Method for differentiating hematopoietic stem / progenitor cells into macrophages

Abstract

The invention relates to a method for inducing proliferation and / or differentiation of stem cells and / or hematopoietic stem / progenitor cells to macrophages. The method comprises the step of activating gamma-aminobutyric acid pathways of the stem cells and / or hematopoietic stem / progenitor cells. The invention further provides a culture medium for inducing the hematopoietic stem / progenitor cells to proliferate and / or differentiate into the macrophages and application of the culture medium.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for differentiating hematopoietic stem / progenitor cells into macrophages. Background Art

[0002] Macrophages are phagocytic immune cells that are widely distributed in the blood and tissues and originate from monocytes. Macrophages possess remarkable functional diversity: they participate in the recognition, phagocytosis, and degradation of cellular debris and pathogens, acting as first responders to infection. They also present antigens to T cells and induce other antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. Furthermore, they play a crucial role in the early stages of inflammation by releasing cytokines and chemokines, which in turn recruit other immune cells to the site of inflammation.

[0003] Hematopoietic stem cells (HSPCs) are adult stem cells in the blood that have the ability to differentiate and proliferate into one or more blood cell lineages. Macrophages are difficult to gene-edit, and macrophages derived from HSPCs can be considered genetically modified macrophages. Therefore, macrophages derived from HSPCs provide a powerful solution for human macrophage research. However, to date, there are no reported methods for in vitro differentiation of HSPCs into macrophages. Summary of the Invention

[0004] This application provides a method for differentiating stem cells and / or hematopoietic stem / progenitor cells into macrophages. The differentiation method provided herein incorporates the gamma-aminobutyric acid (GABA) signaling pathway, which can enhance the differentiation ability of hematopoietic stem / progenitor cells and promote their differentiation into macrophage-granulocyte progenitors (GMPs). The differentiation method provided herein can further incorporate the Notch signaling pathway to effectively enhance the differentiation effect of macrophages.

[0005] In one aspect, the present application provides a method for inducing stem cells to proliferate and / or differentiate into macrophage-granulocyte progenitors (GMPs), which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0006] On the other hand, the present application provides a method for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into macrophage-granulocyte progenitors (GMPs), which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0007] On the other hand, the present application provides a method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into macrophages, which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0008] In certain embodiments, the method comprises activating the GABAA and / or GABAC signaling pathway in the stem cell or hematopoietic stem / progenitor cell.

[0009] In certain embodiments, the method comprises activating a GABAA and / or GABAC receptor of the stem cell or hematopoietic stem / progenitor cell.

[0010] In certain embodiments, the method comprises administering a GABA pathway activator to the stem cells or hematopoietic stem / progenitor cells.

[0011] In certain embodiments, the GABA pathway activator comprises a GABAA pathway activator and / or a GABAC pathway activator.

[0012] In certain embodiments, the GABA pathway activator comprises a GABA receptor agonist.

[0013] In certain embodiments, the GABA receptor agonist comprises a GABAA receptor agonist and / or a GABAC receptor agonist.

[0014] In certain embodiments, the GABAA receptor agonist comprises one or more selected from the group consisting of Abecarnil, Barbiturates, Eszopiclone, Bamaluzole, Fengabine, GABA, Gabamide, GABOB, Gaboxadol, Ibotenic acid, Isoguvacine, Isonipecotic acid, Muscimol, Phenibut, Picamilon, Progabide, Propofol, Quisqualamine, SL75102, Thiomuscimol, Topiramate, and Zolpidem.

[0015] In certain embodiments, the GABAC receptor agonist comprises one or more selected from the group consisting of cis-4-aminocrotonic acid (CACA), (+)-cis-2-(aminomethyl)cyclopropanecarboxylic acid (CAMP), γ-aminobutyric acid (GABA), γ-amino-β-hydroxybutyric acid (GABOB), N4-Chloroacetylcytosine arabinoside, Picamilon, Progabide, and Tolgabide.

[0016] In certain embodiments, the GABA pathway activator comprises a positive allosteric enhancer (PAM) of the GABA receptor.

[0017] In certain embodiments, the positive allosteric modulator of the GABA receptor comprises a positive allosteric modulator of the GABAA receptor and / or a positive allosteric modulator of the GABAC receptor.

[0018] In certain embodiments, the positive allosteric modulator of the GABAA receptor comprises one or more selected from the group consisting of Alcohols, Avermectins, Barbiturates, Benzodiazepines, Bromides, Carbamates, Chloral hydrate, chloralose, petrichloral and other 2,2,2-trichloroethanolprodrugs, Chlormezanone, Clomethiazole, Dihydroergolines, Etazepine, Etifoxine, 2-Substituted phenols, Imidazoles, Kavalactones, Loreclezole, Neuroactive steroids, Nonbenzodiazepines, Propofol, Piperidinediones, Propanidid, Pyrazolopyridines, Quinazolinones, Skullcap constituents, Stiripentol, Disulfonylalkanes, Valerianconstituents and Volatile organic compounds.

[0019] In certain embodiments, the GABA pathway activator comprises GABA and / or a GABA derivative.

[0020] In certain embodiments, the GABA pathway activator comprises one or more selected from the group consisting of trans-4-aminocrotonic acid (TACA), muscitol, (±)-trans-2-(aminomethyl)cyclopropanecarboxylic acid (TAMP), trans-2-methyl-4-aminocrotonic acid (2-MeTACA), 3-(aminomethyl)-1-oxo-1-hydroxyphosphane (3-AMOHP), 3-(amino)-1-oxo-1-hydroxyphosphane (3-AOHP), 3-(guanidino)-1-oxo-1-hydroxy-phosphate (3-GOHP), 4-aminocyclopent-1-enecarboxamide (4-ACPAM) and 4-amino-N-hydroxycyclopent-1-enecarboxamide (4-ACPHA).

[0021] In certain embodiments, the GABA pathway activator comprises one or more selected from the group consisting of Muscimol, (R)-Baclofen, (RS)-Baclofen, SKF 97541, Acamprosate calcium, Thiomuscimol, Flurazepam, Flumazenil, 3-Aminopropylphosphonic Acid, AWD 131-138, Isoguvacine, TB21007, Kojic Amine, Progabide, SL 75102, 3-APSA, MRK 016, TP 003, L-838,417, MK 0343, RuBi GABA trimethylphosphine, RuBi-GABA, Abecarnil, Barbiturate, Eszopiclone, Bamaluzole, Gabamide, Ibotenic acid, Isonipecotic acid, Phenibut, Picamilon, Propofol, Quisqualamine, Topiramate, Zolpidem, 1,4-Butanediol, γ-Butyrolactone, γ-Hydroxybutyric acid, γ-Hydroxyvaleric acid, γ-Valerolactone, Lesogaberan, Phenibut, 4-Fluorophenibut, Tolgabide, N4-Chloroacetylcytosine arabinoside, Methionine, Gabapentin, Piperazine, Gabapentin HCl, Etomidate, 6-Hydroxyflavone, 3,4,5-Trimethoxycinnamic acid(TMCA), Glabridin, Afloqualone and Oxiracetam.

[0022] In certain embodiments, in the method, the concentration of the GABA pathway activator is about 0.1-1000 nM.

[0023] In certain embodiments, in the method, the concentration of the GABA pathway activator is about 10-20 nM.

[0024] In certain embodiments, the method comprises increasing the expression, function and / or activity of a GABA receptor in the stem cell or hematopoietic stem / progenitor cell.

[0025] In certain embodiments, the method comprises increasing the expression, function and / or activity of a GABA receptor in the hematopoietic stem / progenitor cells by gene regulation.

[0026] In certain embodiments, the gene regulation comprises one or more of the following methods: constructing an exogenous gene sequence for expression in a cell, and gene editing to regulate the upregulation and / or activation of an endogenous gene.

[0027] In certain embodiments, the method further comprises activating the Notch signaling pathway in the stem cell or hematopoietic stem / progenitor cell.

[0028] In certain embodiments, the method comprises administering a Notch signaling pathway activator to the stem cells or hematopoietic stem / progenitor cells.

[0029] In certain embodiments, the Notch signaling pathway activator comprises a Notch ligand.

[0030] In certain embodiments, the Notch ligand is selected from one or more of the following groups: DLL1 recombinant protein or its variant, DLL3 recombinant protein or its variant, DLL4 recombinant protein or its variant, Jagged-1 recombinant protein or its variant, Jagged-2 recombinant protein or its variant.

[0031] In certain embodiments, the Notch signaling pathway activator comprises a small molecule that induces Notch signaling.

[0032] In certain embodiments, the small molecule inducing Notch signal transduction is selected from one or more of the following groups: Valproic acid (VPA), Valproic Acid (NSC 93819) sodium salt.

[0033] In certain embodiments, the Notch signaling pathway activator includes RhNF-κB, which promotes the expression of Jagged1 and indirectly activates the Notch pathway.

[0034] In certain embodiments, activating the Notch signaling pathway of the stem cells or hematopoietic stem / progenitor cells comprises directly or indirectly activating the Notch signaling pathway of the stem cells or hematopoietic stem / progenitor cells.

[0035] In certain embodiments, the method comprises activating the Notch signaling pathway by gene regulation.

[0036] In certain embodiments, the gene regulation method comprises one or more of the following methods: transferring the Notch receptor intracellular domain (NICD) gene into cells for overexpression, upregulating Notch receptor, Notch ligand, CSL-DNA binding protein, cutting enzyme (Furin protease, ADAM metalloproteinase, γ-secretase) and / or Notch regulatory factor genes.

[0037] In certain embodiments, the method comprises overexpressing the Hes7 gene in the stem cell or hematopoietic stem / progenitor cell.

[0038] In certain embodiments, the method comprises overexpressing the Hes7 gene in the macrophage-granulocyte progenitor cells.

[0039] In certain embodiments, the method comprises culturing the stem cells or hematopoietic stem / progenitor cells using macrophage differentiation medium I, wherein the macrophage differentiation medium I comprises a basal medium and a GABA pathway activator.

[0040] In certain embodiments, the macrophage differentiation medium I further comprises a Notch pathway activator.

[0041] In certain embodiments, the macrophage differentiation medium I further comprises one or more selected from the group consisting of glutamine, β-mercaptoethanol, M-CSF and IL-3.

[0042] In certain embodiments, the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0043] In certain embodiments, the concentration of glutamine is about 1-5 mmol / L.

[0044] In certain embodiments, the concentration of β-mercaptoethanol is about 0.01-0.1 mmol / L.

[0045] In certain embodiments, the concentration of IL-3 is about 10-50 ng / mL.

[0046] In certain embodiments, the concentration of M-CSF is about 50-100 ng / mL.

[0047] In certain embodiments, the method further comprises using macrophage differentiation medium II, wherein the macrophage differentiation medium II comprises basal medium, glutamine, and M-CSF.

[0048] In certain embodiments, the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0049] In certain embodiments, the macrophage differentiation medium II further comprises a GABA pathway activator.

[0050] In certain embodiments, the macrophage differentiation medium II further comprises a Notch pathway activator.

[0051] In certain embodiments, the concentration of glutamine is about 1-5 mmol / L.

[0052] In certain embodiments, the concentration of M-CSF is about 50-100 ng / mL.

[0053] In certain embodiments, the method further comprises using a macrophage M1 polarization medium or a macrophage M2 polarization medium.

[0054] In certain embodiments, the macrophage M1 polarization medium comprises the macrophage differentiation medium II, LPS and IFN-g.

[0055] In certain embodiments, the concentration of LPS is about 10-200 ng / mL.

[0056] In certain embodiments, the concentration of IFN-g is about 1-20 ng / mL.

[0057] In certain embodiments, the macrophage M2 polarization medium comprises the macrophage differentiation medium II, IL-4, and IL-13.

[0058] In certain embodiments, the concentration of IL-4 is about 10-50 ng / mL.

[0059] In certain embodiments, the concentration of IL-13 is about 10-50 ng / mL.

[0060] In certain embodiments, the macrophage M1 polarization medium or the macrophage M2 polarization medium further comprises a GABA pathway activator.

[0061] In certain embodiments, the macrophage M1 polarization medium or the macrophage M2 polarization medium further comprises a Notch pathway activator.

[0062] In certain embodiments, the method comprises the following steps: 1) inoculating stem cells or hematopoietic stem / progenitor cells in the macrophage differentiation medium I for culture to obtain granulocyte-macrophage progenitor cells; 2) continuing culture using the macrophage differentiation medium II to obtain macrophages M0; 3) continuing culture using the macrophage M1 polarization medium or the macrophage M2 polarization medium to obtain M1 macrophages or M2 macrophages.

[0063] In certain embodiments, the method comprises the following steps: 1) inoculating stem cells or hematopoietic stem / progenitor cells in the macrophage differentiation medium I and culturing for about 1-2 weeks to obtain granulocyte-macrophage progenitor cells; 2) continuing to culture using the macrophage differentiation medium II for about 5-7 days to obtain macrophages M0; 3) continuing to culture using the macrophage M1 polarization medium or the macrophage M2 polarization medium for about 2 days to obtain M1 macrophages or M2 macrophages.

[0064] In certain embodiments, the culture conditions in step 1) are about 35-39° C. and about 3-7% CO 2 .

[0065] In certain embodiments, the hematopoietic stem / progenitor cells are derived from induced pluripotent stem cells.

[0066] In certain embodiments, the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0067] In certain embodiments, the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0068] In certain embodiments, the hematopoietic stem / progenitor cells are derived from bone marrow.

[0069] In certain embodiments, the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0070] On the other hand, the present application also provides a culture medium comprising a GABA pathway activator and a macrophage differentiation medium.

[0071] In certain embodiments, the GABA pathway activator comprises a GABA receptor agonist and / or a positive allosteric modulator of the GABA receptor.

[0072] In certain embodiments, the GABA pathway activator comprises GABA and / or a GABA derivative.

[0073] In certain embodiments, the culture medium further comprises a Notch pathway activator.

[0074] In certain embodiments, the macrophage differentiation medium comprises macrophage differentiation medium I, and the macrophage differentiation medium I comprises basal medium.

[0075] In certain embodiments, the macrophage differentiation medium I further comprises one or more selected from the group consisting of glutamine, β-mercaptoethanol, M-CSF and IL-3.

[0076] In certain embodiments, the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0077] In certain embodiments, the concentration of glutamine is about 1-5 mmol / L.

[0078] In certain embodiments, the concentration of β-mercaptoethanol is about 0.01-0.1 mmol / L.

[0079] In certain embodiments, the concentration of M-CSF is about 50-100 ng / mL.

[0080] In certain embodiments, the concentration of IL-3 is about 10-50 ng / mL.

[0081] In certain embodiments, the macrophage differentiation medium comprises macrophage differentiation medium II, which comprises basal medium, glutamine, and M-CSF.

[0082] In certain embodiments, the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0083] In certain embodiments, the concentration of glutamine is about 1-5 mmol / L.

[0084] In certain embodiments, the concentration of M-CSF is about 50-100 ng / mL.

[0085] In certain embodiments, the culture medium comprises macrophage M1 polarization medium or macrophage M2 polarization medium.

[0086] In certain embodiments, the macrophage M1 polarization medium comprises the macrophage differentiation medium II, LPS and IFN-g.

[0087] In certain embodiments, the concentration of LPS is about 10-200 ng / mL.

[0088] In certain embodiments, the concentration of IFN-g is about 1-20 ng / mL.

[0089] In certain embodiments, the macrophage M2 polarization medium comprises the macrophage differentiation medium II, IL-4, and IL-13.

[0090] In certain embodiments, the concentration of IL-4 is about 10-50 ng / mL.

[0091] In certain embodiments, the concentration of IL-13 is about 10-50 ng / mL.

[0092] On the other hand, the present application also provides use of the culture medium in generating macrophages by differentiating stem cells or hematopoietic stem / progenitor cells.

[0093] On the other hand, the present application also provides a composition comprising stem cells or hematopoietic stem / progenitor cells, and the culture medium.

[0094] In another aspect, the present application also provides modified stem cells or hematopoietic stem / progenitor cells, wherein the expression and / or activity of the GABA receptor is increased compared to unmodified stem cells or hematopoietic stem / progenitor cells.

[0095] In certain embodiments, the GABA receptor comprises a GABAA receptor and / or a GABAC receptor.

[0096] In certain embodiments, the Notch signaling pathway of the modified stem cells or hematopoietic stem / progenitor cells is activated.

[0097] On the other hand, the present application also provides the use of a GABA pathway activator in preparing a reagent for macrophage differentiation.

[0098] In certain embodiments, the reagent for macrophage differentiation further comprises a Notch pathway activator.

[0099] On the other hand, the present application also provides the use of a GABA pathway activator in producing a culture medium for differentiating stem cells or hematopoietic stem / progenitor cells into macrophages.

[0100] In certain embodiments, the culture medium further comprises a Notch pathway activator.

[0101] On the other hand, the present application also provides a culture platform for obtaining macrophages derived from hematopoietic stem / progenitor cells, which comprises the method, the culture medium, and / or hematopoietic stem / progenitor cells.

[0102] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0104] Figure 1 Shown is the detection of the effect of GABA pathway activators on macrophage proliferation on day 7 and day 14 after culture as described in the present application.

[0105] Figure 2 Shown is the detection of the effects of GABA pathway activators and / or Notch signaling pathway activators on macrophage proliferation on day 7 and day 14 after culture as described in the present application.

[0106] Figure 3A and Figure 3B Shown are graphs showing the results of detecting the effects of the GABA pathway activator and / or Notch signaling pathway activator described in the present application on macrophage differentiation after 7 days of culture.

[0107] Figure 4A and Figure 4B Shown is a graph showing the test results of the effects of the GABA pathway activator and / or Notch signaling pathway activator described in the present application on macrophage differentiation after 14 days of culture. DETAILED DESCRIPTION

[0108] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0109] Definition of terms

[0110] In this application, the term "derivative" generally refers to a chemical substance that is structurally related to another chemical substance, or a chemical substance that can be prepared from another chemical substance (i.e., a chemical substance from which the chemical substance is derived), for example, by chemical or enzymatic modification. In this application, the term "derivative" can refer to a substance that has a structure derived from a parent compound and is similar to the structure of the parent compound. A derivative can exhibit functions and / or activities similar to those of the parent compound.

[0111] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0112] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0113] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0114] In this application, the term "proliferation" generally refers to increasing the number of cells of a particular cell type or types from a starting population of cells, which may be different or the same. The starting cells used for proliferation need not be the same as the cells resulting from proliferation. For example, the proliferated cells can arise from the growth and differentiation of the starting population of cells.

[0115] In this application, the term "differentiation" generally refers to the process by which a non-specific or less specific cell acquires the characteristics of a specific cell. A differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.

[0116] In this application, the term "marker phenotype" generally refers to identifying markers or antigens on cells to determine their phenotype (e.g., differentiation state and / or cell type). For example, immunophenotyping can be used, which uses antibodies to identify antigens presented on cells. Antibodies can be monoclonal or polyclonal, and are generally selected to have smaller cross-reactions with other cell markers. These markers that determine the same cell type between species can be identified based on the same markers, and there may be differences in the structure of these markers (e.g., amino acid sequence) between species. Cell markers can include cell differentiation markers, as well as gene expression markers. Gene expression markers can include expressed genes that can indicate cell type or differentiation state.

[0117] In the present application, the term "GABAA" receptor generally refers to a pentameric protein that forms a membrane ion channel. The "GABAA" receptor described in the present application also encompasses its variants, homologues, analogs and / or derivatives.

[0118] In this application, the term "GABAC" receptor may also be referred to as GABA-ρ receptor, GABA-r, GABRR, and is generally a homomer ligand-gated ion channel composed of ρ subunits. The "GABAC" receptor described in this application also encompasses variants, homologues, analogs and / or derivatives thereof.

[0119] In this application, the term "stem cell" generally refers to a self-replicating and multipotent cell that has one or more of the following properties: (1) long-term self-renewal, or the ability to produce at least one identical copy of the original cell, (2) differentiation into multiple, and in some cases, only one specialized cell type at the single-cell level, and (3) functional regeneration of tissue in vivo. Stem cells are subdivided into totipotent, pluripotent, multipotent, and oligo / unipotent according to their developmental potential.

[0120] In this application, the term "progenitor cells" generally also have the ability to self-renew and differentiate into more mature cells, but are committed to a certain lineage (e.g., hematopoietic progenitor cells are committed to the blood lineage; myeloid progenitor cells are committed to the bone marrow lineage; lymphoid progenitor cells are committed to the lymphoid lineage), while stem cells do not necessarily have such limitations.

[0121] Hematopoietic stem cells (HSCs) produce committed hematopoietic progenitor cells (HPCs) that can form a pool of mature blood cells throughout the life of an organism. The term "hematopoietic stem cell" or "HSC" refers to a multipotent stem cell that produces all blood cell types of an organism, including myeloid lineages (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-cells), as well as other lineages known in the art.

[0122] In this application, the term "induced pluripotent stem cells" may be generally abbreviated as iPS cells or iPSCs, and generally refers to a type of pluripotent stem cells artificially prepared from non-pluripotent cells. For example, the artificial method may be the introduction of reprogramming factors. For example, the non-pluripotent cells may be adult somatic cells or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.

[0123] In this application, the term "GABA pathway activator" generally refers to any substance that can activate or enhance the intracellular gamma-aminobutyric acid (GABA) signaling pathway, for example, small molecules, nucleic acids, etc. The "GABA signaling pathway" of this application may include any signal processor involved in the GABA-related signaling pathway, including its upstream signaling pathway and / or its downstream signaling pathway. Exemplary GABA pathway activators include GABA molecules and may also include derivatives of GABA. For example, the GABA pathway activator may include a GABA receptor agonist. In this application, the term "agonist" generally refers to an agent that causes an increase in the expression and / or activity of a target gene or protein. An agonist can bind to its cognate receptor in some form and activate it, which can directly or indirectly bring about a physiological effect on the target gene or protein.

[0124] In this application, the term "cytokine" generally refers to a compound or component (e.g., an autoimmune factor) produced by a cell and affecting the physiological state of the cell (self) or other cells that produces the cytokine. Cytokines also include any compound or component produced by recombinant or synthetic processing, the products of which have similar structures and / or biological activities to naturally occurring forms. For example, the cytokine also encompasses truncated forms, functionally active fragments, homologues, analogs and variants thereof.

[0125] The term "composition" as used herein generally refers to a product comprising the specified ingredients in the specified amounts, as well as any product produced directly or indirectly by the combination of the specified ingredients in the specified amounts. In this application, the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0126] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0127] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0128] In this application, the term "in vivo" generally refers to within a living organism. For example, in some cases, "in vivo" can refer to a specific location in a subject tissue or organ.

[0129] In this application, the term "Notch signaling pathway" may encompass all molecules and pathways that can affect the Notch signaling pathway. For example, the Notch signaling pathway may include a Notch receptor, a Notch ligand, a CSL DNA binding protein, downstream target genes, and the like. For example, the Notch signaling pathway may be activated by a Notch receptor. For example, the Notch signaling pathway may be activated by a Notch ligand. For example, the Notch signaling pathway may also be activated indirectly. Detailed Description of the Invention

[0131] method

[0132] In one aspect, the present application provides a method for inducing stem cells to proliferate and / or differentiate into macrophage-granulocyte progenitors (GMPs), which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0133] On the other hand, the present application provides a method for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into macrophage-granulocyte progenitors (GMPs), which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0134] On the other hand, the present application provides a method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into macrophages, which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0135] In the present application, the method may include differentiating hematopoietic stem / progenitor cells into macrophage-granulocyte progenitor cells, and / or differentiating macrophage-granulocyte progenitor cells into macrophages. In the present application, the method may further include polarization of macrophages.

[0136] In the present application, the method may comprise a process of differentiating stem cells into macrophage-granulocyte progenitor cells, and / or a process of differentiating macrophage-granulocyte progenitor cells into macrophages. In the present application, the method may further comprise a process of polarizing macrophages.

[0137] In the present application, the concentration of the GABA pathway activator is about 0.1-1000 mM. For example, the concentration of the GABA pathway activator is about 0.1-900 mM, about 0.1-900 mM, about 0.1-800 mM, about 0.1-700 mM, about 0.1-600 mM, about 0.1-500 mM, about 0.1-400 mM, about 0.1-300 mM, about 0.1-200 mM, about 0.1-100 mM, about 1-50 mM, about 5-20 mM, about 10-100 mM, about 20- In some embodiments, the present invention relates to a novel nanostructured liquid or a nanostructured liquid. In some embodiments, the nanostructured liquid or the nanostructured liquid is at least about 200 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, or about 900 mM.

[0138] In the present application, the GABA signaling pathway in the method may include the GABAA and / or GABAC signaling pathway.

[0139] For example, the cell is a hematopoietic stem cell. For example, the cell is a hematopoietic progenitor cell. For example, the cell is a stem cell. For example, the cell is a hematopoietic stem / progenitor cell. For example, the GABA signaling pathway is a GABAA signaling pathway. For example, the GABA signaling pathway is a GABAC signaling pathway.

[0140] In the present application, the activation of the GABA pathway may be achieved by regulating the expression, function and / or activity of upstream and downstream related proteins of the GABA pathway.

[0141] In the present application, the method of inducing proliferation and / or differentiation of hematopoietic stem / progenitor cells into macrophages may comprise increasing the expression, function and / or activity of GABA receptors in the hematopoietic stem / progenitor cells.

[0142] For example, the expression of the GABA receptor in the hematopoietic stem / progenitor cells is upregulated by about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, or about 300% compared to unmodified hematopoietic stem / progenitor cells.

[0143] For example, the activity of the GABA receptor in the hematopoietic stem / progenitor cells is upregulated by about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, or about 300% compared to unmodified hematopoietic stem / progenitor cells.

[0144] For example, the function of the GABA receptor in the hematopoietic stem / progenitor cells is enhanced by about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, or about 300% compared to unmodified hematopoietic stem / progenitor cells.

[0145] In the present application, the GABA signaling pathway activated in hematopoietic stem / progenitor cells may be activated at the gene level, at the transcription level, at the translation level, and / or at the protein level.

[0146] For example, activation can be achieved by administering chemical regulatory means or by genetic regulatory means.

[0147] For example, the chemical modulation method may include administering a GABA pathway activator. The GABA pathway activator may directly or indirectly activate the GABA pathway. For example, the GABA pathway activator may act directly on GABA receptors. For example, the GABA pathway activator may indirectly activate the GABA pathway by acting on other pathways in the cell.

[0148] For example, the gene regulation means may include using CRISPR to regulate endogenous gene activation and / or upregulation of expression. For example, the CRISPR / Cas9 system may be used to regulate endogenous gene activation and / or upregulation of expression.

[0149] For example, this can be achieved by introducing an exogenous protein or an exogenous nucleic acid molecule encoding the protein, or by causing increased expression of an endogenous protein or an endogenous gene encoding the protein. For example, upregulation of GABA receptor expression may be caused by a mutation in the regulatory region of the gene encoding the receptor. In some cases, upregulation of GABA receptor expression can be achieved by altering the function of one or more components in the translation and / or transcription process.

[0150] For example, the gene regulation approach can be through small activating RNA (saRNA) approach.

[0151] In the present application, the method may include the following steps: 1) inoculating stem cells or hematopoietic stem / progenitor cells in the macrophage differentiation medium I for culture to obtain granulocyte-macrophage progenitor cells; 2) continuing culture using macrophage differentiation medium II to obtain macrophages M0; 3) continuing culture using macrophage M1 polarization medium or macrophage M2 polarization medium to obtain M1 macrophages or M2 macrophages.

[0152] In the present application, the method may include the following steps: 1) inoculating stem cells or hematopoietic stem / progenitor cells in the macrophage differentiation medium I and culturing for about 1-2 weeks to obtain granulocyte-macrophage progenitor cells; 2) continuing to culture using the macrophage differentiation medium II for about 5-7 days to obtain macrophages M0; 3) continuing to culture using the macrophage M1 polarization medium or the macrophage M2 polarization medium for about 2 days to obtain M1 macrophages or M2 macrophages.

[0153] For example, the culturing condition of step 1) may be about 35-39°C, for example, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C.

[0154] For example, the culture condition of step 1) may be about 3-7% CO2, such as about 3% CO2, about 4% CO2, about 5% CO2, about 6% CO2, or about 7% CO2.

[0155] In certain embodiments, the hematopoietic stem / progenitor cells are derived from induced pluripotent stem cells.

[0156] In certain embodiments, the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0157] In certain embodiments, the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0158] In certain embodiments, the hematopoietic stem / progenitor cells are derived from bone marrow.

[0159] In certain embodiments, the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0160] culture medium

[0161] In the present application, the method may include culturing the stem cells or hematopoietic stem / progenitor cells using Macrophage Differentiation Medium I. For example, the Macrophage Differentiation Medium I may include a basal medium and a GABA pathway activator. For example, the Macrophage Differentiation Medium I may further include a Notch pathway activator. For example, the Macrophage Differentiation Medium I may further include one or more selected from the group consisting of glutamine, β-mercaptoethanol, M-CSF, and IL-3.

[0162] In the present application, the method may further comprise using a macrophage M1 polarization medium or a macrophage M2 polarization medium.

[0163] In the present application, the macrophage M1 polarization medium may comprise the macrophage differentiation medium II, LPS and IFN-g.

[0164] In the present application, the macrophage M2 polarization medium may contain the macrophage differentiation medium II, IL-4 and IL-13.

[0165] For example, the macrophage differentiation medium II may comprise a basal medium. For example, the macrophage differentiation medium II may further comprise glutamine and / or M-CSF.

[0166] In the present application, the basal culture medium may include Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0167] In the present application, the concentration of glutamine may be about 1-5 mmol / L. For example, the concentration of glutamine is about 1 mmol / L. For example, the concentration of glutamine is about 1.5 mmol / L. For example, the concentration of glutamine is about 2 mmol / L. For example, the concentration of glutamine is about 2.5 mmol / L. For example, the concentration of glutamine is about 3 mmol / L. For example, the concentration of glutamine is about 3.5 mmol / L. For example, the concentration of glutamine is about 5 mmol / L. For example, the concentration of glutamine is about 5 mmol / L.

[0168] In the present application, the concentration of the β-mercaptoethanol may be about 0.01-0.1 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.01 mmol / L. For example, the concentration of the β-mercaptoethanol is 0.02 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.03 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.04 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.05 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.06 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.07 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.08 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.09 mmol / L. For example, the concentration of the β-mercaptoethanol is about 0.1 mmol / L.

[0169] In the present application, the concentration of IL-3 may be about 10-50 ng / mL. For example, the concentration of IL-3 is about 10 ng / mL. For example, the concentration of IL-3 is about 15 ng / mL. For example, the concentration of IL-3 is about 20 ng / mL. For example, the concentration of IL-3 is about 25 ng / mL. For example, the concentration of IL-3 is about 30 ng / mL. For example, the concentration of IL-3 is about 35 ng / mL. For example, the concentration of IL-3 is about 40 ng / mL. For example, the concentration of IL-3 is about 45 ng / mL. For example, the concentration of IL-3 is about 50 ng / mL.

[0170] In the present application, the concentration of M-CSF may be about 50-100 ng / mL. For example, the concentration of M-CSF is about 50 ng / mL. For example, the concentration of M-CSF is about 60 ng / mL. For example, the concentration of M-CSF is about 70 ng / mL. For example, the concentration of M-CSF is about 80 ng / mL. For example, the concentration of M-CSF is about 90 ng / mL. For example, the concentration of M-CSF is about 100 ng / mL.

[0171] In the present application, the concentration of LPS may be about 10-200 ng / mL. For example, the concentration of LPS may be about 10 ng / mL. For example, the concentration of LPS may be about 20 ng / mL. For example, the concentration of LPS may be about 50 ng / mL. For example, the concentration of LPS may be about 70 ng / mL. For example, the concentration of LPS may be about 90 ng / mL. For example, the concentration of LPS may be about 100 ng / mL. For example, the concentration of LPS may be about 120 ng / mL. For example, the concentration of LPS may be about 130 ng / mL. For example, the concentration of LPS may be about 150 ng / mL. For example, the concentration of LPS may be about 160 ng / mL. For example, the concentration of LPS may be about 180 ng / mL. For example, the concentration of LPS may be about 200 ng / mL.

[0172] In the present application, the concentration of IFN-g may be about 1-20 ng / mL. For example, the concentration of IFN-g may be about 1 ng / mL. For example, the concentration of IFN-g may be about 5 ng / mL. For example, the concentration of IFN-g may be about 8 ng / mL. For example, the concentration of IFN-g may be about 10 ng / mL. For example, the concentration of IFN-g may be about 12 ng / mL. For example, the concentration of IFN-g may be about 14 ng / mL. For example, the concentration of IFN-g may be about 15 ng / mL. For example, the concentration of IFN-g may be about 20 ng / mL.

[0173] In the present application, the concentration of IL-4 is about 10-50 ng / mL. For example, the concentration of IL-4 is about 10 ng / mL. For example, the concentration of IL-4 is about 15 ng / mL. For example, the concentration of IL-4 is about 20 ng / mL. For example, the concentration of IL-4 is about 25 ng / mL. For example, the concentration of IL-4 is about 30 ng / mL. For example, the concentration of IL-4 is about 40 ng / mL. For example, the concentration of IL-4 is about 50 ng / mL.

[0174] In the present application, the concentration of IL-13 is about 10-50 ng / mL. For example, the concentration of IL-13 is about 10 ng / mL. For example, the concentration of IL-13 is about 15 ng / mL. For example, the concentration of IL-13 is about 20 ng / mL. For example, the concentration of IL-13 is about 25 ng / mL. For example, the concentration of IL-13 is about 30 ng / mL. For example, the concentration of IL-13 is about 40 ng / mL. For example, the concentration of IL-13 is about 50 ng / mL.

[0175] In the present application, each culture medium may further independently contain a GABA pathway activator. In the present application, each culture medium may further independently contain a Notch signaling pathway activator.

[0176] For example, the macrophage differentiation medium may contain a GABA pathway activator. For example, the macrophage differentiation medium may contain a Notch signaling pathway activator. For example, the macrophage differentiation medium I may contain a GABA pathway activator. For example, the macrophage differentiation medium I may contain a Notch signaling pathway activator. The macrophage differentiation medium II may contain a GABA pathway activator. For example, the macrophage differentiation medium II may contain a Notch signaling pathway activator.

[0177] For example, the macrophage M1 polarization medium or the macrophage M2 polarization medium may contain a GABA pathway activator. The macrophage M1 polarization medium or the macrophage M2 polarization medium may contain a Notch signaling pathway activator.

[0178] GABA pathway activators

[0179] In the present application, the GABA pathway activator may encompass substances that can activate the GABA pathway. For example, the substance may be a compound, a nucleic acid molecule, and / or a protein. For example, the substance may be a macromolecular substance or a small molecule substance. For example, the substance may be an organic compound or an inorganic compound.

[0180] In the present application, the GABA pathway activator may include a GABAA pathway activator.

[0181] In the present application, the GABA pathway activator may include a GABAC pathway activator.

[0182] In the present application, the GABA pathway activator may include a GABA receptor agonist. For example, the GABA receptor agonist may include a GABAA receptor agonist. For example, the GABA receptor agonist may include a GABAC receptor agonist.

[0183] In the present application, the GABA receptor agonist may comprise GABA or a derivative thereof. In the present application, the GABA receptor agonist may comprise non-GABA or a derivative thereof, and such compounds may also activate the GABA pathway. For example, the GABAA receptor agonist may comprise one or more selected from the group consisting of Abecarnil, Barbiturates, Eszopiclone, Bamaluzole, Fengabine, GABA, Gabamide, GABOB, Gaboxadol, Ibotenic acid, Isoguvacine, Isonipecotic acid, Muscimol, Phenibut, Picamilon, Progabide, Propofol, Quisqualamine, SL75102, Thiomuscimol, Topiramate, and Zolpidem.

[0184] For example, the GABAC receptor agonist may comprise one or more selected from the group consisting of cis-4-aminocrotonic acid (CACA), (+)-cis-2-(aminomethyl)cyclopropanecarboxylic acid (CAMP), γ-aminobutyric acid (GABA), γ-amino-β-hydroxybutyric acid (GABOB), N4-Chloroacetylcytosine arabinoside, Picamilon, Progabide and Tolgabide.

[0185] In the present application, the GABA pathway activator may further comprise a positive allosteric modulator (PAM) of the GABA receptor. For example, the positive allosteric modulator of the GABA receptor may comprise phenolic, ketone, imidazole and / or pyrazole compounds.

[0186] For example, the positive allosteric modulator of the GABA receptor may comprise a positive allosteric modulator of the GABAA receptor. For example, the positive allosteric modulator of the GABA receptor may comprise a positive allosteric modulator of the GABAC receptor. For example, the positive allosteric modulator of the GABAA receptor may comprise one or more selected from the group consisting of: Alcohols, Avermectins, Barbiturates, Benzodiazepines, Bromides, Carbamates, Chloral hydrate, chloralose, petrichloral and other 2,2,2-trichloroethanol prodrugs, Chlormezanone, Clomethiazole, Dihydroergolines, Etazepine, Etifoxine, 2-Substituted phenols, Imidazoles, Kavalactones, Loreclezole, Neuroactive steroids, Nonbenzodiazepines, Propofol, Piperidinediones, Propanidid, Pyrazolopyridines, Quinazolinones, Skullcap constituents, Stiripentol, Disulfonylalkanes, Valerian constituents and Volatile organic compounds.

[0187] In the present application, the derivative of the GABA pathway activator may include one or more selected from the following groups: trans-4-aminocrotonic acid (TACA), muscyl alcohol, (±)-trans-2-(aminomethyl)cyclopropanecarboxylic acid (TAMP), trans-2-methyl-4-aminocrotonic acid (2-MeTACA), 3-(aminomethyl)-1-oxo-1-hydroxyphosphane (3-AMOHP), 3-(amino)-1-oxo-1-hydroxyphosphane (3-AOHP), 3-(guanidino)-1-oxo-1-hydroxy-phosphate (3-GOHP), 4-aminocyclopent-1-enecarboxamide (4-ACPAM) and 4-amino-N-hydroxycyclopent-1-enecarboxamide (4-ACPHA).

[0188] In the present application, the GABA pathway activator may comprise one or more selected from the group consisting of Muscimol, (R)-Baclofen, (RS)-Baclofen, SKF 97541, Acamprosate calcium, Thiomuscimol, Flurazepam, Flumazenil, 3-Aminopropylphosphonic Acid, AWD 131-138, Isoguvacine, TB 21007, Kojic Amine, Progabide, SL 75102, 3-APSA, MRK 016, TP 003, L-838,417, MK 0343, RuBiGABA trimethylphosphine, RuBi-GABA, Abecarnil, Barbiturate, Eszopiclone, Bamaluzole, Gabamide, Ibotenic acid, Isonipecotic acid, Phenibut, Picamilon, Propofol, Quisqualamine, Topiramate, Zolpidem, 1,4-Butanediol, γ-Butyrolactone, γ-Hydroxybutyric acid, γ-Hydroxyvaleric acid, γ-Valerolactone, Lesogaberan, Phenibut, 4-Fluorophenibut, Tolgabide, N4-Chloroacetylcytosine arabinoside, Methionine, Gabapentin, Piperazine, Gabapentin HCl, Etomidate, 6-Hydroxyflavone, 3,4,5-Trimethoxycinnamic acid(TMCA), Glabridin, Afloqualone and Oxiracetam.

[0189] In the present application, the concentration of the GABA pathway activator can be about 0.1-1000 mM. For example, the concentration of the GABA pathway activator is about 0.1-900 mM, about 0.1-900 mM, about 0.1-800 mM, about 0.1-700 mM, about 0.1-600 mM, about 0.1-500 mM, about 0.1-400 mM, about 0.1-300 mM, about 0.1-200 mM, about 0.1-100 mM, about 1-50 mM, about 5-20 mM, about 10-20 mM, about 10-100 mM. , about 20-200 mM, about 20-300 mM, about 30-400 mM, about 30-500 mM, about 30-600 mM, about 50-700 mM, about 100-800 mM, about 200-800 mM, about 300-900 mM, about 400-900 mM, about 500-900 mM, about 600-1000 mM, about 700-1000 mM, about 800-900 mM or about 900-1000 mM.

[0190] Notch pathway activators

[0191] In the present application, the Notch signaling pathway activator may include substances that can activate the Notch signaling pathway. For example, the substance can be a compound, a nucleic acid molecule and / or a protein. For example, the substance can be a macromolecular substance or a small molecule substance. For example, the substance can be an organic compound or an inorganic compound.

[0192] In the present application, the Notch signaling pathway activator may include a Notch ligand. For example, the Notch ligand may be a DLL1 recombinant protein or a variant thereof. For example, the Notch ligand may be a DLL3 recombinant protein or a variant thereof. For example, the Notch ligand may be a DLL4 recombinant protein or a variant thereof. For example, the Notch ligand may be a Jagged-1 recombinant protein or a variant thereof. For example, the Notch ligand may be a Jagged-2 recombinant protein or a variant thereof.

[0193] In the present application, the Notch signaling pathway activator may include a small molecule that induces Notch signal transduction. For example, the small molecule that induces Nocth signal transduction may be Valproic acid (VPA). For example, the small molecule that induces Nocth signal transduction may be Valproic Acid (NSC 93819) sodium salt.

[0194] In the present application, the Notch signaling pathway activator may include RhNF-κB. For example, the RhNF-κB may promote the expression of Jagged1 and indirectly activate the Notch pathway.

[0195] In the present application, the method may include directly acting on the Notch signaling pathway of the hematopoietic stem / progenitor cells, and may also include indirectly acting on the Notch signaling pathway of the hematopoietic stem / progenitor cells.

[0196] For example, the Notch signaling pathway can be regulated by acting on upstream and downstream molecules of the Notch signaling pathway. For example, the Notch signaling pathway can be activated by gene regulation.

[0197] In the present application, the gene regulation method may include one or more of the following methods: transferring the Notch receptor intracellular domain (NICD) gene into cells for overexpression, upregulating Notch receptor, Notch ligand, CSL-DNA binding protein, cutting enzyme (Furin protease, ADAM metalloproteinase, γ-secretase) and / or Notch regulatory factor genes.

[0198] In the present application, the method may include overexpressing the Hes7 gene in the stem cells or hematopoietic stem / progenitor cells.

[0199] In the present application, the method may include overexpressing the Hes7 gene in the macrophage-granulocyte progenitor cells.

[0200] In the present application, the concentration of the Nocth signaling pathway activator can be about 0.1-1000mM. For example, the concentration of the Nocth signaling pathway activator is about 0.1-900mM, about 0.1-900mM, about 0.1-800mM, about 0.1-700mM, about 0.1-600mM, about 0.1-500mM, about 0.1-400mM, about 0.1-300mM, about 0.1-200mM, about 0.1-100mM, about 1-50mM, about 5-20mM, about 10-20mM, about 10-100mM In some embodiments, the present invention relates to a 5- to 6-μm aqueous solution of at least one amino acid sequence of the present invention. The 5- to 6-μm aqueous solution of the present invention may be present in an amount of at least one amino acid sequence of the present invention.

[0201] cell

[0202] In the present application, the hematopoietic stem / progenitor cells may be derived from induced pluripotent stem cells.

[0203] In the present application, the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0204] In the present application, the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0205] In the present application, the hematopoietic stem / progenitor cells are derived from bone marrow.

[0206] In the present application, the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0207] In the present application, the differentiation pathway of the hematopoietic stem / progenitor cells may include differentiation from hematopoietic stem / progenitor cells to macrophage-granulocyte progenitor cells, and then differentiation from macrophage-granulocyte progenitor cells to macrophages.

[0208] On the other hand, the present application also provides a macrophage, which is obtained by differentiating from hematopoietic stem / progenitor cells by activating the GABA pathway and / or the Notch signaling pathway.

[0209] On the other hand, the present application also provides macrophage-granulocyte progenitor cells, which are obtained by differentiation from hematopoietic stem / progenitor cells by activating the GABA pathway and / or the Notch signaling pathway.

[0210] In another aspect, the present application also provides modified stem cells or hematopoietic stem / progenitor cells, wherein the expression and / or activity of the GABA receptor is increased compared to unmodified stem cells or hematopoietic stem / progenitor cells.

[0211] In the present application, the GABA receptor may include a GABAA receptor and / or a GABAC receptor.

[0212] In the present application, the Notch signaling pathway of the modified stem cells or hematopoietic stem / progenitor cells can be activated.

[0213] In the present application, the cells and / or the state of the cells can be determined by cell markers. For example, the type of cells and / or the state of the cells can be determined by marker phenotype.

[0214] In certain embodiments, the cells described herein are isolated.

[0215] In the present application, described cell can be prepared pharmaceutically according to any conventional method.For example, carrier, excipient or diluent can be used to mix or dilute active ingredient.The example of suitable carrier, excipient or diluent is lactose, dextrose, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.Described preparation can comprise for example filler, anti-agglomeration agent, lubricant, wetting agent, flavoring agent, emulsifier, preservative etc. additionally.Prepare compositions of the present invention to provide quick, lasting or delayed release active ingredient after giving patient by using any well-known method in this area.

[0216] The cells of the present application can be administered by injection (e.g., intramuscular, intravenous, peritoneal, subcutaneous), or by other methods such as infusion to ensure that they enter the bloodstream in an effective form. The cells can also be administered via routes within the tumor, around the tumor, within the lesion, or around the lesion to exert local and systemic therapeutic effects. For example, they can be administered topically or intravenously.

[0217] In the present application, the administration dosage of the cells may be a single dose or multiple doses. For example, the actual administration amount of the cells may be determined based on a variety of relevant factors, such as the type of disease; the route of administration; the age, sex, and / or weight of the patient; and the severity of the patient's symptoms.

[0218] Compositions, reagents, and uses

[0219] On the other hand, the present application also provides a composition comprising hematopoietic stem / progenitor cells and the culture medium.

[0220] On the other hand, the present application also provides a reagent for macrophage differentiation, which comprises a GABA pathway activator and / or a Notch signaling pathway activator.

[0221] In another aspect, the present application further provides the use of a GABA pathway activator and / or a Notch signaling pathway activator in the preparation of a reagent for macrophage differentiation. In another aspect, the present application further provides the use of a GABA pathway activator and / or a Notch signaling pathway activator in the differentiation and / or expansion of hematopoietic stem / progenitor cells into macrophages.

[0222] On the other hand, the present application also provides the use of a GABA pathway activator and / or a Notch signaling pathway activator in producing a culture medium for differentiating stem cells into macrophages.

[0223] On the other hand, the present application also provides a culture platform for obtaining macrophages derived from hematopoietic stem / progenitor cells, which comprises the method, culture medium, culture medium set and / or hematopoietic stem / progenitor cells described in the present application.

[0224] This application also provides the following implementation methods:

[0225] 1. A method for inducing stem cells to proliferate and / or differentiate into macrophage-granulocyte progenitors (GMPs), comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0226] 2. A method for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into macrophage-granulocyte progenitors (GMPs), comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0227] 3. A method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into macrophages, comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0228] 4. The method according to any one of embodiments 1-3, comprising activating the GABAA and / or GABAC signaling pathway of the stem cells or hematopoietic stem / progenitor cells.

[0229] 5. The method according to any one of embodiments 1 to 4, comprising activating the GABAA and / or GABAC receptors of the stem cells or hematopoietic stem / progenitor cells.

[0230] 6. The method of any one of embodiments 1-5, comprising administering a GABA pathway activator to the stem cells or hematopoietic stem / progenitor cells.

[0231] 7. The method according to embodiment 6, wherein the GABA pathway activator comprises a GABAA pathway activator and / or a GABAC pathway activator.

[0232] 8. The method of any one of embodiments 6-7, wherein the GABA pathway activator comprises a GABA receptor agonist.

[0233] 9. The method according to embodiment 8, wherein the GABA receptor agonist comprises a GABAA receptor agonist and / or a GABAC receptor agonist.

[0234] 10. The method of any one of embodiments 8-9, wherein the GABAA receptor agonist comprises one or more selected from the group consisting of Abecarnil, Barbiturates, Eszopiclone, Bamaluzole, Fengabine, GABA, Gabamide, GABOB, Gaboxadol, Ibotenic acid, Isoguvacine, Isonipecotic acid, Muscimol, Phenibut, Picamilon, Progabide, Propofol, Quisqualamine, SL75102, Thiomuscimol, Topiramate, and Zolpidem.

[0235] 11. The method of any one of embodiments 9-10, wherein the GABAC receptor agonist comprises one or more selected from the group consisting of cis-4-aminocrotonic acid (CACA), (+)-cis-2-(aminomethyl)cyclopropanecarboxylic acid (CAMP), γ-aminobutyric acid (GABA), γ-amino-β-hydroxybutyric acid (GABOB), N4-Chloroacetylcytosinearabinoside, Picamilon, Progabide, and Tolgabide.

[0236] 12. The method of any one of embodiments 6-11, wherein the GABA pathway activator comprises a positive allosteric enhancer (PAM) of the GABA receptor.

[0237] 13. The method according to embodiment 12, wherein the positive allosteric modulator of the GABA receptor comprises a positive allosteric modulator of the GABAA receptor and / or a positive allosteric modulator of the GABAC receptor.

[0238] 14. The method of embodiment 13, wherein the positive allosteric modulator of the GABAA receptor comprises one or more selected from the group consisting of: Alcohols, Avermectins, Barbiturates, Benzodiazepines, Bromides, Carbamates, Chloral hydrate, chloralose, petrichloral and other 2,2,2-trichloroethanol prodrugs, Chlormezanone, Clomethiazole, Dihydroergolines, Etazepine, Etifoxine, 2-Substituted phenols, Imidazoles, Kavalactones, Loreclezole, Neuroactive steroids, Nonbenzodiazepines, Propofol, Piperidinediones, Propanidid, Pyrazolopyridines, Quinazolinones, Skullcapconstituents, Stiripentol, Disulfonylalkanes, Valerian constituents and Volatileorganic compounds.

[0239] 15. The method of any one of embodiments 6-14, wherein the GABA pathway activator comprises GABA and / or a GABA derivative.

[0240] 16. A method according to any one of embodiments 6 to 15, wherein the GABA pathway activator comprises one or more selected from the group consisting of trans-4-aminocrotonic acid (TACA), muscitol, (±)-trans-2-(aminomethyl)cyclopropanecarboxylic acid (TAMP), trans-2-methyl-4-aminocrotonic acid (2-MeTACA), 3-(aminomethyl)-1-oxo-1-hydroxyphosphane (3-AMOHP), 3-(amino)-1-oxo-1-hydroxyphosphane (3-AOHP), 3-(guanidino)-1-oxo-1-hydroxy-phosphate (3-GOHP), 4-aminocyclopent-1-enecarboxamide (4-ACPAM) and 4-amino-N-hydroxycyclopent-1-enecarboxamide (4-ACPHA).

[0241] 17. The method of any one of embodiments 6-16, wherein the GABA pathway activator comprises one or more selected from the group consisting of Muscimol, (R)-Baclofen, (RS)-Baclofen, SKF 97541, Acamprosatecalcium, Thiomuscimol, Flurazepam, Flumazenil, 3-Aminopropylphosphonic Acid, AWD131-138, Isoguvacine, TB 21007, Kojic Amine, Progabide, SL 75102, 3-APSA, MRK 016, TP003, L-838,417, MK 0343, RuBi GABA trimethylphosphine, RuBi-GABA, Abecarnil, Barbiturate, Eszopiclone, Bamaluzole, Gabamide, Ibotenic acid, Isonipecotic acid, Phenibut, Picamilon, Propofol, Quisqualamine, Topiramate, Zolpidem, 1,4-Butanediol, γ-Butyrolactone, γ-Hydroxybutyric acid, γ-Hydroxyvaleric acid, γ-Valerolactone, Lesogaberan, Phenibut, 4-Fluorophenibut, Tolgabide, N4-Chloroacetylcytosine arabinoside, Methionine, Gabapentin, Piperazine, GabapentinHCl, Etomidate, 6-Hydroxyflavone, 3,4,5-Trimethoxycinnamic acid(TMCA), Glabridin, Afloqualone and Oxiracetam.

[0242] 18. The method of any one of embodiments 6-17, wherein the concentration of the GABA pathway activator is about 0.1-1000 nM.

[0243] 19. The method of any one of embodiments 6-18, wherein the concentration of the GABA pathway activator is about 10-20 nM.

[0244] 20. The method of any one of embodiments 1-19, comprising increasing the expression, function and / or activity of a GABA receptor in the stem cells or hematopoietic stem / progenitor cells.

[0245] 21. The method according to any one of embodiments 1-20, comprising increasing the expression, function and / or activity of GABA receptors in the hematopoietic stem / progenitor cells by gene regulation.

[0246] 22. The method according to embodiment 21, wherein the gene regulation comprises one or more of the following methods: constructing an exogenous gene sequence for expression in the cell, and gene editing to regulate the upregulation and / or activation of endogenous genes.

[0247] 23. The method according to any one of embodiments 1-22, further comprising activating the Notch signaling pathway of the stem cells or hematopoietic stem / progenitor cells.

[0248] 24. The method of any one of embodiments 1-23, comprising administering a Notch signaling pathway activator to the stem cells or hematopoietic stem / progenitor cells.

[0249] 25. A method according to embodiment 24, wherein the Notch signaling pathway activator comprises a Notch ligand.

[0250] 26. The method according to embodiment 25, wherein the Notch ligand is selected from one or more of the following groups: DLL1 recombinant protein or its variant, DLL3 recombinant protein or its variant, DLL4 recombinant protein or its variant, Jagged 1 recombinant protein or its variant, Jagged 2 recombinant protein or its variant.

[0251] 27. A method according to any one of embodiments 24-26, wherein the Notch signaling pathway activator comprises a small molecule that induces Notch signaling transduction.

[0252] 28. The method according to embodiment 27, wherein the small molecules inducing Notch signal transduction are selected from one or more of the following groups: Valproic acid (VPA), Valproic Acid (NSC 93819) sodium salt.

[0253] 29. The method according to any one of embodiments 24-28, wherein the Notch signaling pathway activator comprises RhNF-κB, and the RhNF-κB promotes the expression of Jagged1 and indirectly activates the Notch pathway.

[0254] 30. The method according to any one of embodiments 23-29, comprising directly or indirectly activating the Notch signaling pathway of the stem cells or hematopoietic stem / progenitor cells.

[0255] 31. A method according to any one of embodiments 21-30, wherein the gene regulation method comprises one or more of the following methods: transferring the intracellular segment (NICD) gene of the Notch receptor into cells for overexpression, upregulating the Notch receptor, Notch ligand, CSL-DNA binding protein, cutting enzyme (Furin protease, ADAM metalloproteinase, γ-secretase) and / or Notch regulatory factor genes.

[0256] 32. The method according to any one of embodiments 1-31, comprising overexpressing the Hes7 gene of the stem cells or hematopoietic stem / progenitor cells.

[0257] 33. The method of any one of embodiments 1-32, comprising overexpressing the Hes7 gene in the macrophage-granulocyte progenitor cells.

[0258] 34. The method according to any one of embodiments 1-33, comprising culturing the stem cells or hematopoietic stem / progenitor cells using macrophage differentiation medium I, wherein the macrophage differentiation medium I comprises a basal medium and a GABA pathway activator.

[0259] 35. The method according to embodiment 34, wherein the macrophage differentiation medium I further comprises a Notch pathway activator.

[0260] 36. The method according to any one of embodiments 34-35, wherein the macrophage differentiation medium I further comprises one or more selected from the group consisting of glutamine, β-mercaptoethanol, M-CSF and IL-3.

[0261] 37. The method according to embodiment 36, wherein the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0262] 38. The method of any one of embodiments 36-37, wherein the concentration of glutamine is about 1-5 mmol / L.

[0263] 39. The method of any one of embodiments 36-38, wherein the concentration of β-mercaptoethanol is 0.01-0.1 mmol / L.

[0264] 40. The method of any one of embodiments 36-39, wherein the concentration of M-CSF is about 50-100 ng / mL.

[0265] 41. The method of any one of embodiments 36-40, wherein the concentration of IL-3 is about 10-50 ng / mL.

[0266] 42. The method according to any one of embodiments 1-41, further comprising using a macrophage differentiation medium II, wherein the macrophage differentiation medium II comprises a basal medium, glutamine and M-CSF.

[0267] 43. The method according to embodiment 42, wherein the base culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0268] 44. The method according to any one of embodiments 42-43, wherein the macrophage differentiation medium II further comprises a GABA pathway activator.

[0269] 45. The method according to any one of embodiments 42-44, wherein the macrophage differentiation medium II further comprises a Notch pathway activator.

[0270] 46. ​​The method of any one of embodiments 42-45, wherein the concentration of glutamine is about 1-5 mmol / L.

[0271] 47. The method of any one of embodiments 42-46, wherein the concentration of M-CSF is about 50-100 ng / mL.

[0272] 48. The method of any one of embodiments 1-47, further comprising using a macrophage M1 polarization medium or a macrophage M2 polarization medium.

[0273] 49. The method according to embodiment 48, wherein the macrophage M1 polarization medium comprises the macrophage differentiation medium II, LPS and IFN-g.

[0274] 50. The method of embodiment 49, wherein the concentration of LPS is about 10-200 ng / mL.

[0275] 51. The method of any one of embodiments 49-50, wherein the concentration of IFN-g is about 1-20 ng / mL.

[0276] 52. The method of embodiment 48, wherein the macrophage M2 polarization medium comprises the macrophage differentiation medium II, IL-4 and IL-13.

[0277] 53. The method of embodiment 52, wherein the concentration of IL-4 is about 10-50 ng / mL.

[0278] 54. The method of any one of embodiments 52-53, wherein the concentration of IL-13 is about 10-50 ng / mL.

[0279] 55. The method of any one of embodiments 48-54, wherein the macrophage M1 polarization medium or the macrophage M2 polarization medium further comprises a GABA pathway activator.

[0280] 56. The method of any one of embodiments 48-55, wherein the macrophage M1 polarization medium or the macrophage M2 polarization medium further comprises a Notch pathway activator.

[0281] 57. The method according to any one of embodiments 1-56 comprises the following steps: 1) inoculating stem cells or hematopoietic stem / progenitor cells into the macrophage differentiation medium I for culture to obtain granulocyte-macrophage progenitor cells; 2) continuing culture using macrophage differentiation medium II to obtain macrophages M0; 3) continuing culture using macrophage M1 polarization medium or macrophage M2 polarization medium to obtain M1 macrophages or M2 macrophages.

[0282] 58. The method according to any one of embodiments 1-57 comprises the following steps: 1) inoculating stem cells or hematopoietic stem / progenitor cells into the macrophage differentiation medium I and culturing them for about 1-2 weeks to obtain granulocyte-macrophage progenitor cells; 2) continuing to culture them using the macrophage differentiation medium II for about 5-7 days to obtain macrophages M0; 3) continuing to culture them using the macrophage M1 polarization medium or the macrophage M2 polarization medium for about 2 days to obtain M1 macrophages or M2 macrophages.

[0283] 59. The method according to any one of embodiments 57-58, wherein the culturing conditions of step 1) are about 35-39° C. and about 3-7% CO 2 .

[0284] 60. A method according to any one of embodiments 1-59, wherein the hematopoietic stem / progenitor cells are derived from induced pluripotent stem cells.

[0285] 61. A method according to any one of embodiments 1-60, wherein the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0286] 62. A method according to any one of embodiments 1-61, wherein the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0287] 63. A method according to any one of embodiments 1-62, wherein the hematopoietic stem / progenitor cells are derived from bone marrow.

[0288] 64. A method according to any one of embodiments 1-63, wherein the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0289] 65. A culture medium comprising a GABA pathway activator and a macrophage differentiation medium.

[0290] 66. The culture medium of embodiment 65, wherein the GABA pathway activator comprises a GABA receptor agonist and / or a positive allosteric modulator of the GABA receptor.

[0291] 67. The culture medium of any one of embodiments 65-66, wherein the GABA pathway activator comprises GABA and / or a GABA derivative.

[0292] 68. The culture medium of any one of embodiments 65-67, further comprising a Notch pathway activator.

[0293] 69. The culture medium of any one of embodiments 65-68, wherein the macrophage differentiation medium comprises macrophage differentiation medium I, and the macrophage differentiation medium I comprises a basal medium.

[0294] 70. The culture medium according to embodiment 69, wherein the macrophage differentiation medium I further comprises one or more selected from the group consisting of glutamine, β-mercaptoethanol, M-CSF and IL-3.

[0295] 71. The culture medium according to embodiment 70, wherein the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0296] 72. The culture medium of any one of embodiments 70-71, wherein the concentration of glutamine is about 1-5 mmol / L.

[0297] 73. The culture medium of any one of embodiments 70-72, wherein the concentration of β-mercaptoethanol is about 0.01-0.1 mmol / L.

[0298] 74. The culture medium of any one of embodiments 70-73, wherein the concentration of M-CSF is about 50-100 ng / mL.

[0299] 75. The culture medium of any one of embodiments 70-74, wherein the concentration of IL-3 is about 10-50 ng / mL.

[0300] 76. A culture medium according to any one of embodiments 65-75, wherein the macrophage differentiation medium comprises macrophage differentiation medium II, and the macrophage differentiation medium II comprises basal medium, glutamine and M-CSF.

[0301] 77. The culture medium according to embodiment 76, wherein the basal culture medium comprises Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum) and X-VIVO 15 medium.

[0302] 78. The culture medium of any one of embodiments 76-77, wherein the concentration of glutamine is about 1-5 mmol / L.

[0303] 79. The culture medium of any one of embodiments 76-78, wherein the concentration of M-CSF is about 50-100 ng / mL.

[0304] 80. The culture medium of any one of embodiments 76-79, comprising a macrophage M1 polarization medium or a macrophage M2 polarization medium.

[0305] 81. The culture medium according to embodiment 80, wherein the macrophage M1 polarization medium comprises the macrophage differentiation medium II, LPS and IFN-g.

[0306] 82. The culture medium of embodiment 81, wherein the LPS is at a concentration of about 10-200 ng / mL.

[0307] 83. The culture medium of any one of embodiments 81-82, wherein the concentration of IFN-g is about 1-20 ng / mL.

[0308] 84. A culture medium according to embodiment 83, wherein the macrophage M2 polarization medium comprises the macrophage differentiation medium II, IL-4 and IL-13.

[0309] 85. The culture medium of embodiment 84, wherein the concentration of IL-4 is about 10-50 ng / mL.

[0310] 86. The culture medium of any one of embodiments 84-85, wherein the concentration of IL-13 is about 10-50 ng / mL.

[0311] 87. Use of the culture medium described in any one of embodiments 65-86 in generating macrophages differentiated from stem cells or hematopoietic stem / progenitor cells.

[0312] 88. A composition comprising stem cells or hematopoietic stem / progenitor cells and the culture medium of any one of embodiments 65-87.

[0313] 89. A modified stem cell or hematopoietic stem / progenitor cell wherein the expression and / or activity of a GABA receptor is increased compared to an unmodified stem cell or hematopoietic stem / progenitor cell.

[0314] 90. The modified stem cell or hematopoietic stem / progenitor cell of embodiment 89, wherein the GABA receptor comprises a GABAA receptor and / or a GABAC receptor.

[0315] 91. The modified stem cell or hematopoietic stem / progenitor cell according to any one of embodiments 89-90, wherein the Notch signaling pathway is activated.

[0316] Use of a GABA pathway activator in the preparation of a reagent for macrophage differentiation.

[0317] 93. The use according to embodiment 92, wherein the reagent for macrophage differentiation further comprises a Notch pathway activator.

[0318] Use of a GABA pathway activator in producing a culture medium for differentiation of stem cells or hematopoietic stem / progenitor cells into macrophages.

[0319] 95. The use according to embodiment 94, wherein the culture medium comprises a Notch pathway activator.

[0320] 96. A culture platform for obtaining macrophages derived from hematopoietic stem / progenitor cells, comprising the method described in any one of embodiments 1-64, the culture medium described in any one of embodiments 65-86, and / or hematopoietic stem / progenitor cells.

[0321] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0322] Example

[0323] culture medium

[0324] Basal culture medium: Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum), or X-VIVO 15 medium

[0325] Macrophage Differentiation Medium I: Basal Medium, Glutamine, β-Mercaptoethanol, M-CSF, IL-3

[0326] Macrophage Differentiation Medium II: Basal Medium, Glutamine, M-CSF

[0327] M1 polarization medium: Macrophage Differentiation Medium II, LPS, IFN-g

[0328] M2 polarization medium: Macrophage Differentiation Medium II, IL-4, IL-13

[0329] During cell culture, a GABA pathway activator and / or a Notch signaling pathway activator are added to the above culture medium.

[0330] GABA pathway activators: TACA, GABA

[0331] Notch signaling pathway activator: VPA

[0332] Example 1 Differentiation and culture of macrophages

[0333] Experimental steps:

[0334] Hematopoietic stem and progenitor cells were cultured in Macrophage Differentiation Medium I for 1-2 weeks, with a half-medium change every 3-4 days. Macrophage Differentiation Medium I consists of basal medium supplemented with glutamine, β-mercaptoethanol, M-CSF, and IL-3. Culture conditions were 35-39°C and 3-7% CO2.

[0335] These cells were collected and cultured for 1 to 2 weeks using macrophage differentiation medium II, with a half-medium change every 3 to 4 days, to obtain macrophage M0; the macrophage differentiation medium II was obtained by adding glutamine and M-CSF to the basal differentiation medium.

[0336] The M0 macrophages were cultured in M1 polarization medium or M2 polarization medium for 2 days. The M1 polarization medium was obtained by adding LPS and IFN-g to the macrophage differentiation medium II, and the M2 polarization medium was obtained by adding IL-4 and IL-13 to the macrophage differentiation medium II.

[0337] The basic culture medium includes Advanced Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum), RPMI (containing 10% fetal bovine serum), and X-VIVO 15 culture medium.

[0338] In the macrophage differentiation medium I, the concentration of glutamine is 2 mmol / L; the concentration of β-mercaptoethanol is about 0.055 mmol / L; the concentration of M-CSF is 50-100 ng / mL; and the concentration of IL-3 is 25 ng / mL.

[0339] In the macrophage differentiation medium II, the concentration of glutamine is 2 mmol / L, and the concentration of M-CSF is 100 ng / mL;

[0340] In the M1 polarization medium, the concentration of LPS is 100 ng / mL; the concentration of IFN-g is 20 ng / mL;

[0341] In the M2 polarization medium, the concentration of IL-4 is 20 ng / mL; the concentration of IL-13 is 20 ng / mL.

[0342] Example 2 Detection of the Effects of GABA Pathway Activators and / or Notch Signaling Pathway Activators on Macrophage Proliferation

[0343] Experimental steps:

[0344] This experiment adopted a two-stage differentiation culture strategy. In the first stage, CD34+ hematopoietic stem and progenitor cells were cultured at a rate of 2×10 4 Cells were seeded at a density of 100 μg / well in 96-well plates and supplemented with Macrophage Differentiation Medium I, which consists of RPMI-1640 as the basal medium supplemented with 10% fetal bovine serum (FBS), 2 mmol / L glutamine, 0.055 mmol / L β-mercaptoethanol, 50 ng / mL macrophage colony-stimulating factor (M-CSF), and 25 ng / mL interleukin-3 (IL-3). Cells were cultured at 37°C and 5% CO₂ for 7 days, with a half-volume medium replacement every 2-3 days.

[0345] After 7 days of culture, cells were harvested and entered the second stage of culture, switching to Macrophage Differentiation Medium II. This medium consists of RPMI-1640 supplemented with 10% FBS, 2 mmol / L glutamine, and 100 ng / mL M-CSF. Cells were cultured for an additional 7 days at 37°C and 5% CO2, with half-medium changes performed every 2-3 days.

[0346] On the 7th and 14th days of culture, cells were collected to detect cell proliferation efficiency.

[0347] Experimental results:

[0348] like Figure 1 As shown, the addition of TACA (a) and GABA (b) on the 7th and 14th days after culture can maintain normal cell proliferation efficiency.

[0349] like Figure 2 As shown, on the 7th and 14th days after culture, the addition of VPA (c), TACA+VPA (d), GABA+VPA (e), and 2×DMSO (f) can maintain normal cell proliferation efficiency.

[0350] The above experimental results show that:

[0351] The addition of GABA pathway activators and / or Notch signaling pathway activators had no negative effect on macrophage proliferation.

[0352] Example 3 Detection of the Effects of GABA Pathway Activators and / or Notch Signaling Pathway Activators on Macrophage Differentiation

[0353] Experimental steps:

[0354] This experiment adopted a two-stage differentiation culture strategy. In the first stage, CD34+ hematopoietic stem and progenitor cells were cultured at a rate of 2×10 4 Cells were seeded at a density of 100 μg / well in 96-well plates and supplemented with Macrophage Differentiation Medium I, which consists of RPMI-1640 as the basal medium supplemented with 10% fetal bovine serum (FBS), 2 mmol / L glutamine, 0.055 mmol / L β-mercaptoethanol, 50 ng / mL macrophage colony-stimulating factor (M-CSF), and 25 ng / mL interleukin-3 (IL-3). Cells were cultured at 37°C and 5% CO₂ for 7 days, with a half-volume medium replacement every 2-3 days.

[0355] After 7 days of culture, cells were harvested and entered the second stage of culture, switching to Macrophage Differentiation Medium II. This medium consists of RPMI-1640 supplemented with 10% FBS, 2 mmol / L glutamine, and 100 ng / mL M-CSF. Cells were cultured for an additional 7 days at 37°C and 5% CO2, with half-medium changes performed every 2-3 days.

[0356] After culture for 7 days and 14 days, the cells were collected and analyzed by flow cytometry. The cells were collected by centrifugation at 400g for 5 minutes and washed with FACS buffer (PBS solution) containing 1% FBS. A flow cytometry antibody mixture was added to the washed cells. The antibodies used in this experiment were APC anti-humanCD11b (Biolegend, Cat: 301310, Clone: ​​ICRF44) and FITC anti-human CD14 (Biolegend, Cat: 325604, Clone: ​​HCD14). After incubation for 30 minutes, DAPI was added for staining for 5 minutes. After staining, the precipitate was collected by centrifugation again, resuspended in FACS buffer, and subjected to flow cytometry detection to evaluate the efficiency and degree of differentiation of CD34+ hematopoietic stem cells into macrophages in each group.

[0357] Experimental results:

[0358] like Figure 3A and Figure 3B As shown, on the 7th day of cell culture, the cells were subjected to marker detection. Compared with the control group, the expression of CD11b was significantly increased in TACA (a), GABA (b), VPA (c), TACA+VPA (d), and GABA+VPA (e).

[0359] like Figure 4A and Figure 4B As shown, on the 14th day of cell culture, the cells were tested for markers. Compared with the control group, the expression of CD11b was significantly increased in TACA (a), GABA (b), VPA (c), TACA+VPA (d), and GABA+VPA (e).

[0360] The above experimental results show that:

[0361] Adding GABA pathway activators and / or Notch signaling pathway activators can promote the differentiation of hematopoietic stem cells into monocyte-macrophage cell lineages.

[0362] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A method for inducing stem cells to proliferate and / or differentiate into macrophage-granulocyte progenitors (GMPs), comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

2. A method for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into macrophage-granulocyte progenitors (GMPs), comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

3. A method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into macrophages, comprising activating the γ- GABA signaling pathway. 4 . The method according to claim 1 , comprising activating the GABAA and / or GABAC signaling pathway of the stem cells or hematopoietic stem / progenitor cells.

5. The method according to any one of claims 1 to 3, comprising activating the GABAA and / or GABAC receptors of the stem cells or hematopoietic stem / progenitor cells. 6 . The method according to claim 1 , further comprising activating the Notch signaling pathway of the stem cells or hematopoietic stem / progenitor cells. 7 . The method according to claim 6 , comprising administering a Notch signaling pathway activator to the stem cells or hematopoietic stem / progenitor cells.

8. The method of claim 8, wherein the Notch signaling pathway activator comprises a Notch ligand.

9. A culture medium comprising a GABA pathway activator and a macrophage differentiation medium. The culture medium according to claim 9 , further comprising a Notch pathway activator.

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