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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
  • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0657—Cardiomyocytes; Heart cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/505—CD4; CD8
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

• the present invention relates to a method for producing CD8 positive cells using one or more hematopoietic progenitor cell markers (HPC markers), a cell population expressing one or more HPC markers, and the like.
• HPC markers hematopoietic progenitor cell markers
• T cells play a central role in the immune system against foreign pathogens such as bacteria and viruses and abnormal cells such as cancer cells.
• CTL cytotoxic T cells
• TCR T cell receptors
• CD8 single positive (SP) cells are CD8 single positive (SP) cells.
• the CD8SP cell is an immature cell that does not express TCR and has neither CD4 nor CD8 in the thymus (CD4 / CD8 double negative (in this specification, double negative may be referred to as DN).
• DN double negative
• DP double positives
• T cells play a central role in the immune system, so if T cells can be replenished and regenerated, they are extremely useful for the prevention or treatment of diseases such as tumors, infections, and autoimmune disorders. It becomes an effective means.
• hematopoietic progenitor cells and T cells can be obtained by a method of inducing hematopoietic progenitor cells (HematopoieticgenProgenitor Cell; sometimes referred to as HPC in this specification) from iPS cells or a method of inducing CD4 / CD8DP cells. Attempts have been made to obtain lineage cells.
• Non-Patent Document 1 describes that CD34 / CD43DP cells can be induced by co-culturing iPS cells with feeder cells (specifically, OP9 cells).
• Patent Document 1 and Non-Patent Document 2 describe a method for separating HPC from a cell population containing HPC derived from human pluripotent stem cells using CD43 and CD34, CD31 or CD144 as indicators, and the separated cells. It is described that the properties of induced hematopoietic cells differ depending on the method of differentiation into cells of the T cell lineage and whether or not Activin / Nodal is activated in the early stage of induction when differentiating from pluripotent stem cells to HPC. Has been.
• Patent Document 2 describes a method for producing CD4 / CD8DP under feeder-free conditions.
• Non-patent document 3 describes various methods for inducing blood cells including hematopoietic progenitor cells from human pluripotent stem cells and the reason why clinical use of blood cells induced in vitro has not progressed. Low induction efficiency is mentioned.
• the present invention provides cells of T cell lineage (eg, CD8 positive cells (eg, CD4 / CD8DP cells, CD8SP cells)) at higher and / or higher concentrations in order to stably provide cell therapy ( That is, an object is to provide a method for producing the cells. Moreover, this invention makes it a subject to provide the cell population containing the hematopoietic progenitor cell for solving the said subject.
• CD8 positive cells eg, CD4 / CD8DP cells, CD8SP cells
• the present inventors have identified a marker (HPC marker) that is expressed on the surface of HPC in order to develop a method for producing CD4 / CD8DP cells that solve the above problems. Furthermore, HPC isolated from a cell population containing HPC using one or more of the markers has high efficiency of differentiating into CD8 positive cells (eg, CD4 / CD8DP cells) and has a high proliferation ability, ie, 1 It has been found that by using more than one HPC marker, more CD8 positive cells (eg, CD4 / CD8DP cells) can be obtained and / or at higher concentrations within the cell population. Furthermore, it has been found that the production method of the present invention can provide stable CD8 positive cells (eg, CD4 / CD8DP cells) with little variation in yield, and the present invention has been completed.
• HPC marker a marker that is expressed on the surface of HPC in order to develop a method for producing CD4 / CD8DP cells that solve the above problems.
• Step 1 From a cell population containing hematopoietic progenitor cells, from CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262, and CD325
• a step 2 a step of differentiating the cells separated in step 1 into CD4 / CD8 double positive cells
• the cells selected in step 1 are CD235a And the method according to [1], wherein the cell does not express CD14 and
• step 1 from the cell population containing hematopoietic progenitor cells, CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226
• the first group shown in the step 1 is a group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD200, CD218a, CD7 and CD144.
• step 1 one or more molecules selected from the second group consisting of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102 and CD156c are selected from a cell population containing hematopoietic progenitor cells.
• the method according to [5] wherein the second group shown in Step 1 is a group consisting of CD49f, CD51 and CD102.
• step 7 The method according to any one of [1] to [6], wherein the cell population containing the hematopoietic progenitor cells in Step 1 is a cell population obtained by inducing differentiation of pluripotent stem cells.
• the method of inducing includes, for example, a method comprising culturing CD4 / CD8 double positive cells in the presence of an adrenocortical hormone agent, an antibody and / or a cytokine, preferably the adrenal cortex
• the pluripotent stem cells are human induced pluripotent stem cells.
• [15] Selected from the first group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262 and CD325 Hematopoietic progenitor cells that express one or more molecules and / or one or more molecules selected from the second group consisting of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102 and CD156c A cell population in which the ratio of hematopoietic progenitor cells (the number of hematopoietic progenitor cells / the total number of cells contained in the cell population) is 40% or more.
• the cell population according to [15], wherein [17] The cell population according to [15], wherein the ratio is a ratio of hematopoietic progenitor cells that do not further express one or more molecules selected from the second group consisting of CD49f, CD51, and CD102.
• a reagent for separating hematopoietic progenitor cells comprising an antibody against each of one or more molecules selected from the group consisting of CD62P, CD69, CD102 and CD156c. [19] The reagent according to [18], further comprising one or more antibodies against CD235a, CD14, CD45, CD34 and / or CD43.
• a method for detecting a surface protein of a hematopoietic progenitor cell comprising the hematopoietic progenitor cell, CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7 , CD144, CD56, CD226, CD262, CD325, CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, CD109 or CD156c.
• the substance comprises an antibody or a fragment thereof.
• the substance further contains a fluorophore.
• a method for selecting hematopoietic progenitor cells from a cell population containing hematopoietic progenitor cells, the presence or absence of one or more proteins on the surface of cells by any of the methods [20] to [24] And (i) CD45, CD34 and / or CD43 and the following proteins: CD24, CD62L, C90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262 and CD325: any one or any combination of these are detected on the surface of the cells and / or (ii) When CD14 and / or CD235a is not detected on the surface of the cell, and the following proteins: CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, CD109 and CD156c: are not detected And selecting the cells.
• CD8 positive cells for example, CD4 / CD8DP cells, CD8SP cells
• CD8 positive cells for example, CD4 / CD8DP cells, CD8SP cells
• the production method of the present invention can provide CD8 positive cells (for example, CD4 / CD8DP cells, CD8SP cells) stably with little variation in yield. Accordingly, the production method of the present invention provides an improved method for separating hematopoietic progenitor cells from a cell population. This provides an improved cell source for generating CD8 positive cells.
• This novel production method is surprising to the inventors that certain HPC positive markers and HPC negative markers can be used to greatly improve the quality and / or quantity of a CD8 positive cell population derived from HPC. And based on unexpected findings.
• This beneficial source of CD8 positive cells of the present invention provides an improved CD8 positive cell population for clinical applications (eg, cell therapy, diagnostic purposes, and in vitro applications).
• the present invention is (step 1) a cell expressing one or more hematopoietic progenitor cell markers (sometimes abbreviated as “HPC marker” in the present specification) from a cell population containing hematopoietic progenitor cells, and / or Alternatively, a step of separating cells that do not express one or more HPC markers, and (step 2) a cell separated by step 1 is a CD8 positive cell (for example, CD4 / CD8 double positive cell (hereinafter referred to as “CD4 A method of producing CD8 positive cells (for example, CD4 / CD8DP cells) (hereinafter abbreviated as “production method of the present invention”), which includes a step of differentiation into “/ CD8DP cells”. To do.
• CD8 positive cell for example, CD4 / CD8 double positive cell
• production method of the present invention production method of the present invention
• CD8 positive cell means a T cell expressing CD8.
• the cell include CD4 / CD8DP cell and CD8 single positive cell (herein, “CD8SP cell”). May be abbreviated as ")".
• CD4 / CD8DP cell means a T cell that expresses both CD4 and CD8, and “CD8SP cell” means a T cell that does not express CD4 but expresses CD8. means.
• Examples of CD8SP cells include cytotoxic T cells and their progenitor cells.
• T cell means, for example, a cell expressing an antigen receptor called T cell receptor (TCR) on its surface and its precursor cell (eg, TCR). (Pro T cells not expressing (TCR ⁇ ), pre-T cells in which TCR ⁇ and pre-TCR ⁇ are associated).
• TCR T cell receptor
• hematopoietic progenitor cell means a CD34 positive cell, preferably a CD34 / CD43DP cell.
• the origin of hematopoietic progenitor cells used in the present invention is not limited.
• hematopoietic progenitor cells induced in vitro by known methods for example, the methods described in Patent Documents 1 and 2 and Non-Patent Documents 1 to 3.
• hematopoietic progenitor cells obtained by inducing differentiation of pluripotent stem cells or hematopoietic progenitor cells isolated from living tissue by a known technique may be used.
• pluripotent stem cells examples include embryonic stem cells (embryonic stem cells: ES cells), induced pluripotent stem cells (induced PS cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells) are mentioned, and iPS cells (more preferably human iPS cells) are preferred.
• ES cells embryonic stem cells
• induced PS cells induced pluripotent stem cells
• EC cells embryonic tumor cells
• EG cells embryonic germ stem cells
• iPS cells more preferably human iPS cells
• the pluripotent stem cell is any cell derived from an ES cell or a human embryo, the cell is a cell prepared without destroying the embryo, even if the cell is prepared by destroying the embryo. However, it is preferably a cell produced without destroying the embryo.
• the iPS cell can be produced by introducing a specific reprogramming factor into a somatic cell in the form of DNA or protein, and has almost the same characteristics as an ES cell, such as pluripotency and proliferation ability by self-replication.
• a specific reprogramming factor such as pluripotency and proliferation ability by self-replication.
• the iPS cells may be prepared from somatic cells by a method known per se, or iPS cells that have already been established and stocked may be used.
• the somatic cells from which iPS cells used in the present invention are derived are not limited, but are preferably cells derived from peripheral blood or cells derived from umbilical cord blood.
• a pluripotent stem cell is derived,
• mammals such as a mouse
• Hematopoietic progenitor cells can be produced by subjecting the pluripotent stem cells to a method known per se.
• the pluripotent stem cell is an iPS cell
• hematopoietic progenitor cells can be produced by, for example, the method described in International Publication No. 2017/221975.
• the biological tissue is not limited as long as it contains hematopoietic progenitor cells, and examples thereof include peripheral blood, lymph nodes, bone marrow, thymus, spleen, and umbilical cord blood.
• peripheral blood and umbilical cord blood are preferable from the viewpoint of low invasiveness to animals and easy preparation.
• the hematopoietic progenitor cells and pluripotent stem cells used in the present invention are preferably GMP (Good Manufacturing Practice) standard cells from the viewpoint of application to therapy.
• ⁇ HPC marker '' is a molecule that can be expressed on the surface of hematopoietic progenitor cells, CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, Any molecule of CD7, CD144, CD56, CD226, CD262, and CD325 (in the specification, these markers may be referred to as “HPC positive marker”, “positive HPC selection marker”, or “positive HPC marker”)
• HPC positive marker any molecule of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102 and CD156c, which is a molecule that is not normally expressed on the surface of hematopoietic progenitor cells
• these markers are referred to as “HPC negative Marker ",” negative HPC selectable marker ", or” negative HPC marker ” (in the specification, HPC positive marker and HPC negative marker are
• HPC positive markers of the present invention CD24, CD62L, CD90, CD143, CD263, Notch3, CD200, CD218a, CD200, CD7 or CD144 are preferred, and CD90, CD143, CD218a or CD200 is more preferred.
• CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200 or CD218a are preferred, and CD24, CD62L, CD90, CD143, CD263, Notch3, CD200 or CD218a are preferred. More preferred.
• the cell expressing the HPC positive marker may be referred to as an HPC marker positive cell (or HPC marker positive cell).
• the cell in the case of a cell expressing a CD24 molecule, the cell is referred to as a CD24 positive cell.
• the HPC negative markers of the present invention CD49f, CD51 or CD102 is preferable.
• the cells that do not express the HPC negative marker may be referred to as HPC marker negative cells (or HPC marker negative cells).
• CD8 positive cells preferably CD4 / CD8 double positive cells, express (a) CD34 and CD43 and (b) CD62L, CD24, CD90, CD143 from a cell population comprising hematopoietic progenitor cells. , CD218a, CD263 or Notch3, and (c) by isolating cells that do not express CD235a and CD14.
• the CD8 positive cells preferably CD4 / CD8 double positive cells, express (a) CD34 and CD43 and (b) CD90, CD143, CD200, CD218a from a cell population comprising hematopoietic progenitor cells. Or (c) by isolating cells that do not express CD235a and CD14.
• the CD8 positive cells preferably CD4 / CD8 double positive cells, express (a) CD34 and CD43 and (b) CD7, CD144, CD56, CD226 from a cell population comprising hematopoietic progenitor cells. , And expressing either CD262 or CD325 and (c) isolating cells that do not express CD235a and CD14.
• the CD8 positive cells preferably CD4 / CD8 double positive cells, express (a) CD34 and CD43 and (b) CD49f, CD51, CD426, CD61 from a cell population comprising hematopoietic progenitor cells. , CD62P, CD69 or CD156c, and (c) is produced by isolating cells that do not express CD235a and CD14.
• “express” means that expression can be detected by a method used for separating cells
• “not expressed” means detection by the detection sensitivity of the method used for separating cells. It means you can't.
• Examples of the method used when separating cells include the following method using flow cytometry and mass cytometry, and magnetic cell separation.
• the cells separated by the above step 1 are cells that express one or more HPC positive markers and cells that do not express one or more HPC negative markers.
• the cells separated by the above step 1 are cells expressing CD34 and / or CD43 in addition to one or more HPC positive markers of the present invention, and / or one or more books. It is a cell that does not express the HPC negative marker of the invention and expresses CD34 and / or CD43.
• the cells to be separated are preferably CD235a negative, CD14 negative and / or CD45 positive.
• the step 1 comprises, from a cell population containing hematopoietic progenitor cells, CD235a negative, CD14 negative, CD45 positive, CD34 positive and CD43 positive, and one or more HPCs of the present invention. This is done by isolating positive marker positive and / or one or more HPC negative marker negative cells of the present invention.
• “separate cells” selects a cell that expresses one or more HPC positive markers and / or does not express one or more HPC negative markers, and isolates the selected cells.
• An embodiment and an embodiment in which the selected cell is subjected to the next differentiation step are included.
• each term such as “comprising” or “comprises” optionally includes “consistingconsistof” or “consists of”. May be replaced.
• Flow cytometry or mass cytometry is used as a method for separating cells expressing one or more HPC positive markers and / or not expressing one or more HPC negative markers from a cell population containing hematopoietic progenitor cells.
• Methods, magnetic cell separation methods, etc., and these methods can be carried out using known methods.
• a cell that expresses one or more HPC positive markers and / or a cell that does not express one or more HPC negative markers includes a cell (for example, an antibody) that specifically binds to each HPC marker. It can isolate
• Such substances include those to which a detectable label (eg, GFP) has been added, and those to which no label has been added.
• a detectable label eg, GFP
• the separation can be performed by further using a substance with a detectable label that directly or indirectly recognizes the substance.
• a substance with a detectable label that directly or indirectly recognizes the substance.
• the substance is an antibody
• a fluorescent dye, a metal isotope or a bead is directly or indirectly supported on the antibody, thereby labeling an HPC marker on the cell surface. It is possible to separate cells based on the label. Only one type of antibody or two or more types of antibodies may be used.
• Examples of a method for differentiating cells isolated from cells that contain hematopoietic progenitor cells as cells expressing one or more HPC positive markers of the present invention and / or not expressing HPC negative markers into CD8 positive cells include: Known methods (for example, methods described in International Publication No. 2016/076415, JournalJof Leukocyte Biology 96 (2016) 1165-1175, Cell Reports (2 (2012) 1722-1735, International Publication No. 2017/221975)) Can be mentioned.
• IMDM Iscove's Modified Dulbecco's Medium
• EMEM Eagle's Minimum Essential Medium
• ⁇ MEM Dulbecco's modified Eagle's Medium
• DMEM Dulbecco's modified Eagle's Medium
• Ham's F12 medium Ham's F12 medium
• RPMI 1640 medium Fischer's medium
• Neurobasal Medium Neurobasal Medium (Life Technologies), and mixed media thereof.
• the medium may or may not contain serum.
• the medium contains, for example, vitamin Cs (eg ascorbic acid, vitamin C phosphate), albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids
• vitamin Cs eg ascorbic acid, vitamin C phosphate
• albumin e.g ascorbic acid, vitamin C phosphate
• insulin transferrin
• selenium fatty acids
• trace elements 2-mercaptoethanol
• thiolglycerol lipids
• amino acids amino acids
• substances such as L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines may be included.
• the medium preferably contains a cytokine.
• cytokines include IL-7, FLT-3L, SCF, TPO, and combinations thereof, and FLT-3L and IL-7 are preferred.
• the concentration of IL-7 in the medium ranges from 1 ng / ml to 50 ng / ml (eg, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml).
• the concentration of FLT-3L in the medium ranges from 1 ng / ml to 100 ng / ml (eg, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml).
• the medium preferably contains Notch ligand (eg, Dll1, Dll4, Dll1, or a fusion protein of Dll4 and Fc). When using a Notch ligand, it may be added to the medium or coated on the culture vessel. Alternatively, feeder cells that express a Notch ligand may be used.
• Notch ligand eg, Dll1, Dll4, Dll1, or a fusion protein of Dll4 and Fc.
• the hematopoietic progenitor cells obtained in step 1 may be subjected to adhesion culture or suspension culture.
• the culture vessel may be coated and used, or co-cultured with feeder cells or the like. From the viewpoint of application to treatment, it is preferably feeder-free.
• the substrate for coating a culture vessel include fibronectin fragments (eg, retronectin), pronectin, vitronectin, matrigel (BD), type I collagen, type IV collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and These combinations are mentioned.
• feeder cells to be co-cultured examples include bone marrow stromal cell line OP9 cells, mouse mesenchymal cell line 10T1 / 2 cells, and Tst-4 cells.
• the exchange of the feeder cells can be performed by transferring the target cells in culture onto the feeder cells previously seeded. The exchange is preferably performed every 2 to 5 days.
• the culture temperature condition for inducing CD4 / CD8 DP cells by culturing hematopoietic progenitor cells is not particularly limited, but is preferably about 37 ° C to 42 ° C, more preferably about 37 to 39 ° C.
• the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD4 / CD8DP cells.
• the number of culture days for inducing CD4 / CD8 DP cells from hematopoietic progenitor cells is not particularly limited, but it is 10 days or longer (eg, 12, 14, 16, 18, 20, or More than that), and preferably 60 days or less.
• CD4 / CD8DP cells can be efficiently obtained in large quantities by culturing for 21 to 28 days.
• the obtained CD4 / CD8DP cells may be isolated and used as a cell population containing other cell types (for example, CD8SP cells).
• CD4, CD8, CD3, CD45 molecules and the like can be used as an index.
• the isolation method a method well known to those skilled in the art can be used. For example, the method is performed by labeling with an antibody against a molecule as an index, and using the above-described flow cytometry or mass cytometry, magnetic cell separation method, Or the method of refine
• CD8SP cells can be produced by subjecting the CD4 / CD8DP cells obtained by the production method of the present invention to the step of inducing differentiation into CD8SP cells (a method for producing CD8SP cells in this way). May be abbreviated as “the method for producing the CD8SP cell of the present invention”). For the method of producing CD4 / CD8DP cells, see 1. As described in.
• basal medium and medium used in the method for producing CD8SP cells of the present invention the same basal medium and medium as described in 1. can be mentioned.
• the medium may contain a corticosteroid.
• corticosteroids include glucocorticoids and derivatives thereof, and the glucocorticoids include, for example, cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, Beclomethasone propionate is mentioned. Of these, dexamethasone is preferable.
• a method for producing CD8 single positive cells comprises culturing CD4 / CD8 double positive cells in the presence of an adrenocortical hormone agent, preferably dexamethasone, to obtain said CD4 / CD8 double positive cells. Inducing CD8 single positive cells.
• an adrenocortical hormone agent preferably dexamethasone
• dexamethasone When dexamethasone is used as a corticosteroid, its concentration in the culture solution is preferably 1 nM to 100 nM (eg, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM) Of these, 10 nM is preferable.
• the medium may contain antibodies (eg, anti-CD3 antibody, anti-CD28 antibody, anti-CD2 antibody), cytokines (eg, IL-7, IL-2, IL-15) and the like.
• antibodies eg, anti-CD3 antibody, anti-CD28 antibody, anti-CD2 antibody
• cytokines eg, IL-7, IL-2, IL-15
• the anti-CD3 antibody used in the present invention is not particularly limited as long as it is an antibody that specifically recognizes CD3, and examples thereof include an antibody produced from an OKT3 clone.
• the anti-CD3 antibody may be one to which magnetic beads or the like are bound, and instead of adding the anti-CD3 antibody to the medium, the TCD is cultured on a culture container in which the anti-CD3 antibody is bound to the surface. Stimulation may be performed by culturing lymphocytes for a certain period of time.
• the concentration of the anti-CD3 antibody in the medium is 10 ng / ml to 1000 ng / ml (eg, 10 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1000 ng / ml) are preferable, and 500 ng / ml is particularly preferable.
• the concentration of other antibodies can also be appropriately determined by those skilled in the art based on culture conditions and the like.
• a method for producing CD8 single positive cells comprises culturing CD4 / CD8 double positive cells in the presence of an antibody, preferably an anti-CD3 antibody, to cultivate said CD4 / CD8 double positive cells to CD8. Inducing to single positive cells.
• the concentration of IL-2 used in the present invention in the medium is 10 U / ml to 1000 U / ml (example: 10 U / ml, 20 U / ml, 30 U / ml, 40 U / ml, 50 U / ml). 60 U / ml, 70 U / ml, 80 U / ml, 90 U / ml, 100 U / ml, 500 U / ml, 1000 U / ml), with 100 U / ml being preferred.
• the concentration of IL-7 or IL15 used in the present invention in the medium is 1 ng / ml to 100 ng / ml (eg, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng).
• a method for producing CD8 single positive cells comprises culturing CD4 / CD8 double positive cells in the presence of a cytokine, preferably IL-2, to convert said CD4 / CD8 double positive cells to CD8. Inducing to single positive cells.
• a cytokine preferably IL-2
• the temperature condition for culturing CD4 / CD8DP cells is not particularly limited, but is preferably about 37 ° C to 42 ° C, more preferably about 37 to 39 ° C.
• the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD8-positive T cells.
• the number of days is not particularly limited as long as CD8SP cells can be obtained, but it is preferably 1 day or more, 3 days or more, 7 days or more, and preferably 60 days or less.
• the CD8SP cells produced by the production method of the present invention are not limited to cells obtained by differentiating from CD4 / CD8DP cells (for example, cells obtained by the above 2.).
• a CD4 / CD8DP cell from a cell isolated from a cell population containing hematopoietic progenitor cells that expresses one or more HPC positive markers of the present invention and / or does not express one or more HPC negative markers.
• CD8SP cells induced without passing through are also included in the CD8SP cells produced by the production method of the present invention.
• Cell population containing hematopoietic progenitor cells expressing one or more types of HPC markers The present invention expresses one or more types of HPC positive markers and / or does not express one or more types of HPC negative markers. Is a high-frequency cell population (hereinafter abbreviated as “cell population of the present invention”).
• the cell population is, for example, a cell population containing hematopoietic progenitor cells, wherein cells that express one or more HPC positive markers and / or do not express one or more HPC negative markers are used according to the separation method described above. Can be obtained by separating them.
• the definition of HPC markers, specific molecules, and the definition of hematopoietic progenitor cells are as described above.
• the cell population of the present invention is a ratio of hematopoietic progenitor cells expressing one or more HPC positive markers and / or not expressing one or more HPC negative markers (the number of hematopoietic progenitor cells / total cells contained in the cell population). Number) is higher than the proportion contained in the hematopoietic progenitor cell population obtained by conventional methods (eg, CD34 positive, CD34 positive and CD43 positive, or CD43 positive and CD34, CD31 or CD144 positive) However, it is preferably 40% or more (eg, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100%).
• the cell population of the present invention can be obtained by the method described in Step 1 of the production method of the present invention.
• the percentage of hematopoietic progenitor cells that express one or more HPC positive markers and / or do not express one or more HPC negative markers can be measured by flow cytometry.
• the cell population of the present invention is compared to a cell population that expresses one or more HPC positive markers and / or does not isolate cells that do not express one or more HPC negative markers.
• the efficiency of differentiation into CD4 / CD8DP cells and CD8SP cells is high, and the proliferation ability is also excellent.
• Reagent for separation of "cell population of the present invention” The present invention also provides an HPC marker of the present invention, i.e., CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262, CD325, CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102 and CD156c, preferably CD24, CD62L, CD90, CD143, CD263, Notch3, CD200CD218a, CD7, CD144
• a reagent for separating a cell population of the present invention comprising an antibody against each of one or more markers selected from the group consisting of CD49f, CD51 and CD102 provide.
• the reagent of the present invention contains antibodies against two or more HPC markers
• the reagent can be provided as a reagent kit containing each antibody in a separate reagent.
• the antibody contained in the reagent of the present invention can be provided in a form bound to, for example, a fluorescent dye, a metal isotope, or a bead (eg, magnetic bead) according to the separation means used in Step 1 of the production method of the present invention. .
• the reagent of the present invention may be an anti-CD4 antibody and / or an anti-CD8 antibody and optionally other cell surface markers known to be expressed or not expressed in CD4 / CD8DP cells and / or CD8SP cells (for example, , CD3, CD45, CD235a, CD14, CD45, CD34, CD43) as a reagent for separating CD4 / CD8DP cells and CD8SP cells induced to differentiate from the cell population of the present invention. Can be used.
• the reagent of the present invention may further contain reagents (eg, basal medium, medium additive, etc.) for inducing differentiation of hematopoietic progenitor cells into CD4 / CD8DP cells and further into CD8SP cells. As described above, 1. And 2. The substances exemplified in (1) are similarly mentioned.
• Example 1 (Examination using TKT3V1-7 strain) As a cell population containing hematopoietic progenitor cells (hereinafter sometimes referred to as HPC), iPS cells (TKT3V1-7 strain) are prepared by known methods (for example, Cell Reports 2 (2012) 1722-1735 and International Publication No. 2017/221975). The floating cell population differentiated according to the method described in No. 1) was used.
• HPC hematopoietic progenitor cells
• TKT3V1-7 strain was seeded at 3 ⁇ 10 5 cells / well on a 6-well plate treated with ultra-low adhesion (Day 0), and EB medium (10 ⁇ g / ml human insulin, 5.5 ⁇ g / well in StemPro34) ml human transferrin, 5 ng / ml sodium selenite, 2 mM L-glutamine, 45 mM ⁇ -monothioglycerol, and 50 ⁇ g / ml ascorbic acid), 10 ng / ml BMP4, 5 ng / ml bFGF, 15 ng / ml VEGF, 2 [mu] M SB431542, added were 5-day culture at a low oxygen conditions (5% 0 2) (Day5 ).
• EB medium 10 ⁇ g / ml human insulin, 5.5 ⁇ g / well in StemPro34
• ml human transferrin 5 ng / ml sodium selenite, 2
• the cell population subjected to the above staining was subjected to sorting by FACSAria.
• the cell fraction obtained from each sample group is shown below.
• fractions A to G each indicate the following.
• Fraction A CD235a negative, CD14 negative, CD34 positive, CD43 positive
• Fraction B CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD62L positive
• Fraction C CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD62L Negative Fraction D: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD24 positive
• Fraction E CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD24 negative
• Fraction E CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD24 negative
• Fraction F CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD90 positive
• Fraction G CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD90 negative
• Fraction H CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD143 positive
• Fraction I CD235a negative, CD14 negative CD34 positive, CD43 positive,
• the cells contained in the fractions A to O can be obtained from lymphocytes according to known methods (for example, the method described in Journal of Leukocyte Biology 96 (2016) 1165-1175 and International Publication No. 2017/221975). Differentiated into cells. Specifically, each cell population of fractions A to O was seeded at 48 cells / well in a 48-well-plate coated with Recombinat h-DLL4 / Fc chimera (Sino Biological) and Retrotrontin (Takara Bio). The cells were cultured under the conditions of 5% CO 2 and 37 ° C. During the culture period, the medium was changed every 2 or 3 days.
• OP9 medium (15% FBS, 2 mM L) supplemented with 50 ng / ml SCF, 50 ng / ml IL-7, 50 ng / ml Flt3L, 100 ng / ml TPO, 15 ⁇ M SB203580, 30 ng / ml SDF-1 ⁇ (Includes glutamine, 100 Uml penicillin, 100 ng / ml streptomycin, 55 ⁇ 2-mercaptoethanol, 50 ⁇ g / ml ascorbic acid, 10 ⁇ g / ml human insulin, 5.5 ⁇ g / ml human transferrin, 5 ng / ml sodium selenite) was used.
• the cells were subcultured to 48-well-plates with the same coating. On the 21st day from the start of the culture, all cells were collected, counted using a hemocytometer, and then stained using the following antibody set.
• the stained cell population was subjected to FACSAria sorting.
• the cell fraction obtained by the sorting is shown below.
• the lymphocyte fraction is the same as the method described in Cytometry (Communications in Clinical Cytometry) 18 (1994) 199-208, using FSC (forward scatter) and SSC (side scatter) as indices.
• the separated fraction Fraction P: Lymphocyte fraction
• Fraction Q Lymphocyte fraction, CD3 positive, CD45 positive, CD5 positive, CD7 positive
• Fraction R Lymphocyte fraction, CD3 positive, CD45 positive, CD7 positive, CD8a positive, CD4 Positive fraction
• S Lymphocyte fraction, CD3 positive, CD45 positive, CD7 positive, CD8a positive, CD4 negative
• Table 4 shows the number of cells on the 21st day of culture in each sample derived from fractions A to O (ratio with the number of cells in the sample derived from fraction A).
• Table 4 shows that fraction B (CD62L positive), fraction D (CD24 positive), fraction F (CD90 positive), and fraction H (CD143 positive) compared to the cell population contained in the sample derived from fraction A
• fraction J CD218a positive
• fraction L CD263 positive
• fraction N Notch3 positive
• Table 5 shows the number of cells in cell fraction P to S obtained by sorting with FACS Aria on the 21st day of culture (ratio with the number of cells in cell fraction P to S in the sample derived from fraction A).
• CD4 / CD8 double positive cells or CD8 positive cells
• CD90, CD143 or CD218a a high proportion of CD4 / CD8 double positive cells (or CD8 positive cells) by isolating cells that express CD90, CD143 or CD218a as a cell population containing hematopoietic progenitor cells and differentiating the isolated cells It has been shown that cell populations comprising can be produced.
• Example 2 (Study using Ff-I01s04 strain) As a cell population containing hematopoietic progenitor cells (hereinafter sometimes referred to as HPC), a floating cell population obtained by subjecting iPS cells (Ff-I01s04 strain) to the method described in Example 1 was used.
• the cell population containing HPC is the antibody set 3, 4, 5, 6 shown in Example 1 for each sample group (in this example, expressed as 3b, 4b, 5b, 6b, respectively) or the antibodies shown below Stained with set 8b.
• the cell population subjected to the above staining was subjected to sorting by FACSAria.
• the cell fraction obtained from each sample group is shown below.
• fractions f to m, t and u each indicate the following.
• Fraction f CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD90 positive
• Fraction g CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD90 negative
• Fraction h CD235a negative, CD14 negative, CD34 positive, CD43 Positive, CD143 positive
• Fraction i CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD143 negative
• Fraction j CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD218a positive
• Fraction k CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD218a negative
• Fraction l CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD263 positive
• Fraction m CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD263 negative
• Fraction t CD235a negative , CD14 negative, CD34 positive, CD43
• the cells contained in the above fractions f to m, t and u were subjected to the method described in Example 1 to be differentiated into lymphocyte cells. On day 28 of the start of culture, all cells were collected and stained using the following antibody set.
• the stained cell population was subjected to FACSAria sorting.
• the cell fraction obtained by the sorting is shown below.
• Fraction p Lymphocyte fraction fraction q: Lymphocyte fraction, CD45 positive, CD5 positive, CD7 positive fraction r: Lymphocyte fraction, CD45 positive, CD7 positive, CD8a positive, CD4 positive fraction s: Lymph Spherical fraction, CD45 positive, CD7 positive, CD8a positive, CD4 negative
• Table 9 shows the number of cells on the 28th day from the start of culture in each sample derived from fractions f to m, t and u (ratio with the number of cells at the start of culture).
• fraction f is fractioned with respect to fraction g, fraction h with respect to fraction i, fraction j with respect to fraction k, and fraction l with respect to fraction m. It was found that t was excellent in proliferation ability for fraction u. That is, it was found that all cell populations contained in samples derived from CD90 positive, CD143 positive, CD218a positive, CD263 positive or CD200 positive were excellent in proliferating ability.
• Table 10 shows the number of cells in cell fraction p-s obtained by sorting by FACS Aria on the 28th day of culture when cells contained in fractions fm, t, and u were seeded at 1000 cells / well. Show.
• CD45 positive and CD5 positive when cells derived from each marker positive cell were used compared to when cells derived from each marker negative cell were used.
• CD7-positive, CD8a-positive, and CD4-positive cells or
• CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-negative cells can be prepared with high yield.
• Example 3 (Examination using Ff-I01s04 strain) As a cell population containing hematopoietic progenitor cells (hereinafter sometimes referred to as HPC), a floating cell population obtained by subjecting iPS cells (Ff-I01s04 strain) to the method described in Example 1 was used. The floating cell population containing HPC was stained using the following antibody set for each sample group.
• HPC hematopoietic progenitor cells
• the cell population subjected to the above staining was subjected to sorting by FACSAria.
• the cell fraction obtained from each sample group is shown below.
• fractions Ac to Pc indicate the following.
• Fraction Ac CD235a negative, CD14 negative, CD34 positive, CD43 positive
• Fraction Bc CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD7 positive
• Fraction Cc CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD144 Positive
• Fraction Dc CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD56 positive
• Fraction Ec CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD226 positive
• Fc CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD262 positive
• Fraction Gc CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD325 positive
• Fraction Hc CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD49f positive
• Fraction Ic CD235a negative, CD14 negative CD34 positive, CD43 positive, CD51 positive
• Fraction Jc CD235a negative, CD14 negative,
• the cells contained in the fractions Ac to Pc were subjected to the method described in Example 1 to differentiate into lymphocyte cells. On the 21st day from the start of the culture, all cells were collected, counted using a hemocytometer, and then stained using the following antibody set.
• the stained cell population was subjected to FACSAria sorting.
• the cell fraction obtained by the sorting is shown below.
• Fraction Qc Lymphocyte fraction
• Fraction Rc Lymphocyte fraction, CD3 positive, CD45 positive, CD5 positive, CD7 positive
• Fraction Sc Lymphocyte fraction, CD3 positive, CD45 positive, CD7 positive, CD8a positive
• Tc Lymphocyte fraction, CD3 positive, CD45 positive, CD7 positive, CD8a positive, CD4 negative
• Table 14 shows the number of cells on the 21st day from the start of culture in each sample derived from fractions Ac to Pc (ratio with the number of cells in the sample derived from fraction Ac).
• fraction Bc (CD7 positive), fraction Cc (CD144 positive), fraction Dc (CD56 positive), fraction Ec (CD226 positive) It was found that the cell populations contained in the samples derived from fraction Fc (CD262 positive) and fraction Gc ⁇ (CD325 positive) all have excellent proliferation ability. Also, fraction Hc (CD49f positive), fraction Ic (CD51 positive), fraction Kc (CD42b positive), fraction Lc (CD61 positive), fraction Mc (CD62P positive), fraction Nc (CD69 positive) and It was found that all the cell populations contained in the sample derived from the fraction Pc (CD156c positive) were inferior in proliferation ability.
• Table 15 shows the number of cells in the cell fraction Qc to Tc obtained by sorting by FACS Aria on the 21st day of culture (ratio with the number of cells in the cell fraction Qc to Tc in the sample derived from the fraction Ac).
• fraction Ac derived from fraction Hc, fraction Ic, fraction Jc, fraction Kc, fraction Lc, fraction Mc, fraction Nc, fraction Oc, or fraction Pc. It is shown that a cell population containing CD3 positive CD45 positive CD7 positive CD8 positive CD4 positive cell or CD3 positive CD45 positive CD7 positive CD8a positive CD4 negative cell can be prepared compared to It was done.
• a cell population containing hematopoietic progenitor cells cells expressing any one or more of CD7, CD144, CD56, CD226, CD262, and CD325, or CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102
• more and / or higher concentration of immature T cells and mature T cells can be obtained, and the cells thus obtained can prevent diseases such as tumors, infectious diseases, autoimmune disorders, etc. Or it is useful for treatment.

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