Hematopoietic progenitor cell markers

Abstract

To provide a method for obtaining a larger number of cells of T-cell series and / or at higher concentration in order to stably provide cell therapy.SOLUTION: A method for producing a CD4 / CD8 double positive cell includes the following processes: a process 1 of separating a cell expressing one or more kinds of molecules selected from the group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200 and CD218a from a cell population including a hemopoietic precursor cell; and a process 2 of differentiating the cell separated in the process 1 into a CD4 / CD8 double positive cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing CD8 positive cells using one or more hematopoietic progenitor cell markers (HPC markers) and a cell population expressing one or more HPC markers.

[0002] BACKGROUND OF THEINVENTION 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. Among them, cytotoxic T cells (CTLs) recognize antigen peptides derived from viruses, tumors, etc., that are presented together with class 1 major histocompatibility antigens on antigen-presenting cells via T cell receptors (TCRs) present on the cell surface, and exert specific cytotoxic activity against cells presenting the antigen peptides, which are foreign substances.

[0003] Here, most CTLs are CD8 single positive (SP) cells. It is known that the CD8SP cells differentiate in the thymus from immature cells that do not express TCR and have neither CD4 nor CD8 (CD4 / CD8 double negative (herein, double negative is sometimes abbreviated as DN) cells), via cells that express CD8 and CD4 (CD4 / CD8 double positive (herein, double positive is sometimes abbreviated as DP) cells).

[0004] As described above, T cells play a central role in the immune system, and therefore, if T cells can be replenished or regenerated, it will be an extremely effective means for preventing or treating diseases such as tumors, infectious diseases, and autoimmune disorders. For this reason, attempts have been made to obtain hematopoietic progenitor cells and cells of the T cell lineage by a method of inducing hematopoietic progenitor cells (hereinafter sometimes referred to as HPCs) from iPS cells, and further by a method of inducing CD4 / CD8DP cells. For example, 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 isolating HPC from a cell population containing HPC induced from human pluripotent stem cells using the expression of CD43 and CD34, CD31, or CD144 as an index, a method for differentiating the isolated cells into T cell lineage cells, and the fact that the properties of the induced hematopoietic cells differ depending on whether or not Activin / Nodal is allowed to act at the initial stage of induction when differentiating pluripotent stem cells into HPC. Patent Document 2 describes a method for producing CD4 / CD8DP under feeder-free conditions. Non-Patent Document 3 describes various methods for inducing blood system cells including hematopoietic progenitor cells from human pluripotent stem cells, and cites low induction efficiency as a reason why the clinical use of blood system cells induced in vitro has not progressed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 075222 [Patent Document 2] International Publication No. 2017 / 221975 [Non-patent literature]

[0006] [Non-Patent Document 1] Choi KD et al., Stem Cells, 27 (2009);559-567 [Non-Patent Document 2] Kennedy M. et al., Cell Reports, 2 (2012);1722-1735 [Non-Patent Document 3] Liu S. et al., Cytotherapy, 17 (2015);344-358 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to obtain a larger amount and / or a higher concentration of T cell lineage cells (e.g., CD8 positive cells (e.g., CD4 / CD8DP cells, CD8SP cells)) in order to provide stable cell therapy (i.e., to provide a method for producing the cells). Another objective of the present invention is to provide a cell population containing hematopoietic progenitor cells in order to solve the above-mentioned problems. [Means for solving the problem]

[0008] In order to develop a method for producing CD4 / CD8DP cells that solves the above problems, the present inventors identified a marker (HPC marker) expressed on the surface of HPC. Furthermore, they found that HPCs separated from a cell population containing HPCs using one or more of the markers are highly efficient at differentiating into CD8 positive cells (e.g., CD4 / CD8DP cells) and have high proliferation capacity, that is, by using one or more HPC markers, it is possible to obtain more CD8 positive cells (e.g., CD4 / CD8DP cells) and / or obtain them at a higher concentration in a cell population. Furthermore, they found that the production method of the present invention can stably provide CD8 positive cells (e.g., CD4 / CD8DP cells) with little variation in yield, and thus completed the present invention.

[0009] That is, the present invention is as follows. [1] A method for producing CD4 / CD8 double positive cells, comprising the steps of: Step 1: separating cells expressing one or more molecules selected from a first group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262, and CD325, and / or cells not expressing one or more molecules selected from a second group consisting of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c from a cell population containing hematopoietic progenitor cells; Step 2: Differentiating the cells separated in step 1 into CD4 / CD8 double positive cells [2] The method according to [1], wherein the cells selected in step 1 do not express CD235a and CD14, and express CD45, CD34 and CD43. [3] The method according to [1] or [2], wherein step 1 is a step of separating cells expressing one or more molecules 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 from a cell population containing hematopoietic progenitor cells. [4] The method according to [3], wherein the first group shown in step 1 is the group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD200, CD218a, CD7 and CD144. [5] The method according to [1] or [2], wherein step 1 is a step of separating cells that do not express one or more molecules selected from the second group consisting of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102 and CD156c from a cell population containing hematopoietic progenitor cells. [6] The method according to [5], wherein the second group shown in step 1 is the group consisting of CD49f, CD51 and CD102. [7] The method according to any of [1] to [6], wherein the cell population containing hematopoietic progenitor cells in step 1 is a cell population obtained by inducing differentiation of pluripotent stem cells. [8] The method according to [7], wherein the pluripotent stem cells are human induced pluripotent stem cells. [9-1] A method for producing CD8 single-positive cells, comprising the step of inducing CD4 / CD8 double-positive cells obtained by any of the methods [1] to [8] into CD8 single-positive cells. [9-2] The method according to [9-1], wherein the inducing step comprises, for example, culturing CD4 / CD8 double positive cells in the presence of a corticosteroid, an antibody and / or a cytokine, and preferably, the corticosteroid is dexamethasone, and / or the antibody is an anti-CD3 antibody, and / or the cytokine is IL-2.

[10] The method according to [9-1] or [9-2], wherein the CD8 single positive cells are cytotoxic T cells.

[11] A cell population obtained by separating, from a cell population containing hematopoietic progenitor cells, cells that express one or more molecules 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, and / or cells that do not express one or more molecules selected from the second group consisting of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c.

[12] The cell population described in

[11] , wherein the separated cells do not express CD235a and CD14, and express CD45, CD34, and CD43.

[13] The cell population described in

[11] or

[12] , wherein the cell population containing hematopoietic progenitor cells is a cell population obtained by inducing differentiation of pluripotent stem cells.

[14] The cell population described in

[13] , wherein the pluripotent stem cells are human induced pluripotent stem cells.

[15] A cell population in which the proportion of hematopoietic progenitor cells expressing one or more molecules 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, and / or hematopoietic progenitor cells not expressing one or more molecules selected from the second group consisting of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c (the number of hematopoietic progenitor cells / the total number of cells contained in the cell population) is 40% or more.

[16] The cell population described in

[15] , wherein the percentage is a percentage of hematopoietic progenitor cells that further express one or more molecules selected from the first group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD200, CD218a, CD7, and CD144.

[17] The cell population described in

[15] , wherein the percentage is the percentage of hematopoietic progenitor cells that do not further express one or more molecules selected from the second group consisting of CD49f, CD51, and CD102.

[18] A reagent for separating hematopoietic progenitor cells, comprising an antibody against one or more molecules selected from the group consisting of 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.

[19] The reagent described in

[18] , further comprising one or more antibodies against CD235a, CD14, CD45, CD34 and / or CD43.

[20] A method for detecting a surface protein of a hematopoietic progenitor cell, the method comprising the step of contacting the hematopoietic progenitor cell with a substance that specifically binds to 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.

[21] The method according to

[20] , wherein the substance comprises an antibody or a fragment thereof.

[22] The method according to

[20] or

[21] , wherein the substance further comprises a fluorophore.

[23] The method according to any of

[20] to

[22] , further comprising the step of contacting the hematopoietic progenitor cells with a substance that specifically binds to CD34, CD43, CD14, or CD235a.

[24] A method for detecting a plurality of proteins on the surface of a hematopoietic progenitor cell, the method comprising the step of contacting the hematopoietic progenitor cell with a substance that specifically binds to each of the plurality of proteins, the plurality of proteins being selected from the group consisting of CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD5 6, any combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, or twenty-eight of the following: CD226, CD262, CD325, CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, CD109, and CD156c.

[25] A method for selecting hematopoietic progenitor cells from a cell population containing hematopoietic progenitor cells, comprising the steps of: detecting the presence or absence of one or more proteins on the surface of the cells by any of the methods of

[20] to

[24] ; and selecting the cells based on the presence or absence of the one or more proteins, the method comprising: (i) detecting the presence or absence of any of the following proteins: CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD2 and / or (ii) selecting the cell if any one or any combination of the following proteins: CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, CD109, and CD156c are not detected on the surface of the cell.

[26] The method according to

[25] , wherein the cell is selected if any one or any combination of the following proteins are detected on the surface of the cell: CD24, CD62L, CD90, CD143, CD263, Notch3, CD200, CD218a, CD7 and CD144.

[27] The method according to

[25] or

[26] , wherein the cells are selected if any one or any combination of the following proteins: CD49f, CD51 and CD102 are not detected on the surface of the cells.

[28] A method for selecting hematopoietic progenitor cells from a cell population containing hematopoietic progenitor cells, comprising the steps of: detecting the presence or absence of one or more proteins on the surface of the cells by any of the methods of

[20] to

[24] ; and, based on the presence or absence of the one or more proteins, (i) detecting CD45, CD34 and / or CD43 and the following proteins: CD24, CD62L, C90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD2 and / or (ii) selecting the cells if (i) any one or any combination of: CD14, CD18a, CD7, CD144, CD56, CD226, CD262 and CD325 is detected on the surface of the cells, and / or (ii) CD14 and / or CD235a is not detected on the surface of the cells, and the following proteins: CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, CD109 and CD156c are not detected. Effect of the Invention

[0010] The production method of the present invention using one HPC marker can provide more CD8 positive cells (e.g., CD4 / CD8DP cells, CD8SP cells) and / or produce CD8 positive cells at a higher concentration. Furthermore, the production method of the present invention can stably provide CD8 positive cells (e.g., CD4 / CD8DP cells, CD8SP cells) with less variability in yield. Thus, the production method of the present invention provides an improved method for isolating hematopoietic progenitor cells from a cell population. This provides an improved cell source for producing CD8 positive cells. This novel production method is based on the surprising and unexpected finding by the inventors that certain HPC positive markers and HPC negative markers can be used to greatly improve the quality and / or quantity of the CD8 positive cell population derived from HPC. This valuable source of CD8 positive cells of the present invention provides an improved CD8 positive cell population for clinical applications (e.g., cell therapy, diagnostic purposes, and in vitro applications).

[0011] Detailed Description of the Invention The present invention provides a method for producing CD8 positive cells (e.g., CD4 / CD8DP cells) (hereinafter abbreviated as the "production method of the present invention") comprising: (Step 1) a step of separating cells expressing one or more types of hematopoietic progenitor cell markers (sometimes abbreviated as "HPC markers" herein) and / or cells not expressing one or more types of HPC markers from a cell population containing hematopoietic progenitor cells; and (Step 2) a step of differentiating the cells separated in Step 1 into CD8 positive cells (e.g., CD4 / CD8 double positive cells (sometimes abbreviated as "CD4 / CD8DP cells" herein).

[0012] In the present invention, "CD8 positive cells" refer to T cells expressing CD8, and examples of such cells include CD4 / CD8DP cells and CD8 single positive cells (sometimes abbreviated as "CD8SP cells" in the present specification). In the present invention, "CD4 / CD8DP cells" refer to T cells expressing both CD4 and CD8, and "CD8SP cells" refer to T cells expressing CD8 but not CD4. Examples of CD8SP cells include cytotoxic T cells and their precursor cells. In addition, in the present invention, "T cells" refer to, for example, cells expressing an antigen receptor called a T cell receptor (T cell receptor, TCR) on the surface, and their precursor cells (e.g., pro-T cells not expressing TCR (TCRαβ), and pre-T cells in which TCRβ and pre-TCRα are associated).

[0013] In the present invention, the term "hematopoietic progenitor cells" refers to CD34-positive cells, preferably CD34 / CD43DP cells. The origin of the hematopoietic progenitor cells used in the present invention is not limited, and may be, for example, hematopoietic progenitor cells induced in vitro by known techniques (e.g., techniques described in Patent Documents 1 and 2, and Non-Patent Documents 1 to 3) (e.g., hematopoietic progenitor cells obtained by inducing differentiation of pluripotent stem cells), or hematopoietic progenitor cells isolated from living tissues by known techniques.

[0014] Examples of the pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), and embryonic germ stem cells (EG cells), and are preferably iPS cells (more preferably human iPS cells). When the pluripotent stem cells are ES cells or any cells derived from a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, and are preferably cells produced without destroying an embryo.

[0015] The iPS cells are artificial stem cells derived from somatic cells that have almost the same characteristics as ES cells, such as pluripotency and the ability to proliferate by self-replication, and can be produced by introducing a specific reprogramming factor into somatic cells in the form of DNA or protein (e.g., Takahashi K. and Yamanaka S. (2006) Cell, 126; 663-676: Takahashi K. et al. (2007) Cell, 131; 861-872: Yu J. et al. (2007) Science, 318; 1917-1920: Nakagawa M. et al. (2008) Nat. Biotechnol. 26; 101-106). When using iPS cells, the iPS cells may be produced from somatic cells by a method known per se, or iPS cells that have already been established and stocked may be used. There is no limitation on the somatic cells from which the iPS cells used in the present invention are derived, but they are preferably cells derived from peripheral blood or umbilical cord blood. There are no limitations on the animals from which pluripotent stem cells can be derived, and examples include mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans, with humans being preferred.

[0016] Hematopoietic progenitor cells can be produced by subjecting the pluripotent stem cells to a method known per se. When the pluripotent stem cells are iPS cells, the hematopoietic progenitor cells can be produced by, for example, the method described in International Publication No. 2017 / 221975.

[0017] 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. Among these, peripheral blood and umbilical cord blood are preferred from the viewpoints of low invasiveness to animals and ease of preparation.

[0018] From the viewpoint of therapeutic application, the hematopoietic progenitor cells and pluripotent stem cells used in the present invention are preferably cells that comply with GMP (Good Manufacturing Practice) standards.

[0019] In the present invention, the term "HPC marker" refers to any one of the following molecules that can be expressed on the surface of hematopoietic progenitor cells: CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262, and CD325 (these markers may be referred to as "HPC positive markers," "positive HPC selection markers," or "positive HPC markers" in the specification). or any of the molecules CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c (herein, these markers may be referred to as "HPC negative markers", "negative HPC selection markers", or "negative HPC markers"), which are molecules not normally expressed on the surface of hematopoietic progenitor cells (herein, HPC positive markers and HPC negative markers may be collectively referred to as "HPC markers of the present invention"). Among the HPC positive markers of the present invention, CD24, CD62L, CD90, CD143, CD263, Notch3, CD200, CD218a, CD200, CD7, or CD144 is preferred, and CD90, CD143, CD218a, or CD200 is more preferred. In another embodiment, 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 more preferred. In the present invention, cells expressing the HPC positive marker may be referred to as HPC marker positive cells (or HPC marker positive cells), and for example, cells expressing the CD24 molecule may be referred to as CD24 positive cells. Among the HPC negative markers of the present invention, CD49f, CD51 or CD102 are preferred. In the present invention, cells not expressing the HPC negative marker may be referred to as HPC marker negative cells (or HPC marker negative cells).

[0020] In a typical embodiment, CD8 positive cells, preferably CD4 / CD8 double positive cells, are produced by isolating from a cell population comprising hematopoietic progenitor cells, cells that (a) express CD34 and CD43, (b) express any of CD62L, CD24, CD90, CD143, CD218a, CD263 or Notch3, and (c) do not express CD235a and CD14.

[0021] In an exemplary embodiment, CD8 positive cells, preferably CD4 / CD8 double positive cells, are produced by isolating from a cell population comprising hematopoietic progenitor cells, cells that (a) express CD34 and CD43, (b) express any of CD90, CD143, CD200, CD218a or CD263, and (c) do not express CD235a or CD14.

[0022] In an exemplary embodiment, CD8 positive cells, preferably CD4 / CD8 double positive cells, are produced by isolating from a cell population comprising hematopoietic progenitor cells, cells that (a) express CD34 and CD43, (b) express any of CD7, CD144, CD56, CD226, CD262 or CD325, and (c) do not express CD235a and CD14.

[0023] In an exemplary embodiment, CD8 positive cells, preferably CD4 / CD8 double positive cells, are produced by isolating from a cell population comprising hematopoietic progenitor cells, cells that (a) express CD34 and CD43, (b) do not express CD49f, CD51, CD426, CD61, CD62P, CD69 or CD156c, and (c) do not express CD235a and CD14.

[0024] In the present invention, "expressed" means that the expression can be detected by the method used to separate the cells, and "not expressed" means that the expression cannot be detected with the detection sensitivity of the method used to separate the cells. Methods used to separate the cells include the methods using flow cytometry or mass cytometry described below, and magnetic cell separation methods.

[0025] The cells separated by the step 1 include cells expressing one or more HPC positive markers and cells not expressing one or more HPC negative markers. In one embodiment of the present invention, the cells separated by the step 1 are cells expressing CD34 and / or CD43 in addition to one or more HPC positive markers of the present invention, and / or cells not expressing one or more HPC negative markers of the present invention and expressing CD34 and / or CD43. Furthermore, the cells separated are preferably CD235a negative, CD14 negative, and / or CD45 positive. Thus, in a preferred embodiment of the present invention, the step 1 is carried out by separating cells that are CD235a negative, CD14 negative, CD45 positive, CD34 positive, and CD43 positive, and positive for one or more HPC positive markers of the present invention and / or negative for one or more HPC negative markers of the present invention from a cell population containing hematopoietic progenitor cells.

[0026] In the present invention, "separating cells" includes aspects of selecting cells that express one or more HPC positive markers and / or do not express one or more HPC negative markers and isolating the selected cells, and aspects of subjecting the selected cells to the next differentiation step.

[0027] As used herein, terms such as "comprising" or "comprises" may optionally be replaced with "consisting of" or "consists of".

[0028] Methods 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 include methods using flow cytometry or mass cytometry, magnetic cell separation, etc., and these methods can be performed using known methods. For example, cells expressing one or more HPC positive markers and / or cells not expressing one or more HPC negative markers can be separated by a method including a step of contacting the cells with a substance (e.g., an antibody) that specifically binds to each HPC marker. The above-mentioned substances include those that have a detectable label (e.g., GFP) attached thereto, and those that have no label attached thereto. When the above-mentioned substances have no label attached thereto, the separation can be achieved by further using a substance that has a detectable label attached thereto that directly or indirectly recognizes the substance. For example, when the substance is an antibody, a fluorescent dye, a metal isotope, or beads (e.g., magnetic beads) can be directly or indirectly attached to the antibody, thereby labeling the HPC marker on the cell surface, and the cells can be separated based on the label. The antibody used in this case may be one type only, or two or more types of antibodies.

[0029] Methods for differentiating cells separated from a cell population containing hematopoietic progenitor cells as cells that express one or more HPC positive markers of the present invention and / or do not express an HPC negative marker into CD8 positive cells include, for example, known methods (e.g., the methods described in WO 2016 / 076415, Journal of Leukocyte Biology 96(2016)1165-1175, Cell Reports 2(2012)1722-1735, and WO 2017 / 221975).

[0030] 1. Method for producing CD4 / CD8DP cells Examples of the basal medium for cell culture used in step 2 of the method of the present invention include Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixed media thereof. The medium may or may not contain serum. If necessary, the medium may contain one or more substances such as vitamin C (e.g., ascorbic acid, vitamin C phosphate), albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiol glycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines.

[0031] The medium preferably contains a cytokine. Examples of cytokines include IL-7, FLT-3L, SCF, TPO, and combinations thereof, with FLT-3L and IL-7 being preferred. The concentration of IL-7 in the medium is preferably 1 ng / ml to 50 ng / ml (e.g., 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml), with 5 ng / ml being particularly preferred. The concentration of FLT-3L in the medium is preferably 1 ng / ml to 100 ng / ml (e.g., 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml), and more preferably 10 ng / ml. The concentrations of other cytokines can also be appropriately determined by those skilled in the art based on the culture conditions, etc. In addition, it is preferable that the medium contains a Notch ligand (e.g., Dll1, Dll4, fusion protein of Dll1 or Dll4 and Fc). When a Notch ligand is used, it may be added to the medium or may be coated on the culture vessel. In addition, feeder cells expressing a Notch ligand may be used.

[0032] In the method of the present invention, the hematopoietic progenitor cells obtained in step 1 may be cultured in an adherent or suspended state. In the case of adherent culture, the culture vessel may be coated and used, or may be co-cultured with feeder cells or the like. From the viewpoint of application to treatment, it is preferable to be feeder-free. Examples of the substrate for coating the culture vessel include fibronectin fragments (e.g., retronectin), pronectin, vitronectin, matrigel (BD), type I collagen, type IV collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Examples of the feeder cells to be co-cultured include bone marrow stromal cell line OP9 cells, mouse mesenchymal cell line 10T1 / 2 cells, and Tst-4 cells. When feeder cells are used, it is preferable to appropriately replace the feeder cells and perform the culture. The feeder cells can be replaced by transferring the target cells being cultured onto the feeder cells that have been seeded in advance. The replacement is preferably performed every 2 to 5 days.

[0033] In the present invention, the culture temperature conditions when hematopoietic progenitor cells are cultured to induce CD4 / CD8 DP cells are not particularly limited, but are preferably about 37°C to 42°C, and more preferably about 37°C to 39°C. In addition, a person skilled in the art can appropriately determine the culture period while monitoring the number of CD4 / CD8 DP cells. As long as CD4 / CD8 DP cells can be obtained, the number of culture days for inducing CD4 / CD8 DP cells from hematopoietic progenitor cells is not particularly limited, but is preferably 10 days or more (e.g., 12 days, 14 days, 16 days, 18 days, 20 days or more), and is preferably 60 days or less. As shown in the examples described later, in a specific embodiment, CD4 / CD8 DP cells can be efficiently obtained in large quantities by culture for 21 to 28 days.

[0034] In the present invention, the obtained CD4 / CD8DP cells may be isolated and used, or may be used as a cell population containing other cell types (e.g., CD8SP cells). When isolating, CD4, CD8, CD3, CD45 molecules, etc. can be used as an indicator. The isolation method can be a method well known to those skilled in the art, and examples of the method include labeling with an antibody against the indicator molecule and purifying using the above-mentioned flow cytometry or mass cytometry, magnetic cell separation, or an affinity column on which a desired antigen is immobilized, etc.

[0035] 2. Method for producing CD8 single positive cells CD8SP cells can be produced by subjecting the CD4 / CD8DP cells obtained by the method of the present invention to a step of inducing differentiation into CD8SP cells (such a method of producing CD8SP cells may be abbreviated as "the method of producing CD8SP cells of the present invention"). The method of producing CD4 / CD8DP cells is as described in 1.

[0036] The basal medium and medium used in the method for producing CD8SP cells of the present invention include the same basal medium and medium as described in 1.

[0037] The medium may contain a corticosteroid. Examples of the corticosteroid include glucocorticoids and their derivatives. Examples of the glucocorticoid include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. Among them, dexamethasone is preferred. Thus, in one embodiment, the method for producing CD8 single positive cells includes a step of inducing the CD4 / CD8 double positive cells into CD8 single positive cells by culturing the CD4 / CD8 double positive cells in the presence of a corticosteroid, preferably dexamethasone.

[0038] When dexamethasone is used as the adrenal cortical hormone, its concentration in the culture medium is preferably 1 nM to 100 nM (e.g., 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM), and of these, 10 nM is preferable.

[0039] 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.

[0040] The anti-CD3 antibody used in the present invention is not particularly limited as long as it specifically recognizes CD3, and examples thereof include antibodies produced from OKT3 clones. The anti-CD3 antibody may be bound to magnetic beads or the like, and instead of adding the anti-CD3 antibody to the medium, the T lymphocytes may be stimulated by culturing the T lymphocytes for a certain period of time on a culture vessel having an anti-CD3 antibody bound to its surface. The concentration of the anti-CD3 antibody in the medium is preferably 10 ng / ml to 1000 ng / ml (e.g., 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), and 500 ng / ml is particularly preferred. The concentrations of other antibodies can also be appropriately determined by those skilled in the art based on the culture conditions, etc. Thus, in one embodiment, a method for producing CD8 single positive cells comprises a step of inducing CD4 / CD8 double positive cells into CD8 single positive cells by culturing the CD4 / CD8 double positive cells in the presence of an antibody, preferably an anti-CD3 antibody.

[0041] The concentration of IL-2 used in the present invention in the culture medium is preferably 10 U / ml to 1000 U / ml (e.g., 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), and of these, 100 U / ml is preferred. The concentration of IL-7 or IL15 in the medium used in the present invention is preferably 1 ng / ml to 100 ng / ml (e.g., 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml), and more preferably 10 ng / ml. Thus, in one embodiment, the method for producing CD8 single positive cells comprises the step of inducing CD4 / CD8 double positive cells into CD8 single positive cells by culturing the CD4 / CD8 double positive cells in the presence of a cytokine, preferably IL-2.

[0042] In the method for producing CD8SP cells of the present invention, the temperature conditions for culturing CD4 / CD8DP cells are not particularly limited, but are preferably about 37° C. to 42° C., and more preferably about 37° C. to 39° C. In addition, those skilled in the art can appropriately determine the culture period while monitoring the number of CD8-positive T cells, etc. The number of days is not particularly limited as long as CD8SP cells can be obtained, but is preferably 1 day or more, 3 days or more, or 7 days or more, and is preferably 60 days or less.

[0043] The CD8SP cells produced by the method of the present invention are not limited to cells obtained by differentiation from CD4 / CD8DP cells (e.g., cells obtained by the method 2 above). Thus, for example, CD8SP cells induced from cells separated from a cell population containing hematopoietic progenitor cells as cells that express one or more HPC positive markers and / or do not express one or more HPC negative markers of the present invention, without passing through CD4 / CD8DP cells, are also included in the CD8SP cells produced by the method of the present invention.

[0044] 3. A cell population containing hematopoietic progenitor cells expressing one or more HPC markers The present invention provides a cell population containing hematopoietic progenitor cells at a high frequency that express one or more HPC positive markers and / or do not express one or more HPC negative markers (hereinafter abbreviated as "cell population of the present invention"). The cell population can be obtained, for example, by separating cells that express one or more HPC positive markers and / or do not express one or more HPC negative markers from a cell population containing hematopoietic progenitor cells using the separation method described above. The definition of HPC markers, specific molecules, and the definition of hematopoietic progenitor cells are as described above.

[0045] The cell population of the present invention is not particularly limited as long as the percentage 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 number of cells contained in the cell population) is higher than the percentage contained in a hematopoietic progenitor cell population obtained by a conventional method (e.g., a method of separating using CD34 positivity, CD34 positivity and CD43 positivity, or CD43 positivity and CD34, CD31 or CD144 positivity as an indicator), but is preferably 40% or more (e.g., 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 expressing one or more HPC positive markers and / or not expressing one or more HPC negative markers can be measured by flow cytometry.

[0046] As shown in the Examples below, the cell population of the present invention has higher differentiation efficiency into CD4 / CD8DP cells and CD8SP cells and superior proliferation ability compared to a cell population in which cells that express one or more HPC positive markers and / or do not express one or more HPC negative markers have not been separated.

[0047] 4. Reagents for separating the "cell population of the present invention" The present invention also provides a reagent for separating the cell population of the present invention (hereinafter abbreviated as "the reagent of the present invention") comprising an antibody against each of one or more markers selected from the group consisting of the HPC markers 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, CD49f, CD51 and CD102. When 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 beads (e.g., magnetic beads), depending on the separation means used in step 1 of the production method of the present invention.

[0048] Furthermore, the reagent of the present invention may further contain an anti-CD4 antibody and / or an anti-CD8 antibody, and, if necessary, one or more antibodies against other cell surface markers (e.g., CD3, CD45, CD235a, CD14, CD45, CD34, CD43) known to be expressed or not expressed in CD4 / CD8DP cells and / or CD8SP cells, and can be used as a reagent for separating CD4 / CD8DP cells or CD8SP cells that have been induced to differentiate from the cell population of the present invention. In addition, the reagent of the present invention may further contain reagents (e.g., basal medium, medium additives, etc.) for inducing differentiation of hematopoietic progenitor cells into CD4 / CD8DP cells and further into CD8SP cells, and examples of such reagents include the substances exemplified in 1. and 2. above.

[0049] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and the present invention is not limited to these. EXAMPLES

[0050] Example 1 (Study using TKT3V1-7 strain) A suspension cell population containing hematopoietic progenitor cells (hereinafter sometimes referred to as HPCs) was used, which was prepared by differentiating iPS cells (TKT3V1-7 line) according to known methods (e.g., the methods described in Cell Reports 2 (2012) 1722-1735 and International Publication No. 2017 / 221975). Specifically, 3 x 10 TKT3V1-7 line was cultured in an ultra-low adhesion treated 6-well plate. 5 The cells were seeded at 10 cells / well (Day 0), and cultured in EB medium (StemPro34 supplemented with 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 2 mM L-glutamine, 45 mM α-monothioglycerol, and 50 μg / ml ascorbic acid) with 10 ng / ml BMP4, 5 ng / ml bFGF, 15 ng / ml VEGF, and 2 μM SB431542 under hypoxic conditions (5% O2) for 5 days (Day 5). Next, 50 ng / ml SCF, 30 ng / ml TPO, and 10 ng / ml Flt3L were added, and the cells were cultured for another 5 to 9 days (~Day 14) to obtain a floating cell population. Note that the medium was changed every 2 or 3 days during the culture period. The above floating cell population including HPCs was stained with the following antibody sets for each sample group.

[0051] [Table 1]

[0052] The stained cell populations were sorted using FACSAria. The cell fractions obtained from each sample group are shown below.

[0053] [Table 2]

[0054] In the above table, fractions A to G respectively represent 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 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 N: CD235a negative, CD14 negative, CD34 positive, CD43 positive, Notch3 positive Fraction O: CD235a negative, CD14 negative, CD34 positive, CD43 positive, Notch3 negative

[0055] The cells contained in the above fractions A to O were differentiated into lymphocyte cells according to known methods (for example, the methods described in Journal of Leukocyte Biology 96 (2016) 1165-1175 and International Publication No. 2017 / 221975). Specifically, each cell population of fractions A to O was seeded at 2000 cells / well on a 48-well plate coated with Recombinat h-DLL4 / Fc chimera (Sino Biological) and Retronectin (Takara Bio), and cultured under conditions of 5% CO2 and 37°C. During the culture period, the medium was changed every 2 or 3 days. The medium used was OP9 medium (containing 15% FBS, 2 mM L-glutamine, 100 Uml penicillin, 100 ng / ml streptomycin, 55 μM 2-mercaptoethanol, 50 μg / ml ascorbic acid, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, and 5 ng / ml sodium selenite) supplemented with 50 ng / ml SCF, 50 ng / ml IL-7, 50 ng / ml Flt3L, 100 ng / ml TPO, 15 μM SB203580, and 30 ng / ml SDF-1α. On days 7 and 14 after the start of culture, the cells were subcultured onto similarly coated 48-well plates. On day 21 after the start of culture, all the cells were collected, the number of cells was counted using a hemocytometer, and stained with the following antibody set.

[0056] [Table 3]

[0057] The stained cell population was subjected to sorting using FACSAria. The cell fractions obtained by the above sorting are shown below. In this specification, the lymphocyte fraction refers to a fraction separated using FSC (forward scatter) and SSC (side scatter) as indicators, similar to the method described in Cytometry (Communications in Clinical Cytometry) 18 (1994) 199-208. 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

[0058] Table 4 shows the cell counts in each sample derived from fractions A to O on day 21 of culture (proportions relative to the cell count in the sample derived from fraction A).

[0059] [Table 4]

[0060] As shown in Table 4, compared to the cell population contained in the sample derived from fraction A, the cell populations contained in the samples derived from fraction B (CD62L positive), fraction D (CD24 positive), fraction F (CD90 positive), fraction H (CD143 positive), fraction J (CD218a positive), fraction L (CD263 positive) or fraction N (Notch3 positive) all had superior proliferation ability.

[0061] Table 5 shows the cell numbers of cell fractions P to S obtained by sorting with FACS Aria on the 21st day of culture (the ratio to the cell numbers of cell fractions P to S in the sample derived from fraction A).

[0062] [Table 5]

[0063] As a result, it was shown that by using cells derived from fraction F, it is possible to prepare a cell population containing (a) CD3-positive, CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-positive cells, or (b) CD3-positive, CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-negative cells at a higher percentage than when cells derived from fraction G or fraction A are used. Similarly, it was shown that by using cells derived from fraction H, it is possible to prepare a cell population containing (a) CD3-positive, CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-positive cells, or (b) CD3-positive, CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-negative cells at a higher percentage than when cells derived from fraction I or fraction A are used. It was also shown that by using cells derived from fraction J, it was possible to prepare a cell population containing CD3-positive, CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-positive cells at a higher rate than when cells derived from fraction K or fraction A were used. That is, it was shown that a cell population containing hematopoietic progenitor cells and containing CD4 / CD8 double-positive cells (or CD8-positive cells) at a higher rate could be produced by isolating cells expressing CD90, CD143, or CD218a as a cell population containing hematopoietic progenitor cells and differentiating the isolated cells.

[0064] Example 2 (Study using Ff-I01s04 strain) As a cell population containing hematopoietic progenitor cells (hereinafter sometimes referred to as HPCs), a floating cell population obtained by subjecting iPS cells (Ff-I01s04 line) to the method described in Example 1 was used. Cell populations including HPCs were stained for each sample group using antibody sets 3, 4, 5, and 6 shown in Example 1 (referred to as 3b, 4b, 5b, and 6b, respectively, in this example), or antibody set 8b shown below.

[0065] [Table 6]

[0066] The stained cell populations were sorted using FACSAria. The cell fractions obtained from each sample group are shown below.

[0067] [Table 7]

[0068] In the above table, fractions f~m, t and u respectively represent 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 positive, CD200 positive Fraction u: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD200 negative

[0069] The cells contained in the above fractions f to m, t, and u were differentiated into lymphoid cells by the method described in Example 1. On day 28 after the start of culture, all the cells were collected and stained with the following antibody set.

[0070] [Table 8]

[0071] The stained cell population was subjected to sorting using a FACSAria. The cell fractions obtained by the above sorting are shown below. Fraction p: Lymphocyte fraction Fraction Q: Lymphocyte fraction, CD45 positive, CD5 positive, CD7 positive Fraction: Lymphocyte fraction, CD45 positive, CD7 positive, CD8a positive, CD4 positive Fractions: Lymphocyte fraction, CD45 positive, CD7 positive, CD8a positive, CD4 negative

[0072] Table 9 shows the cell numbers on day 28 of culture in each sample derived from fractions f to m, t, and u (ratio to the cell numbers at the start of culture).

[0073] [Table 9]

[0074] From Table 9, it was found that fraction f has superior proliferation ability to fraction g, fraction h to fraction i, fraction j to fraction k, fraction l to fraction m, and fraction t to fraction u. In other words, it was found that the cell populations contained in the samples derived from CD90-positive, CD143-positive, CD218a-positive, CD263-positive, or CD200-positive all have superior proliferation ability.

[0075] Table 10 shows the cell numbers of cell fractions p to s obtained by sorting using FACS Aria on day 28 of culture when the cells contained in fractions f to m, t, and u were seeded at 1,000 cells / well.

[0076] [Table 10]

[0077] The FACS analysis on day 28 of culture shown in Table 10 demonstrated that, when cells derived from each marker-positive cell were used, it was possible to prepare (a) CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-positive cells, or (b) CD45-positive, CD5-positive, CD7-positive, CD8a-positive, and CD4-negative cells in higher yields than when cells derived from each marker-negative cell were used. These results indicate that by isolating cells expressing CD90, CD143, CD200, CD218a or CD263 as a cell population containing hematopoietic progenitor cells and differentiating the isolated cells, it is possible to produce a cell population containing a high percentage of CD8 positive cells (e.g., CD4 / CD8DP cells, CD8SP cells).

[0078] Example 3 (Study using Ff-I01s04 strain) As a cell population containing hematopoietic progenitor cells (hereinafter sometimes referred to as HPCs), a floating cell population obtained by subjecting iPS cells (Ff-I01s04 line) to the method described in Example 1 was used. The floating cell population containing HPCs was stained with the following antibody sets for each sample group.

[0079] [Table 11-1]

[0080] [Table 11-2]

[0081] The stained cell populations were sorted using FACSAria. The cell fractions obtained from each sample group are shown below.

[0082] [Table 12]

[0083] In the above table, fractions Ac to Pc respectively 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 Fractionated Fc: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD262 positive Fractionated 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, CD34 positive, CD43 positive, CD102 positive Fraction Kc: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD42b positive Fraction Lc: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD61 positive Fraction Mc: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD62P positive Nc fraction: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD69 positive Fraction Oc: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD102 positive Fraction PC: CD235a negative, CD14 negative, CD34 positive, CD43 positive, CD156c positive

[0084] The cells contained in the above fractions Ac to Pc were differentiated into lymphoid cells by the method described in Example 1. On the 21st day after the start of culture, all the cells were collected and the cell numbers were counted using a hemocytometer, and then stained with the following antibody set.

[0085] [Table 13]

[0086] The stained cell population was subjected to sorting using a FACSAria. The cell fractions obtained by the above sorting are 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, CD4 positive Differential Tc: Lymphocyte fraction, CD3 positive, CD45 positive, CD7 positive, CD8a positive, CD4 negative

[0087] Table 14 shows the cell counts in each sample derived from fractions Ac to Pc on day 21 of culture (proportions to the cell counts in the sample derived from fraction Ac).

[0088] [Table 14]

[0089] From Table 14, it was found that the cell populations contained in the samples derived from fraction Bc (CD7 positive), fraction Cc (CD144 positive), fraction Dc (CD56 positive), fraction Ec (CD226 positive), fraction Fc (CD262 positive) and fraction Gc (CD325 positive) all have superior proliferation ability compared to the cell populations contained in the samples derived from fraction Ac. It was also found that the cell populations contained in the samples derived from 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 fraction Pc (CD156c positive) all have inferior proliferation ability.

[0090] Table 15 shows the cell numbers of cell fractions Qc to Tc obtained by sorting with FACS Aria on day 21 of culture (the ratio to the cell numbers of cell fractions Qc to Tc in the sample derived from fraction Ac).

[0091] [Table 15]

[0092] As a result, it was shown that by using cells derived from fraction Bc, fraction Cc, fraction Dc, fraction Ec, fraction Fc, or fraction Gc, it is possible to prepare a cell population containing a higher percentage of CD3+CD45+CD7+CD8a+CD4+ cells or CD3+CD45+CD7+CD8a+CD4+ cells than when cells derived from fraction Ac were used. It was also shown that by using cells derived from fraction Hc, fraction Ic, fraction Jc, fraction Kc, fraction Lc, fraction Mc, fraction Nc, fraction Oc, or fraction Pc, it is possible to prepare a cell population containing a lower percentage of CD3+CD45+CD7+CD8a+CD4+ cells or CD3+CD45+CD7+CD8a+CD4+ cells than when cells derived from fraction Ac were used. In other words, it was shown that a cell population containing hematopoietic progenitor cells can be produced by isolating cells expressing one or more of CD7, CD144, CD56, CD226, CD262, and CD325, or cells not expressing one or more of CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c, and differentiating the isolated cells, thereby producing a cell population containing a high percentage of CD4 / CD8 double positive cells (or CD8 single positive cells).

[0093] This application is based on patent application No. 2017-087723 filed in Japan (filing date: April 26, 2017), the contents of which are incorporated in their entirety into this specification by reference herein. [Industrial Applicability]

[0094] According to the present invention, immature T cells and mature T cells can be obtained in larger quantities and / or at higher concentrations, and the cells thus obtained are useful for preventing or treating diseases such as tumors, infectious diseases, and autoimmune disorders.

Claims

1. A cell population obtained by separating cells from a cell population containing hematopoietic progenitor cells, which express CD45, CD34 and CD43, and one or more molecules selected from the group consisting of CD62L, CD143, CD263, Notch3, CD200, CD218a, CD226, CD262 and CD325, but do not express CD235a or CD14.

2. The cell population described in claim 1, wherein the cell population containing the hematopoietic progenitor cells is a cell population obtained by inducing differentiation of pluripotent stem cells.

3. The cell population described in claim 2, wherein the pluripotent stem cells are human induced pluripotent stem cells.

4. A cell population which expresses CD45, CD34 and CD43, and also expresses one or more molecules selected from the group consisting of CD62L, CD143, CD263, Notch3, CD200, CD218a, CD226, CD262 and CD325, and in which the proportion of hematopoietic progenitor cells which do not express CD235a and CD14 (the number of hematopoietic progenitor cells / total number of cells contained in the cell population) is 40% or more.

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