Method for differentiating hematopoietic stem / progenitor cells into granulocytes

Abstract

The present application relates to a method of inducing proliferation and / or differentiation of stem cells and / or hematopoietic stem / progenitor cells to granulocytes comprising activating a gamma-aminobutyric acid (GABA) signaling pathway of the hematopoietic stem / progenitor cells. The invention further provides a culture medium for inducing the hematopoietic stem / progenitor cells to proliferate and / or differentiate into granulocytes 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 granulocytes. Background Art

[0002] Granulocytes are white blood cells containing special staining granules. According to the staining results, they can be divided into neutrophils, eosinophils, and basophils. Among them, neutrophils are the most numerous, accounting for about 60% of the circulating white blood cells in the human body. Neutrophils are rich in lysosomal enzymes, which help to decompose microbial pathogens and tissue fragments that invade cells. Chemotaxis causes neutrophils to accumulate at the site of inflammation, activates immune regulation, and helps the body resist the invasion of pathogenic microorganisms. The half-life of mature neutrophils in the blood circulation is only 6-8 hours.

[0003] Hematopoietic stem / progenitor cells (HSPCs) are adult stem / progenitor cells in the blood that have the ability to differentiate and proliferate toward one or more blood cell lineages. Neutrophils obtained by further differentiation of hematopoietic stem / progenitor cells can be considered as genetically modified neutrophils.

[0004] Providing a more effective method for inducing granulocyte differentiation is an urgent problem to be solved. Summary of the invention

[0005] The present application provides a method for differentiating stem cells and / or hematopoietic stem / progenitor cells into granulocytes. The differentiation method provided by the present application introduces a γ-aminobutyric acid (GABA) signaling pathway, which can enhance the differentiation ability of hematopoietic stem / progenitor cells and promote their differentiation into granulocyte-macrophage progenitors (GMP).

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

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

[0008] On the other hand, the present application provides a method for inducing proliferation and / or differentiation of granulocyte-macrophage progenitor cells into granulocytes, which comprises activating the gamma-aminobutyric acid (GABA) signaling pathway of the granulocyte-macrophage progenitor cells.

[0009] In certain embodiments, the method comprises activating the GABAA and / or GABAC signaling pathway in the stem cell, hematopoietic stem / progenitor cell, or granulocyte-macrophage progenitor cell.

[0010] In certain embodiments, the method comprises activating a GABAA and / or GABAC receptor of the stem cell, hematopoietic stem / progenitor cell, or granulocyte-macrophage progenitor cell.

[0011] In certain embodiments, the method comprises administering a GABA pathway activator to the stem cells, hematopoietic stem / progenitor cells, or granulocyte-macrophage progenitor cells.

[0012] In certain embodiments, the GABA pathway activator comprises a GABAA pathway activator and / or a GABAC pathway activator.

[0013] In certain embodiments, the GABA pathway activator comprises a GABA receptor agonist.

[0014] In certain embodiments, the GABA receptor agonist comprises a GABAA receptor agonist and / or a GABAC receptor agonist.

[0015] 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.

[0016] 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.

[0017] In certain embodiments, the GABA pathway activator comprises a positive allosteric enhancer (PAM) of the GABA receptor.

[0018] 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.

[0019] 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.

[0020] In certain embodiments, the GABA pathway activator comprises GABA and / or a GABA derivative.

[0021] In certain embodiments, the GABA pathway activator includes 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).

[0022] In some embodiments, the GABA pathway activator includes 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.

[0023] In certain embodiments, the concentration of the GABA pathway activator is about 0.1-1000 nM.

[0024] In certain embodiments, the concentration of the GABA pathway activator is about 10-20 nM.

[0025] In certain embodiments, the method comprises culturing the hematopoietic stem / progenitor cells using an initial differentiation medium, wherein the initial differentiation medium comprises basal medium I and the GABA pathway activator.

[0026] In certain embodiments, the initial differentiation medium further comprises one or more of the following cytokines: SCF, IL3, IL7, IL15 and Flt3-L.

[0027] In certain embodiments, the concentration of SCF is about 5-50 ng / mL.

[0028] In certain embodiments, the concentration of IL3 is about 5-50 ng / mL.

[0029] In certain embodiments, the concentration of IL7 is about 5-50 ng / mL.

[0030] In certain embodiments, the concentration of IL15 is about 5-50 ng / mL.

[0031] In certain embodiments, the concentration of Flt3-L is about 5-50 ng / mL.

[0032] In certain embodiments, the basal medium I comprises StemSpan TM SFEMII medium.

[0033] In certain embodiments, the method further comprises using a granulocyte differentiation medium I, wherein the granulocyte differentiation medium I comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, TPO, G-CSF, IL3, IL5 and IL6.

[0034] In certain embodiments, the concentration of SCF is about 5-50 ng / mL.

[0035] In certain embodiments, the concentration of IL3 is about 5-50 ng / mL.

[0036] In certain embodiments, the concentration of TPO is about 5-50 ng / mL.

[0037] In certain embodiments, the concentration of IL5 is about 5-50 ng / mL.

[0038] In certain embodiments, the concentration of IL6 is about 5-50 ng / mL.

[0039] In certain embodiments, the concentration of G-CSF is about 5-50 ng / mL.

[0040] In certain embodiments, the basal medium II comprises IMDM medium and fetal calf serum FCS (fetal calf serum).

[0041] In certain embodiments, the granulocyte differentiation medium I further comprises a GABA pathway activator.

[0042] In certain embodiments, the method further comprises using a granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

[0043] In certain embodiments, the concentration of SCF is about 5-50 ng / mL.

[0044] In certain embodiments, the concentration of IL7 is about 5-50 ng / mL.

[0045] In certain embodiments, the concentration of IL15 is about 5-50 ng / mL.

[0046] In certain embodiments, the concentration of Flt3-L is about 5-50 ng / mL.

[0047] In certain embodiments, the granulocyte differentiation medium II further comprises a GABA pathway activator.

[0048] In certain embodiments, the method comprises the following steps: 1) inoculating hematopoietic stem / progenitor cells in an initial differentiation medium for culture, wherein the initial differentiation medium comprises a GABA pathway activator; 2) after completing step 1), continuing culture using the granulocyte differentiation medium I; and 3) continuing culture using the granulocyte differentiation medium II.

[0049] In certain embodiments, the method comprises the following steps: 1) inoculating hematopoietic stem / progenitor cells in an initial differentiation medium and culturing the medium, wherein the initial differentiation medium comprises a GABA pathway activator; 2) after completing step 1), continuing culturing using the granulocyte differentiation medium I for about 3-5 days, or until granulocytes are obtained, wherein the granulocytes include neutrophils or eosinophils; and 3) continuing culturing using the granulocyte differentiation medium II, performing a half-volume medium replacement using the granulocyte differentiation medium II every about 2-3 days, and culturing for about 22-28 days.

[0050] In certain embodiments, the granulocyte differentiation medium I further comprises a GABA pathway activator.

[0051] In certain embodiments, the granulocyte differentiation medium II further comprises a GABA pathway activator.

[0052] In certain embodiments, in step 1), the culture conditions are about 35-39° C. and about 3-7% CO 2 .

[0053] In certain embodiments, the method comprises increasing the expression, function and / or activity of a GABA receptor in said stem cell, said hematopoietic stem / progenitor cell, or said granulocyte-macrophage progenitor cell.

[0054] In certain embodiments, the GABA receptor comprises a GABAA receptor and / or a GABAC receptor.

[0055] In certain embodiments, the method comprises increasing the expression, function and / or activity of the GABA receptor in the stem cells, the hematopoietic stem / progenitor cells or the granulocyte-macrophage progenitor cells by gene regulation.

[0056] 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 endogenous genes.

[0057] In certain embodiments, the hematopoietic stem / progenitor cells are derived from induced pluripotent stem cells.

[0058] In certain embodiments, the hematopoietic stem / progenitor cells are derived from embryonic stem cells.

[0059] In certain embodiments, the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0060] In certain embodiments, the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0061] In certain embodiments, the hematopoietic stem / progenitor cells are derived from bone marrow.

[0062] In certain embodiments, the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0063] In certain embodiments, the granulocytes are neutrophils or eosinophils.

[0064] On the other hand, the present application also provides a culture medium, which comprises a GABA pathway activator and a granulocyte differentiation medium.

[0065] In certain embodiments, the GABA pathway activator comprises a GABA receptor agonist and / or a positive allosteric modulator of a GABA receptor.

[0066] In certain embodiments, the GABA pathway activator comprises GABA and / or a GABA derivative.

[0067] In certain embodiments, the granulocyte differentiation medium comprises an initial differentiation medium and one or more cytokines.

[0068] In certain embodiments, the initial differentiation medium comprises basal medium I and a GABA pathway activator.

[0069] In certain embodiments, the cytokines include one or more of the following cytokines: SCF, IL3, IL7, IL15, and Flt3-L.

[0070] In certain embodiments, the basal medium I comprises StemSpan TM SFEMII medium.

[0071] In certain embodiments, the concentration of SCF is about 5-50 ng / mL.

[0072] In certain embodiments, the concentration of IL3 is about 5-50 ng / mL.

[0073] In certain embodiments, the concentration of IL7 is about 5-50 ng / mL.

[0074] In certain embodiments, the concentration of IL15 is about 5-50 ng / mL.

[0075] In certain embodiments, the concentration of Flt3-L is about 5-50 ng / mL.

[0076] In certain embodiments, the granulocyte differentiation medium further comprises a granulocyte differentiation medium I, wherein the granulocyte differentiation medium I comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, TPO, G-CSF, IL3, IL5 and IL6.

[0077] In certain embodiments, the concentration of SCF is about 5-50 ng / mL.

[0078] In certain embodiments, the concentration of IL3 is about 5-50 ng / mL.

[0079] In certain embodiments, the concentration of TPO is about 5-50 ng / mL.

[0080] In certain embodiments, the concentration of IL5 is about 5-50 ng / mL.

[0081] In certain embodiments, the concentration of IL6 is about 5-50 ng / mL.

[0082] In certain embodiments, the concentration of G-CSF is about 5-50 ng / mL.

[0083] In certain embodiments, the basal medium II comprises IMDM medium and fetal calf serum FCS (fetal calf serum).

[0084] In certain embodiments, the granulocyte differentiation medium I further comprises a GABA pathway activator.

[0085] In certain embodiments, the culture medium further comprises granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

[0086] In certain embodiments, the concentration of SCF is about 5-50 ng / mL.

[0087] In certain embodiments, the concentration of IL7 is about 5-50 ng / mL.

[0088] In certain embodiments, the concentration of IL15 is about 5-50 ng / mL.

[0089] In certain embodiments, the concentration of Flt3-L is about 5-50 ng / mL.

[0090] In certain embodiments, the granulocyte differentiation medium II further comprises a GABA pathway activator.

[0091] On the other hand, the present application also provides the use of the culture medium in generating granulocytes by differentiating hematopoietic stem / progenitor cells.

[0092] On the other hand, the present application also provides a composition comprising stem cells, hematopoietic stem / progenitor cells or granulocyte-macrophage progenitor cells, and the culture medium.

[0093] On the other hand, the present application also provides modified hematopoietic stem / progenitor cells, which have increased expression, function and / or activity of GABA receptors compared to unmodified hematopoietic stem / progenitor cells.

[0094] On the other hand, the present application also provides granulocyte-macrophage progenitor cells, which are obtained by differentiation of hematopoietic stem / progenitor cells by activating the GABA pathway.

[0095] On the other hand, the present application also provides granulocytes, which are obtained by differentiation of hematopoietic stem / progenitor cells by activating the GABA pathway.

[0096] On the other hand, the present application also provides use of a GABA pathway activator in preparing a reagent for granulocyte differentiation.

[0097] On the other hand, the present application also provides the use of a GABA pathway activator in differentiating and / or expanding hematopoietic stem / progenitor cells into granulocytes.

[0098] 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 into granulocytes.

[0099] On the other hand, the present application also provides a culture platform for obtaining granulocytes derived from hematopoietic stem / progenitor cells, which comprises the method, the culture medium and / or hematopoietic stem / progenitor cells.

[0100] Those skilled in the art can easily perceive 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 the present 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 the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0102] Figure 1 Shown is the effect of culturing cells with and without the addition of a GABA pathway activator as described in the present application on cell proliferation.

[0103] Figure 2 Shown is the effect of culturing cells for 18 days in the medium with or without the addition of the GABA pathway activator described in the present application on cell differentiation.

[0104] Figure 3 Shown is the effect of culturing cells for 28 days in the medium with or without the addition of the GABA pathway activator described in the present application on cell differentiation. DETAILED DESCRIPTION

[0105] The following is an explanation of the implementation of the present invention by means of 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.

[0106] Definition of terms

[0107] In this application, the term "derivative" generally refers to another chemical substance that is structurally related to a 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), such as by chemical or enzymatic modification. In this application, the term "derivative" may 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 may exhibit similar functions and / or activities as the parent compound.

[0108] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "for", "consisting of...".

[0109] In the present application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0110] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 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 a specified value.

[0111] In this application, the term "proliferation" generally refers to increasing the number of cells of a particular cell type or multiple types from a starting population of cells, which may be different or identical. The starting cells for proliferation need not be the same as the cells produced by proliferation. For example, the cells that proliferate can be generated from the growth and differentiation of the starting population of cells.

[0112] 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.

[0113] 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 recognize antigens presented on cells. Antibodies can be monoclonal or polyclonal, and are usually selected to have less cross-reaction 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.

[0114] 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.

[0115] In the present application, the term "GABAC" receptor may also be referred to as GABA-ρ receptor, GABA-r, GABRR, which is generally a homopentameric ligand-gated ion channel composed of ρ subunits. The "GABAC" receptor described in the present application also encompasses variants, homologues, analogs and / or derivatives thereof.

[0116] In this application, the term "stem cell" generally refers to a self-replicating and multipotent cell having 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.

[0117] 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.

[0118] Hematopoietic stem cells (HSCs) generate 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 specialized 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.

[0119] In the present application, the term "induced pluripotent stem cell" can be generally abbreviated as iPS cell or iPSC, and generally refers to a type of pluripotent stem cell prepared from non-pluripotent cells in an artificial manner. For example, the artificial manner can be the introduction of reprogramming factors or chemical small molecule induction, etc. For example, the non-pluripotent cells can be adult somatic cells or terminally differentiated cells, for example, fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.

[0120] In the present application, the term "GABA pathway activator" generally refers to any substance that can activate or increase the intracellular γ-aminobutyric acid (GABA) signaling pathway, for example, small molecules, nucleic acids, etc. The "GABA signaling pathway" of the present 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 the present 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.

[0121] In this application, the term "cytokine" generally refers to a compound or composition (e.g., an autoimmune factor) produced by a cell and affecting the physiological state of the cell (self) or other cells that produce the cytokine. Cytokines also include any compound or composition produced by recombinant or synthetic processing, and the products of these processing 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.

[0122] The term "composition" as used herein generally refers to a product comprising a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of specified amounts of a specified ingredient. In the present application, the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0123] In this application, the term "ex vivo" generally refers to operations involving 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 by certain methods, or enter another organism.

[0124] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0125] In this application, the term "in vivo" generally refers to within a living organism. For example, in some cases, "in vivo" may refer to a specific location in a subject tissue or organ. DETAILED DESCRIPTION OF THE INVENTION

[0127] method

[0128] On the one hand, the present application provides a method for inducing proliferation and / or differentiation of hematopoietic stem / progenitor cells into granulocytes, which may include activating the gamma-aminobutyric acid (GABA) signaling pathway of the hematopoietic stem / progenitor cells.

[0129] On the other hand, the present application provides a method for inducing stem cells to proliferate and / or differentiate into granulocytes, which may include activating the γ-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0130] On the other hand, the present application provides a method for inducing proliferation and / or differentiation of granulocyte-macrophage progenitor cells into granulocytes, which may include activating the gamma-aminobutyric acid (GABA) signaling pathway of the granulocyte-macrophage progenitor cells.

[0131] In the present application, the method may include a process of differentiating hematopoietic stem / progenitor cells into granulocyte-macrophage progenitor cells, and / or a process of differentiating granulocyte-macrophage progenitor cells into granulocytes.

[0132] In the present application, the GABA signaling pathway in the method may include the GABAA and / or GABAC signaling pathway.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] For example, the expression level of 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] For example, it can be activated by applying chemical regulatory means or by genetic regulatory means.

[0141] For example, the chemical regulation means 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 directly act on the GABA receptor. For example, the GABA pathway activator may indirectly cause activation of the GABA pathway by acting on other pathways of the cell.

[0142] For example, the gene regulation means can include using CRISPR to regulate endogenous gene activation and / or up-regulation of expression. For example, the CRISPR / Cas9 system can be used to regulate endogenous gene activation and / or up-regulation of expression.

[0143] For example, it can be achieved by introducing an exogenous protein or an exogenous nucleic acid molecule encoding the protein, or by causing an increase in the expression of an endogenous protein or an endogenous gene encoding the protein. For example, the up-regulation of GABA receptor expression may be caused by a mutation in the gene regulatory region encoding the receptor. In some cases, up-regulation of GABA receptor expression can be achieved by changing the function of one or more components in the translation and / or transcription process.

[0144] For example, the gene regulation approach can be through small activating RNA (saRNA) approach.

[0145] In the present application, the method may comprise culturing the hematopoietic stem / progenitor cells using an initial differentiation medium, wherein the initial differentiation medium may comprise basal medium I and the GABA pathway activator.

[0146] In the present application, the method may further comprise the use of granulocyte differentiation medium I, wherein the granulocyte differentiation medium I may comprise basal medium II and one or more cytokines selected from the group consisting of SCF, TPO, G-CSF, IL3, IL5 and IL6.

[0147] In the present application, the method may further comprise using a granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

[0148] In the present application, the method may include the following steps: 1) inoculating hematopoietic stem / progenitor cells in an initial differentiation medium for culture, wherein the initial differentiation medium contains a GABA pathway activator; 2) after completing step 1), continuing culture using the granulocyte differentiation medium I; and 3) continuing culture using the granulocyte differentiation medium II.

[0149] In the present application, the method may include the following steps: 1) inoculating hematopoietic stem / progenitor cells in an initial differentiation medium and culturing the medium, wherein the initial differentiation medium contains a GABA pathway activator; 2) after completing step 1), continuing to culture using the granulocyte differentiation medium I for about 3-5 days, or until granulocytes are obtained, wherein the granulocytes include neutrophils or eosinophils or basophils; and 3) continuing to culture using the granulocyte differentiation medium II, performing a half-volume medium replacement every about 2-3 days, and culturing for about 22-28 days.

[0150] In the present application, in step 1), the culture conditions are about 35-39°C and about 3-7% CO 2 .

[0151] Culture medium

[0152] In another aspect, the present application also provides a culture medium, which may contain a GABA pathway activator and a granulocyte differentiation medium.

[0153] In the present application, the granulocyte differentiation medium may comprise an initial differentiation medium and one or more cytokines.

[0154] In the present application, the initial differentiation medium may contain basal medium I and a GABA pathway activator.

[0155] In the present application, the cytokine may include one or more of the following cytokines: SCF, IL3, IL7, IL15 and Flt3-L.

[0156] In the present application, the basic medium I may include StemSpan TM SFEMII medium.

[0157] In the present application, the granulocyte differentiation medium may further comprise granulocyte differentiation medium I, and the granulocyte differentiation medium I may comprise basal medium II and one or more cytokines selected from the group consisting of SCF, TPO, G-CSF, IL3, IL5 and IL6.

[0158] In the present application, the basic culture medium II may include IMDM culture medium and fetal calf serum FCS (fetal calf serum).

[0159] In the present application, the granulocyte differentiation medium I may further contain a GABA pathway activator.

[0160] In the present application, the culture medium may further comprise granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

[0161] In the present application, the granulocyte differentiation medium II may further contain a GABA pathway activator.

[0162] In the present application, the concentration of the SCF is about 5-50ng / mL. For example, the concentration of the SCF is about 5ng / mL. For example, the concentration of the SCF is about 10ng / mL. For example, the concentration of the SCF is about 15ng / mL. For example, the concentration of the SCF is about 20ng / mL. For example, the concentration of the SCF is about 25ng / mL. For example, the concentration of the SCF is about 30ng / mL. For example, the concentration of the SCF is about 35ng / mL. For example, the concentration of the SCF is about 40ng / mL. For example, the concentration of the SCF is about 45ng / mL. For example, the concentration of the SCF is about 50ng / mL.

[0163] In the present application, the concentration of IL3 is about 5-50ng / mL. For example, the concentration of IL3 is about 5ng / mL. For example, the concentration of IL3 is about 10ng / mL. For example, the concentration of IL3 is about 15ng / mL. For example, the concentration of IL3 is about 20ng / mL. For example, the concentration of IL3 is about 25ng / mL. For example, the concentration of IL3 is about 30ng / mL. For example, the concentration of IL3 is about 35ng / mL. For example, the concentration of IL3 is about 40ng / mL. For example, the concentration of IL3 is about 45ng / mL. For example, the concentration of IL3 is about 50ng / mL.

[0164] In the present application, the concentration of IL7 is about 5-50ng / mL. For example, the concentration of IL7 is about 5ng / mL. For example, the concentration of IL7 is about 10ng / mL. For example, the concentration of IL7 is about 15ng / mL. For example, the concentration of IL7 is about 20ng / mL. For example, the concentration of IL7 is about 25ng / mL. For example, the concentration of IL7 is about 30ng / mL. For example, the concentration of IL7 is about 35ng / mL. For example, the concentration of IL7 is about 40ng / mL. For example, the concentration of IL7 is about 45ng / mL. For example, the concentration of IL7 is about 50ng / mL.

[0165] In the present application, the concentration of IL15 is about 5-50ng / mL. For example, the concentration of IL15 is about 5ng / mL. For example, the concentration of IL15 is about 10ng / mL. For example, the concentration of IL15 is about 15ng / mL. For example, the concentration of IL15 is about 20ng / mL. For example, the concentration of IL15 is about 25ng / mL. For example, the concentration of IL15 is about 30ng / mL. For example, the concentration of IL15 is about 35ng / mL. For example, the concentration of IL15 is about 40ng / mL. For example, the concentration of IL15 is about 45ng / mL. For example, the concentration of IL15 is about 50ng / mL.

[0166] In the present application, the concentration of Flt3-L is about 5-50ng / mL. For example, the concentration of Flt3-L is about 5ng / mL. For example, the concentration of Flt3-L is about 10ng / mL. For example, the concentration of Flt3-L is about 15ng / mL. For example, the concentration of Flt3-L is about 20ng / mL. For example, the concentration of Flt3-L is about 25ng / mL. For example, the concentration of Flt3-L is about 30ng / mL. For example, the concentration of Flt3-L is about 35ng / mL. For example, the concentration of Flt3-L is about 40ng / mL. For example, the concentration of Flt3-L is about 45ng / mL. For example, the concentration of Flt3-L is about 50ng / mL.

[0167] In the present application, the concentration of the TPO is about 5-50ng / mL. For example, the concentration of the TPO is about 5ng / mL. For example, the concentration of the TPO is about 10ng / mL. For example, the concentration of the TPO is about 15ng / mL. For example, the concentration of the TPO is about 20ng / mL. For example, the concentration of the TPO is about 25ng / mL. For example, the concentration of the TPO is about 30ng / mL. For example, the concentration of the TPO is about 35ng / mL. For example, the concentration of the TPO is about 40ng / mL. For example, the concentration of the TPO is about 45ng / mL. For example, the concentration of the TPO is about 50ng / mL.

[0168] In the present application, the concentration of IL5 is about 5-50ng / mL. For example, the concentration of IL5 is about 5ng / mL. For example, the concentration of IL5 is about 10ng / mL. For example, the concentration of IL5 is about 15ng / mL. For example, the concentration of IL5 is about 20ng / mL. For example, the concentration of IL5 is about 25ng / mL. For example, the concentration of IL5 is about 30ng / mL. For example, the concentration of IL5 is about 35ng / mL. For example, the concentration of IL5 is about 40ng / mL. For example, the concentration of IL5 is about 45ng / mL. For example, the concentration of IL5 is about 50ng / mL.

[0169] In the present application, the concentration of IL6 is about 5-50ng / mL. For example, the concentration of IL6 is about 5ng / mL. For example, the concentration of IL6 is about 10ng / mL. For example, the concentration of IL6 is about 15ng / mL. For example, the concentration of IL6 is about 20ng / mL. For example, the concentration of IL6 is about 25ng / mL. For example, the concentration of IL6 is about 30ng / mL. For example, the concentration of IL6 is about 35ng / mL. For example, the concentration of IL6 is about 40ng / mL. For example, the concentration of IL6 is about 45ng / mL. For example, the concentration of IL6 is about 50ng / mL.

[0170] In the present application, the concentration of the G-CSF is about 5-50 ng / mL. For example, the concentration of the G-CSF is about 5 ng / mL. For example, the concentration of the G-CSF is about 10 ng / mL. For example, the concentration of the G-CSF is about 15 ng / mL. For example, the concentration of the G-CSF is about 20 ng / mL. For example, the concentration of the G-CSF is about 25 ng / mL. For example, the concentration of the G-CSF is about 30 ng / mL. For example, the concentration of the G-CSF is about 35 ng / mL. For example, the concentration of the G-CSF is about 40 ng / mL. For example, the concentration of the G-CSF is about 45 ng / mL. For example, the concentration of the G-CSF is about 50 ng / mL.

[0171] In the present application, the concentration of Flt3-L is about 5-50ng / mL. For example, the concentration of Flt3-L is about 5ng / mL. For example, the concentration of Flt3-L is about 10ng / mL. For example, the concentration of Flt3-L is about 15ng / mL. For example, the concentration of Flt3-L is about 20ng / mL. For example, the concentration of Flt3-L is about 25ng / mL. For example, the concentration of Flt3-L is about 30ng / mL. For example, the concentration of Flt3-L is about 35ng / mL. For example, the concentration of Flt3-L is about 40ng / mL. For example, the concentration of Flt3-L is about 45ng / mL. For example, the concentration of Flt3-L is about 50ng / mL.

[0172] GABA pathway activators

[0173] In the present application, the GABA pathway activator may include 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.

[0174] In the present application, the GABA pathway activator may include a GABAA pathway activator.

[0175] In the present application, the GABA pathway activator may include a GABAC pathway activator.

[0176] 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.

[0177] In the present application, the GABA receptor agonist may include GABA or its derivatives. In the present application, the GABA receptor agonist may include non-GABA or its derivatives, and such compounds may also activate the GABA pathway. For example, the GABAA receptor agonist may include 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.

[0178] For example, the GABAC receptor agonist may include 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.

[0179] In the present application, the GABA pathway activator may also include a positive allosteric enhancer (PAM) of a GABA receptor. For example, the positive allosteric enhancer of a GABA receptor may include phenolic, ketone, imidazole and / or pyrazole compounds.

[0180] For example, the positive allosteric modulator of the GABA receptor may include a positive allosteric modulator of the GABAA receptor. For example, the positive allosteric modulator of the GABA receptor may include 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.

[0181] 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), 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).

[0182] In the present application, the GABA pathway activator may include 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.

[0183] 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 20-200mM, about 20-300mM, about 30-400mM, about 30-500mM, about 30-600mM, about 50-700mM, about 100-800mM, about 200-800mM, about 300-900mM, about 400-900mM, about 500-900mM, about 600-1000mM, about 700-1000mM, about 800-900mM or about 900-1000mM.

[0184] cell

[0185] In the present application, the hematopoietic stem / progenitor cells may be derived from induced pluripotent stem cells.

[0186] In the present application, the hematopoietic stem / progenitor cells may be derived from embryonic stem cells.

[0187] In the present application, the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0188] In the present application, the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0189] In the present application, the hematopoietic stem / progenitor cells are derived from bone marrow.

[0190] In the present application, the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0191] In the present application, the differentiation pathway of the hematopoietic stem / progenitor cells may include differentiation from hematopoietic stem / progenitor cells to granulocyte-macrophage progenitor cells, and then differentiation from granulocyte-macrophage progenitor cells to granulocytes.

[0192] On the other hand, the present application also provides a granulocyte, which is obtained by differentiation of hematopoietic stem / progenitor cells by activating the GABA pathway.

[0193] In the present application, the cell and / or the state of the cell can be determined by cell markers. For example, the type of cell and / or the state of the cell can be determined by marker phenotype.

[0194] In certain embodiments, the cells described herein are isolated.

[0195] In the present application, the cell can be prepared pharmaceutically according to any conventional method. For example, carrier, excipient or diluent can be used to mix or dilute the 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. The preparation can additionally include filler, anti-agglomeration agent, lubricant, wetting agent, flavoring agent, emulsifier, preservative etc. Prepare the composition of the present invention by using any known method in the art to provide quick, continuous or delayed release active ingredient after giving the patient.

[0196] The application of the cell of the present application can be by injection (e.g., muscle, vein, peritoneal cavity, subcutaneous), or by other methods such as infusion, to ensure that it enters the bloodstream in an effective form. The cell can also be administered by a route within a tumor, around a tumor, within a lesion, or around a lesion to exert local and systemic therapeutic effects. For example, it can be administered locally or intravenously.

[0197] In the present application, the administration dosage of the cells may also be a single dose or multiple doses. For example, the actual administration amount of the cells may be determined according to 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.

[0198] Composition, reagent, use

[0199] On the other hand, the present application also provides the use of the culture medium in generating granulocytes by differentiating hematopoietic stem / progenitor cells.

[0200] On the other hand, the present application also provides use of the culture medium in generating granulocytes by differentiating stem cells.

[0201] On the other hand, the present application also provides the use of the culture medium in generating granulocytes by differentiating hematopoietic stem / progenitor cells.

[0202] On the other hand, the present application also provides use of the culture medium in generating granulocytes by differentiating granulocyte-macrophage progenitor cells.

[0203] On the other hand, the present application also provides a composition comprising hematopoietic stem / progenitor cells and the culture medium.

[0204] In another aspect, the present application also provides a composition comprising stem cells and the culture medium.

[0205] On the other hand, the present application also provides a composition comprising granulocyte-macrophage progenitor cells and the culture medium.

[0206] On the other hand, the present application also provides granulocytes, which are obtained by differentiation of hematopoietic stem / progenitor cells by activating the GABA pathway.

[0207] In another aspect, the present application also provides an agent for granulocyte differentiation, which comprises a GABA pathway activator.

[0208] On the other hand, the present application also provides use of a GABA pathway activator in preparing a reagent for granulocyte differentiation.

[0209] 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 into granulocytes.

[0210] On the other hand, the present application also provides a culture platform for obtaining granulocytes derived from hematopoietic stem / progenitor cells, which comprises the method, the culture medium, and / or hematopoietic stem / progenitor cells.

[0211] The present application also provides the following implementation modes:

[0212] 1. A method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into granulocytes, comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the hematopoietic stem / progenitor cells.

[0213] 2. A method for inducing stem cells to proliferate and / or differentiate into granulocytes, comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the stem cells.

[0214] 3. A method for inducing proliferation and / or differentiation of granulocyte-macrophage progenitor cells into granulocytes, comprising activating the gamma-aminobutyric acid (GABA) signaling pathway of the granulocyte-macrophage progenitor cells.

[0215] 4. The method according to any one of embodiments 1-3, comprising activating the GABAA and / or GABAC signaling pathway of the stem cells, hematopoietic stem / progenitor cells or granulocyte-macrophage progenitor cells.

[0216] 5. The method according to any one of embodiments 1-4, comprising activating the GABAA and / or GABAC receptors of the stem cells, hematopoietic stem / progenitor cells or granulocyte-macrophage progenitor cells.

[0217] 6. The method according to any one of embodiments 1-5, comprising administering a GABA pathway activator to the stem cells, hematopoietic stem / progenitor cells, or granulocyte-macrophage progenitor cells.

[0218] 7. The method according to embodiment 6, wherein the GABA pathway activator comprises a GABAA pathway activator and / or a GABAC pathway activator.

[0219] 8. The method of any one of embodiments 6-7, wherein the GABA pathway activator comprises a GABA receptor agonist.

[0220] 9. The method according to embodiment 8, wherein the GABA receptor agonist comprises a GABAA receptor agonist and / or a GABAC receptor agonist.

[0221] 10. The method according to embodiment 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.

[0222] 11. A method according to 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.

[0223] 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.

[0224] 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.

[0225] 14. The method according to 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.

[0226] 15. The method of any one of embodiments 6-14, wherein the GABA pathway activator comprises GABA and / or a GABA derivative.

[0227] 16. A method according to any one of embodiments 6-15, wherein the GABA pathway activator includes 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).

[0228] 17. The method according to 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 GABAtrimethylphosphine, 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.

[0229] 18. The method of any one of embodiments 6-17, wherein the concentration of the GABA pathway activator is about 0.1-1000 nM.

[0230] 19. The method of any one of embodiments 6-18, wherein the concentration of the GABA pathway activator is about 10-20 nM.

[0231] 20. The method according to any one of embodiments 1-19, comprising culturing the hematopoietic stem / progenitor cells using an initial differentiation medium, wherein the initial differentiation medium comprises basal medium I and the GABA pathway activator.

[0232] 21. The method according to embodiment 20, wherein the initial differentiation medium further comprises one or more of the following cytokines: SCF, IL3, IL7, IL15 and Flt3-L.

[0233] 22. The method of embodiment 21, wherein the concentration of SCF is about 5-50 ng / mL.

[0234] 23. The method of any one of embodiments 21-22, wherein the concentration of IL3 is about 5-50 ng / mL.

[0235] 24. The method of any one of embodiments 21-23, wherein the concentration of IL7 is about 5-50 ng / mL.

[0236] 25. The method of any one of embodiments 21-24, wherein the concentration of IL15 is about 5-50 ng / mL.

[0237] 26. The method of any one of embodiments 21-25, wherein the concentration of Flt3-L is about 5-50 ng / mL.

[0238] 27. A method according to any one of embodiments 20-26, wherein the basal medium I comprises StemSpanTM SFEMII medium.

[0239] 28. The method according to any one of embodiments 1-27, further comprising the use of granulocyte differentiation medium I, wherein the granulocyte differentiation medium I comprises basal medium II and one or more cytokines selected from the group consisting of SCF, TPO, G-CSF, IL3, IL5 and IL6.

[0240] 29. The method of embodiment 28, wherein the concentration of SCF is about 5-50 ng / mL.

[0241] 30. The method of any one of embodiments 28-29, wherein the concentration of IL3 is about 5-50 ng / mL.

[0242] 31. The method of any one of embodiments 28-30, wherein the concentration of TPO is about 5-50 ng / mL.

[0243] 32. The method of any one of embodiments 28-31, wherein the concentration of IL5 is about 5-50 ng / mL.

[0244] 33. The method of any one of embodiments 28-32, wherein the concentration of IL6 is about 5-50 ng / mL.

[0245] 34. The method of any one of embodiments 28-33, wherein the concentration of G-CSF is about 5-50 ng / mL.

[0246] 35. The method according to any one of embodiments 28-34, wherein the basal medium II comprises IMDM medium and fetal calf serum FCS (fetal calf serum).

[0247] 36. A method according to any one of embodiments 28-35, wherein the granulocyte differentiation medium I further comprises a GABA pathway activator.

[0248] 37. The method according to any one of embodiments 1-36, further comprising using a granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

[0249] 38. The method of embodiment 37, wherein the concentration of SCF is about 5-50 ng / mL.

[0250] 39. The method of any one of embodiments 37-38, wherein the concentration of IL7 is about 5-50 ng / mL.

[0251] 40. The method of any one of embodiments 37-39, wherein the concentration of IL15 is about 5-50 ng / mL.

[0252] 41. The method of any one of embodiments 37-40, wherein the concentration of Flt3-L is about 5-50 ng / mL.

[0253] 42. A method according to any one of embodiments 37-41, wherein the granulocyte differentiation medium II further comprises a GABA pathway activator.

[0254] 43. The method according to any one of embodiments 1-42 comprises the following steps: 1) inoculating hematopoietic stem / progenitor cells and culturing them in an initial differentiation medium, wherein the initial differentiation medium comprises a GABA pathway activator; 2) after completing step 1), continuing culturing using the granulocyte differentiation medium I; and 3) continuing culturing using the granulocyte differentiation medium II.

[0255] 44. The method according to any one of embodiments 1-43 comprises the following steps: 1) inoculating hematopoietic stem / progenitor cells in an initial differentiation medium and culturing the medium, wherein the initial differentiation medium comprises a GABA pathway activator; 2) after completing step 1), continuing culturing using the granulocyte differentiation medium I for about 3-5 days, or until granulocytes are obtained, wherein the granulocytes include neutrophils or eosinophils; and 3) continuing culturing using the granulocyte differentiation medium II, performing a half-volume medium replacement using the granulocyte differentiation medium II every about 2-3 days, and culturing for about 22-28 days.

[0256] 45. A method according to any one of embodiments 28-44, wherein the granulocyte differentiation medium I further comprises a GABA pathway activator.

[0257] 46. ​​A method according to any one of embodiments 37-45, wherein the granulocyte differentiation medium II also contains a GABA pathway activator.

[0258] 47. The method according to any one of embodiments 43-46, wherein in step 1), the culture conditions are about 35-39° C. and about 3-7% CO 2 .

[0259] 48. A method according to any one of embodiments 1-47, comprising increasing the expression, function and / or activity of GABA receptors in the stem cells, the hematopoietic stem / progenitor cells or the granulocyte-macrophage progenitor cells.

[0260] 49. The method of embodiment 48, wherein the GABA receptor comprises a GABAA receptor and / or a GABAC receptor.

[0261] 50. A method according to any one of embodiments 48-49, comprising increasing the expression, function and / or activity of the GABA receptor in the stem cells, the hematopoietic stem / progenitor cells or the granulocyte-macrophage progenitor cells by gene regulation.

[0262] 51. A method according to embodiment 50, 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.

[0263] 52. A method according to any one of embodiments 1-51, wherein the hematopoietic stem / progenitor cells are derived from induced pluripotent stem cells.

[0264] 53. A method according to any one of embodiments 1-52, wherein the hematopoietic stem / progenitor cells are derived from embryonic stem cells.

[0265] 54. A method according to any one of embodiments 1-53, wherein the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

[0266] 55. A method according to any one of embodiments 1-54, wherein the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

[0267] 56. A method according to any one of embodiments 1-55, wherein the hematopoietic stem / progenitor cells are derived from bone marrow.

[0268] 57. A method according to any one of embodiments 1-56, wherein the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0269] 58. A method according to any one of embodiments 1-57, wherein the granulocytes are neutrophils or eosinophils or basophils.

[0270] 59. A culture medium comprising a GABA pathway activator and a granulocyte differentiation medium.

[0271] 60. The culture medium of embodiment 59, wherein the GABA pathway activator comprises a GABA receptor agonist and / or a positive allosteric modulator of the GABA receptor.

[0272] 61. The culture medium of any one of embodiments 59-60, wherein the GABA pathway activator comprises GABA and / or a GABA derivative.

[0273] 62. A culture medium according to any one of embodiments 59-61, wherein the granulocyte differentiation medium comprises an initial differentiation medium and one or more cytokines.

[0274] 63. A culture medium according to embodiment 62, wherein the initial differentiation medium comprises basal medium I and a GABA pathway activator.

[0275] 64. The culture medium of embodiment 63, wherein the cytokines comprise one or more of the following cytokines: SCF, IL3, IL7, IL15, and Flt3-L.

[0276] 65. A culture medium according to any one of embodiments 63-64, wherein the basal culture medium I comprises StemSpanTM SFEMII medium.

[0277] 66. The culture medium of any one of embodiments 64-65, wherein the concentration of SCF is about 5-50 ng / mL.

[0278] 67. The culture medium of any one of embodiments 64-66, wherein the concentration of IL3 is about 5-50 ng / mL.

[0279] 68. The culture medium of any one of embodiments 64-67, wherein the concentration of IL7 is about 5-50 ng / mL.

[0280] 69. The culture medium of any one of embodiments 64-68, wherein the concentration of IL15 is about 5-50 ng / mL.

[0281] 70. The culture medium of any one of embodiments 64-69, wherein the concentration of Flt3-L is about 5-50 ng / mL.

[0282] 71. A culture medium according to any one of embodiments 59-70, wherein the granulocyte differentiation medium further comprises a granulocyte differentiation medium I, wherein the granulocyte differentiation medium I comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, TPO, G-CSF, IL3, IL5 and IL6.

[0283] 72. The culture medium of embodiment 71, wherein the concentration of SCF is about 5-50 ng / mL.

[0284] 73. The culture medium of any one of embodiments 71-72, wherein the concentration of IL3 is about 5-50 ng / mL.

[0285] 74. The culture medium of any one of embodiments 71-73, wherein the concentration of TPO is about 5-50 ng / mL.

[0286] 75. The culture medium of any one of embodiments 71-74, wherein the concentration of IL5 is about 5-50 ng / mL.

[0287] 76. The culture medium of any one of embodiments 71-75, wherein the concentration of IL6 is about 5-50 ng / mL.

[0288] 77. The culture medium of any one of embodiments 71-76, wherein the concentration of G-CSF is about 5-50 ng / mL.

[0289] 78. A culture medium according to any one of embodiments 71-77, wherein the basic culture medium II comprises IMDM culture medium and fetal calf serum FCS (fetal calf serum).

[0290] 79. A culture medium according to any one of embodiments 71-78, wherein the granulocyte differentiation medium I further comprises a GABA pathway activator.

[0291] 80. The culture medium according to any one of embodiments 59-79, further comprising a granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

[0292] 81. The culture medium of embodiment 80, wherein the concentration of SCF is about 5-50 ng / mL.

[0293] 82. The culture medium of any one of embodiments 80-81, wherein the concentration of IL7 is about 5-50 ng / mL.

[0294] 83. The culture medium of any one of embodiments 80-82, wherein the concentration of IL15 is about 5-50 ng / mL.

[0295] 84. The culture medium of any one of embodiments 80-83, wherein the concentration of Flt3-L is about 5-50 ng / mL.

[0296] 85. A culture medium according to any one of embodiments 80-84, wherein the granulocyte differentiation medium II further comprises a GABA pathway activator.

[0297] 86. Use of the culture medium described in any of embodiments 59-85 in producing granulocytes by differentiating hematopoietic stem / progenitor cells.

[0298] 87. A composition comprising stem cells, hematopoietic stem / progenitor cells, or granulocyte-macrophage progenitor cells, and a culture medium according to any one of embodiments 59-85.

[0299] 88. The modified hematopoietic stem / progenitor cells have increased expression, function and / or activity of GABA receptors compared to unmodified hematopoietic stem / progenitor cells.

[0300] 89. Granulocyte-macrophage progenitor cells, which are differentiated from hematopoietic stem / progenitor cells by activating the GABA pathway.

[0301] 90. Granulocytes obtained by differentiation of hematopoietic stem / progenitor cells by activating the GABA pathway.

[0302] 91. Use of a GABA pathway activator in the preparation of a reagent for granulocyte differentiation.

[0303] Application of GABA pathway activators in differentiation and / or expansion of hematopoietic stem / progenitor cells into granulocytes.

[0304] 93. Application of GABA pathway activators in producing culture medium for stem cell differentiation into granulocytes.

[0305] 94. A culture platform for obtaining granulocytes derived from hematopoietic stem / progenitor cells, comprising the method described in any one of embodiments 1-58, the culture medium and / or hematopoietic stem / progenitor cells described in any one of embodiments 59-85.

[0306] 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.

[0307] Example

[0308] Example 1 Differentiation and culture of granulocytes

[0309] 1.1 Hematopoietic stem cells were inoculated into GMP differentiation medium and cultured for 5 days;

[0310] The GMP differentiation medium is a medium obtained by adding cytokines SCF, IL3, IL7, IL15, Flt3-L and a GABA pathway activator to the basic differentiation medium I;

[0311] 1.2 After completing step 1, continue culturing with granulocyte differentiation medium for 18-24 days, or until granulocytes (neutrophils or eosinophils or basophils) are obtained;

[0312] The granulocyte differentiation medium is a medium obtained by adding cytokines SCF, TPO, G-CSF, IL3, IL5, and IL6 to the basic differentiation medium II.

[0313] The basic differentiation medium I includes StemSpan TM SFEMII medium; basic differentiation medium II includes IMDM medium and FCS.

[0314] In the initial differentiation medium, the concentration of SCF is 5-50 ng / mL; the concentration of IL3 is 5-50 ng / mL; the concentration of IL7 is 5-50 ng / mL; the concentration of IL15 is 5-50 ng / mL; and the concentration of Flt3-L is 5-50 ng / mL.

[0315] In the granulocyte differentiation culture, the concentration of SCF is 5-50 ng / mL; the concentration of IL3 is 5-50 ng / mL; the concentration of TPO is 5-50 ng / mL; the concentration of IL5 is 5-50 ng / mL; the concentration of IL6 is 5-50 ng / mL; and the concentration of G-CSF is 5-50 ng / mL.

[0316] In step 1, the culture conditions are 35-39°C and 3-7% CO 2 nourish.

[0317] The experimental results are as follows Figure 1 As shown, under the same culture conditions, compared with the Control group (the culture medium did not add the GABA pathway activator), the number of cell proliferation in the GABA group (the pathway activator of GABA added to the culture medium was GABA) and the TACA group (the pathway activator of GABA added to the culture medium was TACA) was significantly increased, indicating that activating the GABA pathway of cells can promote granulocyte proliferation.

[0318] The experimental results are as follows Figure 2 As shown, on the 18th day of cell culture (mid-differentiation), the cells were subjected to flow cytometry detection. Compared with the Control, the GABA and TACA groups could differentiate more granulocytes, indicating that activating the GABA pathway of cells can promote the differentiation of hematopoietic stem / progenitor cells into granulocytes.

[0319] The experimental results are as follows Figure 3 As shown, on the 28th day of cell culture (late differentiation stage), the cells were subjected to flow cytometry detection. Compared with the Control, the GABA and TACA groups could differentiate more granulocytes, indicating that activating the GABA pathway of cells can promote the differentiation of hematopoietic stem / progenitor cells into granulocytes.

[0320] The above experimental results indicate that activating the γ-aminobutyric acid (GABA) signaling pathway of cells can better induce the proliferation and / or differentiation of hematopoietic stem / progenitor cells into granulocytes, methods for inducing stem cells to proliferate and / or differentiate into granulocytes, and methods for inducing the proliferation and / or differentiation of granulocyte-macrophage progenitor cells into granulocytes.

[0321] 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 currently listed in this application are obvious to those of ordinary skill in the art and are retained within the scope of the appended claims and their equivalents.

Claims

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

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

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

4. The method according to any one of claims 1 to 3, comprising administering a GABA pathway activator to the stem cells, hematopoietic stem / progenitor cells or granulocyte-macrophage progenitor cells.

5. The method according to claim 4, wherein the GABA pathway activator 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, Zolpide, 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, Tolgabide, positive allosteric modulators of GABA receptors (positive allostericenhancer (PAM), 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, Volatileorganic compounds, trans-4-aminocrotonic acid (TACA), muscimol, (±)-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), 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, 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-Chloroacetylcytosinearabinoside, Methionine, Gabapentin, Piperazine, Gabapentin HCl, Etomidate, 6-Hydroxyflavone, 3,4,5-Trimethoxycinnamic acid (TMCA), Glabridin, Afloqualone and Oxiracetam., 6 . The method according to any one of claims 1 to 3 , comprising culturing the hematopoietic stem / progenitor cells using an initial differentiation medium, wherein the initial differentiation medium comprises basal medium I and the GABA pathway activator.

7. The method of claim 6, wherein the initial differentiation medium further comprises one or more of the following cytokines: SCF, IL3, IL7, IL15 and Flt3-L.

8. The method according to any one of claims 1 to 3, further comprising using a granulocyte differentiation medium II, wherein the granulocyte differentiation medium II comprises a basal medium II and one or more cytokines selected from the group consisting of SCF, IL7, IL15 and Flt3-L.

9. The method according to claim 8, wherein the granulocyte differentiation medium II further comprises a GABA pathway activator.

10. The method according to any one of claims 1-3, comprising the following steps: 1) inoculating hematopoietic stem / progenitor cells in an initial differentiation medium for culture, wherein the initial differentiation medium contains a GABA pathway activator; 2) after completing step 1), continuing culture using the granulocyte differentiation medium I; and 3) continuing culture using the granulocyte differentiation medium II.

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