aryl alkoxy group, C1to C4carboxyl group, C1to C6alkyl alkanoate group, or C1to C6alkenyl alkanoate group;R5and R6are independently selected from hydrogen, hydroxy group, carbonyl group, oxime group, or R5and R6are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N); represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time; andwherein the method does not comprise a method of modifying the germline genetic identity of human beings.The method according to claim 13, wherein R1and R2are methoxy group.The method according to claim 13, wherein R3and R4are independently selected from hydroxy group, methoxy group, phenylmethoxy group, acetate group, or methacrylate group.The method according to claim 13, wherein R5and R6are independently selected from hydrogen, carbonyl group, oxime group, or R5and R6are taken together form a heterocyclic ring having structure.The method according to claim 13, wherein the diarylheptanoid-based compound is select from,,,, or.The method according to claim 17, wherein the diarylheptanoid-based compound is.The method according to claim 13, wherein an amount of the diarylheptanoid-based compound ranges from 1 to 10 µM per HSC density of 1x106cells / mL.The method according to claim 13, wherein the HSCs are obtained from mammalian cells.The method according to claim 20, wherein said mammalian cells are human cells.The method according to claim 21, wherein said HSCs are selected from a group comprising human bone marrow, human peripheral blood, or human cord blood.The method according to claim 13, wherein the HSCs are differentiated into erythrocytes, granulocytes, macrophages, megakaryocytes, or multipotent mixed lineage colony type cells.[Rectified under Rule 91, 18.11.2024]A method for inducing anex vivoexpansion of hematopoietic stem cell populations comprising a step of incubating hematopoietic stem cells (HSCs) in a culture medium comprising a diarylheptanoid-based compound of Formula (II)……(II)whereinR7and R8are independently selected from hydrogen (H), hydroxy group (OH), or C1to C2alkane alkoxy group;R9and R10are independently selected from hydrogen, hydroxy group, C1to C12alkane alkoxy group, or C2to C5alkene alkoxy group;R11and R12are independently selected from hydrogen, hydroxy group, carbonyl group, methoxy group, or R11and R12are taken together to form a heterocyclic ring having heteroatoms comprising oxygen (O) and / or nitrogen (N); represents either a single bond or a double bond, and adjacent bonds are not double bonds at the same time; andwherein the method does not comprise a method of modifying the germline genetic identity of human beings.The method according to claim 24, wherein R7and R8are independently selected from hydrogen or methoxy group.The method according to claim 24, wherein R9and R10are independently selected from hydrogen, hydroxy group, or penthoxy group.The method according to claim 24, wherein R11and R12are independently selected from hydrogen, methoxy group, or R11and R12are taken together to form a heterocyclic ring having structures, or.The method according to claim 24, wherein the diarylheptanoid-based compound is select from,, or.The method according to claim 28, wherein the diarylheptanoid-based compound is.The method according to claim 24, wherein an amount of the diarylheptanoid-based compound ranges from 1 to 10 µM per HSC density of 1x106cells / mL.The method according to claim 24, wherein the HSCs are obtained from mammalian cells.The method according to claim 31, wherein said mammalian cells are human cells.The method according to claim 32, wherein said HSCs are selected from a group comprising human bone marrow, human peripheral blood, or human cord blood.The method according to claim 24, wherein the HSCs are differentiated into erythrocytes, granulocytes, macrophages, megakaryocytes, or multipotent mixed lineage colony type cells.

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