Method for producing T progenitor cells

Abstract

The present invention provides: a production method for a T cell progenitor that includes a step in which CD34+ cells are cultivated in a culture medium that includes an aromatic hydrocarbon receptor antagonist (1); a culture medium for differentiation of T cell progenitors; and a T cell progenitor inducer including an aromatic hydrocarbon receptor antagonist.

Description

[Technical Field]

[0001] The present invention relates to a method for producing T progenitor cells, a culture medium for T progenitor cell differentiation, and a T progenitor cell inducer.

[0002] (Background of the invention) Hematopoietic stem cell transplantation (HSCT) is the current standard treatment for many malignant and non-malignant hematopoietic diseases. Before the infusion (transplantation) of hematopoietic stem cells (HSCs), which are donor grafts, patients must undergo a pretreatment regimen that includes chemotherapy or chemoradiotherapy. This pretreatment causes severe damage to the thymic microenvironment, resulting in peripheral CD4 + and CD8 + This delays T cell rearrangement and makes the patient vulnerable to opportunistic infections. The thymic epithelial cells are the most vulnerable component of the thymic microenvironment to damage from chemoradiotherapy, and such damage can lead to long-term impairment of thymic formation after transplantation.

[0003] The above pretreatment can cause problems in thymocyte development. However, it is known that in vitro injection therapy of T cell progenitor (proT) cells derived from hematopoietic stem cell transplantation promotes the recovery of the thymus damaged by radiation. Therefore, in recent years, research and development of cell therapy using T cell progenitor cells (proT therapy) has been progressing with the aim of recovering T cells due to thymic damage after radiation therapy.

[0004] Non-patent document 1 describes how a feeder-free system based on immobilized Delta-like 4 (DLL4) can be used for T cell differentiation, and how this method was used to differentiate and expand T progenitor cells in an allogeneic mouse model of hematopoietic stem cell transplantation, thereby increasing thymic lymphocyte neogenesis.

[0005] Non-patent document 2 contains CD34 +Umbilical cord blood cells are cultured in plates coated with Delta-like-4 (DL-4) in a medium containing IL-7, Flt3-ligand, stem cell factor (SCF), and thrombopoietin, followed by FACS to obtain CD34 - / CD7 - It is described that T progenitor cells were obtained by removing myeloid cells.

[0006] Non-Patent Document 3 describes that CD34 + / CD43 - / CD184 - / CD73 - haemogenic endothelium is cultured on OP-DLL4 stromal cells in a medium containing TPO, Flt-3L, BMP4, IL11, SCF and IL7, whereby the haematopoietic multipotent progenitor CD45 + / RUNX1C hi / MYB hi / TAL1 hi / GATA2 hi is described to be obtainable.

[0007] Non-Patent Document 4 describes that after expanding and culturing CD34 + Hematopoietic Stem / Progenitor Cells (HSPC), T progenitor cells were obtained by co-culturing with OP-DL1 cells in a medium containing IL-7, Flt-3 and SCF.

[0008] Non-Patent Document 5 describes that StemRegenin 1 (SR1), which is an aryl hydrocarbon receptor (AHR) antagonist, promotes the expansion culture of CD34 + cells, and that the AHR antagonist prevents the differentiation of hematopoietic stem cells to proliferate NOD-SCID Repopulating cells (SRC).

[0009] However, considering the future practical application of proT therapy, an industrial method for producing proT with high manufacturing efficiency (yield) is desirable, but no reports on such a manufacturing method have been found to date. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Smith et al., JCI Insight. 2017;2(10):e92056 [Non-Patent Document 2] Riemann et al., Stem Cells, 30 (8), 1771-1780, 2012 [Non-Patent Document 3] Ditadi et al., Nature cell biol., 17 (5), 580-591, 2015 [Non-Patent Document 4] Singh et al., Blood advances, 3 (20), 2934-2948, 2019 [Non-Patent Document 5] Boitano et al., Science, 329 (10), 1345-1348, 2010 [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention aims to provide a method for producing T progenitor cells, a culture medium for T progenitor cell differentiation, and a T progenitor cell inducer that can produce T progenitor cells with high efficiency. [Means for solving the problem]

[0012] The inventors of the present invention have conducted extensive research to achieve the above objective, and as a result, CD34 + Cells (especially CD34) + / CD43 - / CD184- / CD73 - We found that culturing cells in a medium containing SR1, an aromatic hydrocarbon receptor antagonist, can efficiently differentiate them into T cell progenitor cells. Furthermore, we discovered that such a medium is even more desirable if it also contains retronectin, DLL4, and IL-3.

[0013] Based on these findings and further investigations, the present invention provides the following methods for producing T progenitor cells, a culture medium for T progenitor cell differentiation, and a T progenitor cell inducer.

[0014] [1] A method for producing T progenitor cells, (1) CD34 in a medium containing an aromatic hydrocarbon receptor antagonist + A method that includes the step of culturing cells. [2] The method according to [1], wherein the culture medium further comprises at least one selected from the group consisting of fibronectin, fibronectin fragments, Delta-like 4 (DLL4), and interleukin-3 (IL-3). [2a] The method according to [1], wherein the culture is carried out in the presence of at least one selected from the group consisting of fibronectin, fibronectin fragments, Delta-like 4 (DLL4), and interleukin-3 (IL-3). [3] The method according to [1], wherein the culture medium further comprises fibronectin and / or a fragment thereof, and DLL4. [3a] The method according to [1], wherein the culture is carried out in the presence of fibronectin and / or a fragment thereof, and DLL4. [4] The method according to [1], [3] or [3a], wherein the culture medium further comprises IL-3. [5] The CD34 + The cell, CD34 + / CD43 - / CD184 - / CD73 - The method described in any of [1] to [4], wherein the cell is a cell. [5a] The CD34+ The cell, CD34 + / CD7 - / CD43 - / CD184 - / CD73 - The method described in any of [1] to [4], wherein the cell is a cell. [6] The method according to any one of [1] to [5a], wherein the T progenitor cells are derived from pluripotent stem cells. [6a] The CD34 + The method described in any of [1] to [5a], wherein the cells are derived from pluripotent stem cells. [6b] The method according to [6] or [6a], wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells). [7] The method according to any one of [1] to [6b], wherein the aromatic hydrocarbon acceptor antagonist is StemRegenin-1 (SR1). [8] The culture is cultured in the absence of feeder cells, as described in any of [1] to [7]. [8a] (X) Further comprising the step of preparing iPS cells, wherein step (1) involves CD34 derived from the iPS cells + A method using cells, as described in any of [1] to [8]. [8b] (Y) Pluripotent stem cells are CD34 + The process further includes a step of inducing differentiation into cells, wherein step (1) the CD34 + A method using cells, as described in any of [1] to [8a]. [8c] CD34 obtained in the (Z) process (Y) + From cells to hematopoietic vascular endothelial cells (CD34) + / CD43 - / CD184 - / CD73 - The method according to [8b], further comprising the step of selecting cells. [9] A medium for T cell precursor differentiation containing an aromatic hydrocarbon receptor antagonist.

[10] The medium according to [9], further comprising at least one selected from the group consisting of fibronectin, fibronectin fragments, DLL4, and IL-3.

[11] The medium according to [9], further comprising fibronectin and / or a fragment thereof, and DLL4.

[12] The culture medium according to [9] or

[11] , further comprising IL-3.

[13] The culture medium according to any one of [9] to

[12] , wherein the aromatic hydrocarbon receptor antagonist is SR1.

[14] T cell precursor inducers containing aromatic hydrocarbon receptor antagonists.

[15] The agent according to

[14] , further comprising at least one selected from the group consisting of fibronectin, fibronectin fragments, DLL4, and IL-3.

[16] The agent according to

[14] , further comprising fibronectin and / or a fragment thereof, and DLL4.

[17] The agent according to

[14] or

[16] , further comprising IL-3.

[18] The agent according to any one of

[14] to

[17] , wherein the aromatic hydrocarbon receptor antagonist is SR1. T progenitor cells obtained by any of the methods described in [1] to [8c]. A cell transplantation therapy agent comprising the T progenitor cells described in

[20]

[19] .

[21] A kit for T cell precursor differentiation containing an aromatic hydrocarbon receptor antagonist.

[22] The kit according to

[21] , further comprising at least one selected from the group consisting of fibronectin, fibronectin fragments, DLL4, and IL-3.

[23] The kit according to

[21] , further comprising fibronectin and / or a fragment thereof, and DLL4.

[24] The kit described in

[21] or

[23] , further comprising IL-3.

[25] The kit according to any one of

[21] to

[24] , wherein the aromatic hydrocarbon receptor antagonist is SR1. [Effects of the Invention]

[0015] According to the present invention, CD34 can be processed with high efficiency. + It will become possible to produce T progenitor cells from cells. [Brief explanation of the drawing]

[0016] [Figure 1] The results of flow cytometry performed on a cell population containing CD34+ cells differentiated from induced pluripotent stem cells are shown. [Figure 2] The results of flow cytometry performed on a cell population, including T progenitor cells differentiated from induced pluripotent stem cells, are shown. [Figure 3] The results of measuring the number of cells in a cell population including T progenitor cells differentiated from induced pluripotent stem cells are shown (control condition, control condition with IL-3 removed (-IL-3), control condition with SR1 removed (-SR1)). [Figure 4] The results of flow cytometry performed on a cell population including T progenitor cells differentiated from induced pluripotent stem cells are shown (control condition, control condition with IL-3 removed (-IL-3), and control condition with SR1 removed (-SR1)). [Modes for carrying out the invention]

[0017] The embodiments of the present invention will be described in detail below.

[0018] In this specification, "comprise" encompasses both the meanings of "essentially consist of" and "consist of."

[0019] In this specification, “culture” means maintaining and / or growing cells in an in vitro environment. “To culture” means sustaining and / or growing cells outside of tissue or in vitro, for example, in a cell culture dish or flask.

[0020] In this specification, "pluripotent stem cell" refers to embryonic stem cells (ES cells) and cells that possess similar pluripotency, that is, the potential ability to differentiate into various tissues of the body (all of the endoderm, mesoderm, and ectoderm). Examples of cells that possess similar pluripotency to ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification).

[0021] In this specification, “population” means two or more cells of the same or different types. “Population” also means a mass of cells of the same or different types.

[0022] In this specification, "hematopoietic stem cell" refers to a multipotent stem cell capable of differentiating into blood cells. In the human body, hematopoietic stem cells are mainly found in the bone marrow and differentiate into leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages), erythrocytes, platelets, mast cells, dendritic cells, etc. In the present invention, hematopoietic stem cells may be cells isolated from living tissue such as bone marrow, or they may be cells derived from ES cells and iPS cells.

[0023] In this specification, "hematopoietic progenitor cells" are cells capable of differentiating into hematopoietic cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes, and hematopoietic progenitor cells can be recognized by being positive for the surface antigens CD34 and / or CD43.

[0024] In this specification, "positive (+)" means that a protein or gene is expressed in a detectable amount by methods known in the art. Furthermore, in the case of proteins expressed intracellularly but not on the cell surface (e.g., transcription factors or their subunits), the target protein can be detected by expressing a reporter protein together with the target protein and detecting the reporter protein. Gene detection can be performed, for example, using nucleic acid amplification methods and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNA-seq.

[0025] In this specification, "negative (-)" means that the expression level of a protein or gene is below the detection limit of any or all of the known methods described above. The detection limit of protein or gene expression may vary depending on the method. In this specification, "cell surface marker" refers to a protein expressed on the cell surface that can be labeled (stained) with a fluorescent substance, allowing for easy detection, concentration, and isolation of cells expressing the cell marker. The cell marker refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a given cell type, specifically a substance that is produced (positive marker) or not produced (negative marker) as mRNA resulting from the transcription of the gene in the genome, or as a protein resulting from the translation of that mRNA.

[0026] The detection of such cell surface markers can be performed using immunoassays with antibodies specific to the cell surface marker, such as ELISA, immunostaining, and flow cytometry.

[0027] In this specification, “expression” is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter within a cell.

[0028] In this specification, "T progenitor cells (proT)" refers to hematopoietic cells that arise in the process of differentiation from hematopoietic stem cells (HSCs) into CD3-positive T cells in the human body, and are positive for CD34 and CD7 (CD34 + / CD7 + (Cells). Furthermore, in this specification, proT may be negative for CD43, CD1a and / or CD116.

[0029] CD34 used in the present invention + The cells are not particularly restricted, for example, CD34 induced in vitro according to known methods. + CD34 obtained by differentiating cells (for example, hematopoietic progenitor cells (HPCs) from pluripotent stem cells) + It may be a cell, or CD34 isolated from biological tissue by a known method. + It could be a cell.

[0030] By subjecting pluripotent stem cells to a known method, CD34 + Cells can be manufactured. If the pluripotent stem cells are iPS cells, for example, hematopoietic progenitor cells can be differentiated and CD34 cells by the method described in International Publication No. 2017 / 221975, International Publication No. 2018 / 135646 and Cell Reports 2(2012)1722-1735. + Cells can be manufactured.

[0031] Also, the above CD34 + As for biological tissues from which cells are isolated, CD34 + The material is not limited as long as it contains cells, but examples include bone marrow, umbilical cord blood, blood, and thymus.

[0032] CD34 used in the present invention + From the perspective of therapeutic application, it is preferable that the cells and pluripotent stem cells conform to GMP (Good Manufacturing Practice) standards.

[0033] 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 cells (EG cells), with iPS cells being preferred (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 the embryo or cells produced without destroying the embryo, and are preferably cells produced without destroying the embryo.

[0034] For "ES cells," various mouse ES cell lines established by inGenious Targeting Laboratory, RIKEN (the Institute of Physical and Chemical Research), etc., are available for use as mouse ES cells, while various human ES cell lines established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc., are available for use as human ES cells. For example, human ES cell lines that can be used include CHB-1 to CHB-12, RUES1, RUES2, HUES1 to HUES28, etc., distributed by ESI Bio, H1, H9, etc., distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, SSES3, etc., distributed by RIKEN.

[0035] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells." These include iPS cells established by Yamanaka et al. by introducing four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPS cells established by selecting cells based on Nanog expression after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPS cells produced using methods that do not include c-Myc (Nakagawa M, Yamanaka S., et al. iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing the four factors OCT3 / 4, SOX2, NANOG, and LIN28, as created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (Japanese Patent Publication No. 2008-307007) can also be used.

[0036] In addition, all published papers (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, Any induced pluripotent stem cell known in the art described in 795-797) or patents (for example, Japanese Patent Publication No. 2008-307007, Japanese Patent Publication No. 2008-283972, U.S. Patent Application Publication No. 2008 / 2336610, U.S. Patent Application Publication No. 2009 / 047263, International Publication No. 2007 / 069666, International Publication No. 2008 / 118220, International Publication No. 2008 / 124133, International Publication No. 2008 / 151058, International Publication No. 2009 / 006930, International Publication No. 2009 / 006997, International Publication No. 2009 / 007852) may be used.

[0037] Various iPS cell lines established by institutions such as the NIH, RIKEN, and Kyoto University can be used as induced pluripotent stem cell lines. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2, and Kyoto University's Ff-I01s04, QHJI, RWMH, DRXT, RJWI, YZWJ, ILCL, GLKV, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, and 648A1.

[0038] The method for producing T progenitor cells according to the present invention is (1) CD34 in a medium containing an aromatic hydrocarbon receptor antagonist (which may be referred to herein as "medium for T progenitor cell differentiation") + The process of culturing cells It is characterized by including.

[0039] The T cell precursor differentiation medium is a basal medium used for culturing animal cells and contains an aromatic hydrocarbon receptor antagonist. The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB 131 medium, Neurobasal Medium (Life Technologies), StemSpan SFEM II (StemCell Technologies), and mixtures thereof.

[0040] Aromatic hydrocarbon receptors (AHRs) are transcription factors belonging to the Per / ARNT / SIM (PAS) family. AHRs are inactive when unbound to a ligand. When an aromatic hydrocarbon compound binds as a ligand, they translocate to the nucleus, where they form a heterodimer with a molecule called ARNT (AhR Nuclear Translocator). They then activate transcription by binding to a xenobiotic response element (XRE) on DNA. Aromatic hydrocarbon receptor antagonists (AHR antagonists) are substances that inhibit at least some or all of the reactions that occur when an agonist binds to an AHR. AHR antagonists may act directly on AHR or indirectly on AHR through the action of another substance.

[0041] Aromatic hydrocarbon receptor antagonists are not particularly limited and include low molecular weight compounds, high molecular weight compounds, proteins such as antibodies and their fragments, peptides, nucleic acids, etc. Specifically, examples include SR1 (4-(2-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purine-6-yl)amino)ethyl)phenol), α-naphthoflavone, 1,4-dihydroxyanthraquinone, 1,5-dihydroxyanthraquinone, 1,8-dihydroxyanthraquinone, galangin, resveratrol, PD98059, CH-223191, GNF-351, TSU-16, 6,2',4'-trimethoxyflavone, 3',4'-dimethoxyflavone, and compounds listed in International Publication No. 2012 / 015914 as AHR antagonists. Of these, SR1 is preferred.

[0042] The concentration of the AHR antagonist in the culture medium is not particularly limited as long as T precursor cells can be produced, and can be adjusted as appropriate depending on the type of AHR antagonist used. For example, when the AHR antagonist is SR1, the concentration can be 0.1 nM to 300 nM, with 0.1 nM to 100 nM being preferred.

[0043] The T cell precursor differentiation medium preferably further contains at least one selected from the group consisting of fibronectin, fibronectin fragments, DLL4, and IL-3. More preferably, the T cell precursor differentiation medium contains two or more of (i) fibronectin and / or its fragments, (ii) DLL4, and (iii) IL-3, and particularly preferably three. Furthermore, it is desirable that the T cell precursor differentiation medium further contains fibronectin and / or its fragments, as well as DLL4, and further contains IL-3.

[0044] The above fibronectin (FN) fragments are selected from fragments contained in the FN-binding domain, cell adhesion domain, or heparin-binding domain. Examples of fibronectin fragments include III1, III2, III3, III7, III8, III9, III 11 III12 III 13 The fragment may include at least one fragment selected from CS-1, and may also be a fragment in which multiple domains are repeatedly linked. Examples of fibronectin fragments include a cell adhesion domain containing a ligand for VLA-5, a heparin-binding domain, a CS-1 domain which is a ligand for VLA-4, III1, etc. Furthermore, examples of fibronectin fragments include CH-271, CH-296, H-271, H-296, and their derivatives and modifiers, as described in J. Biochem., Vol. 110, pp. 284-291 (1991). CH-296 is commercially available under the trade name RetroNectin. In addition, the C-terminal 2 / 3 polypeptide of III1 is commercially available under the name Fibronectin Fragment III1-C.

[0045] FN and / or its fragments may be used immobilized on a suitable solid phase, such as cell culture equipment, beads, membranes, or glass slides. Immobilization of FN and / or its fragments on a solid phase can be carried out, for example, according to the method described in International Publication No. 00 / 09168. The concentration of FN and / or its fragments in the culture medium is not particularly limited as long as it is possible to produce T progenitor cells, and is preferably, for example, 0.001 μg / mL to 500 μg / mL, and more preferably 0.01 μg / mL to 500 μg / mL. When using immobilized FN and / or its fragments, immobilization on the solid phase can be carried out using a solution of FN and / or its fragments at the above concentrations. Culture of cell populations in the presence of FN and / or its fragments is described in International Publication No. 03 / 080817 and can be carried out by referring to it. DLL4 can also be used by immobilizing it on a cell culture carrier in a similar manner. When FN and / or its fragments, or DLL4, are immobilized on a solid phase and used, they do not need to be included in the T precursor cell differentiation medium. The concentration of DLL4 in the medium is not particularly limited as long as it is possible to produce T precursor cells, and is preferably, for example, 0.001 μg / mL to 500 μg / mL, and more preferably 0.01 μg / mL to 500 μg / mL.

[0046] The concentration of IL-3 in the culture medium can be, for example, 0.5 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 500 ng / mL, and more preferably 1 ng / mL to 300 ng / mL.

[0047] In addition to the components mentioned above, the T cell progenitor differentiation medium may also contain, for example, stem cell factor (SCF), thrombopoietin (TPO), Flt3L (Fms-like tyrosine kinase 3 ligand), IL-6, IL-7, and others.

[0048] The culture medium for T cell progenitor differentiation may contain serum or be serum-free, and is more preferably serum-free.

[0049] The T precursor cell differentiation medium may also contain serum substitutes (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplements, etc.). One or more serum substitutes may be used.

[0050] Furthermore, the culture medium used in the present invention may also contain one or more substances such as lipids, amino acids (including non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts.

[0051] For T cell progenitor differentiation media, it is desirable to use a chemically defined medium that does not contain any unknown components such as serum, as this reduces lot-to-lot variability and allows for the preparation of T cell progenitor cells of consistent quality.

[0052] The pH of the T precursor cell differentiation medium is typically 7.0 to 7.8, preferably 7.2 to 7.6. Before use, the medium is more preferably sterilized by methods such as filtration, UV irradiation, heat sterilization, or radiation irradiation to prevent contamination.

[0053] The T cell precursor differentiation medium can be prepared in solution, dry, or concentrated form (e.g., 2× to 1000×). In the concentrated form, it can be diluted to an appropriate concentration before use. The liquid used to dilute the dry or concentrated medium can be appropriately selected from water, buffer solution, physiological saline solution, etc.

[0054] The present invention further provides a kit for T cell precursor differentiation. The kit for T cell precursor differentiation contains components similar to those of the T cell precursor differentiation medium described above. In one aspect of the present invention, the kit for T cell precursor differentiation contains an aromatic hydrocarbon receptor antagonist, fibronectin and / or a fragment thereof, and DLL4 immobilized on a suitable solid phase (e.g., a cell culture carrier such as cell culture equipment, beads, membranes, or glass slides), and IL-3. In one aspect of the present invention, fibronectin and / or a fragment thereof and DLL4 may already be immobilized on a suitable solid phase, or may be designed to be immobilized on a suitable solid phase at the time of use.

[0055] The culture method used in this invention may be either adherent culture or suspension culture.

[0056] The manufacturing method of the present invention is preferably carried out in the absence of feeder cells. Because the manufacturing method of the present invention allows for culture in the absence of feeder cells, it is possible to stably produce T progenitor cells with uniform properties without contamination by substances of unknown composition.

[0057] In this specification, "without feeder cells" or "feeder-free" essentially means the absence of feeder cells and the use of culture media preconditioned by culturing feeder cells. Therefore, the culture media does not contain substances such as growth factors and cytokines secreted by feeder cells.

[0058] The term "feeder cell" or "feeder" refers to a cell that is co-cultured with another type of cell, providing support and an environment in which that cell can grow. Feeder cells may be of the same species as the cells they support, or of a different species. For example, human dermal fibroblasts or human embryonic stem cells may be used as feeders for human cells, or primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts may be used. Feeder cells can be inactivated by irradiation or treatment with mitomycin C, etc.

[0059] The culture conditions for the manufacturing method of the present invention are not particularly limited, and the culture temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C. The culture period is also not particularly limited, and those skilled in the art can determine it appropriately while monitoring the number of T precursor cells, for example, 10 days or more. In the culture according to the present invention, the number of subculturing cycles required to obtain the desired amount of T precursor cells may be performed, and the culture medium may be added and replaced. The culture according to the present invention can be carried out using a known CO2 incubator.

[0060] CD34 used in the manufacturing method of the present invention + Cells express CD34 (CD34 + These are cells that express CD34 and do not express CD7 (CD34 + / CD7 - ) These are cells. In another embodiment, CD34 +The cells are hematopoietic endothelial cells (HECs). Hematopoietic endothelial cells express CD34 and do not express CD43, CD184, and CD73 (CD34 + / CD43 - / CD184 - / CD73 - ) These are cells (and CD34 + / CD43 - / CD184 - / CD73 - Since the cells do not express CD7, CD34 + / CD43 - / CD184 - / CD73 - The cells are CD34 + / CD7 - / CD43 - / CD184 - / CD73 - (Also referred to as cells). Thus, by using hematopoietic vascular endothelial cells in step (1), it becomes possible to produce T progenitor cells with higher efficiency.

[0061] The manufacturing method of the present invention may further include a step of preparing (X)iPS cells.

[0062] The method described above is one way to prepare iPS cells. The iPS cells prepared here are CD34 + Induce differentiation into cells, and the CD34 + The cells can be used in the culture process in step (1). In this invention, if pluripotent stem cells other than iPS cells are used, the other pluripotent stem cells will be prepared in place of iPS cells in step (X).

[0063] Furthermore, the manufacturing method of the present invention involves (Y) pluripotent stem cells being converted to CD34 + The process may further include a step of inducing differentiation into cells.

[0064] CD34 differentiated here + The cells can be used in the culture process (1). CD34 of pluripotent stem cells.+ Differentiation induction into cells can be performed according to a known method. When the pluripotent stem cells are iPS cells, for example, hematopoietic progenitor cells can be induced to differentiate by the methods described in International Publication No. 2017 / 221975, International Publication No. 2018 / 135646, and Cell Reports 2 (2012) 1722-1735, to obtain CD34 + cells can be produced.

[0065] The production method of the present invention may further comprise a step of selecting, from the CD34 + cells obtained in step (Y) in step (Z), hematopoietic vascular endothelial cells (CD34 + / CD43 - / CD184 - / CD73 - cells).

[0066] CD34 + Separation of hematopoietic vascular endothelial cells from cells can be carried out according to a known method, and examples of such a method include flow cytometry, magnetic cell separation method and the like. By using the hematopoietic vascular endothelial cells sorted here in the culture of step (1), T progenitor cells can be produced with higher efficiency.

[0067] In addition, the T progenitor cell inducing agent of the present invention can use the same components as the aforementioned medium for T progenitor cell differentiation. The medium for T progenitor cell differentiation and the T progenitor cell inducing agent of the present invention can induce differentiation of T progenitor cells by being used in the above production method.

[0068] The production method, the medium for T progenitor cell differentiation, and the T progenitor cell inducing agent of the present invention make it possible to produce T progenitor cells with high efficiency.

[0069] T progenitor cells produced by the manufacturing method of the present invention are useful for treating animals (especially humans) with conditions requiring an increase in T cell count. "Conditions requiring an increase in T cell count" refers to conditions in which the blood T cell count is reduced compared to healthy animals (e.g., conditions after anticancer drug treatment and / or radiation therapy, acquired immunodeficiency syndrome (AIDS)). Furthermore, T progenitor cells produced by the manufacturing method of the present invention are useful in cell therapy aimed at, for example, the recovery of T cells due to thymic damage after radiation therapy. When using the above T progenitor cells in cell therapy, cells into which a desired gene has been introduced can also be used. Using such cells can treat genetic diseases (e.g., recombinase deficiency, recombinase regulatory gene deficiency, severe combined immunodeficiency (SCID)). Therefore, the present invention provides a pharmaceutical composition (especially a cell transplantation therapy agent) containing T progenitor cells produced by the above manufacturing method, and a cell therapy method using said T progenitor cells.

[0070] When performing such cell therapy, from the standpoint of preventing rejection, it is preferable that the subjects from which T cells are isolated have a matching HLA type to the subjects to whom the T progenitor cells are administered, and more preferably, they are the same subjects to whom the T progenitor cells are administered.

[0071] The pharmaceutical composition of the present invention can be prepared as an oral / parenteral formulation by mixing an effective amount of T progenitor cells with a pharmaceutically acceptable carrier according to known means. The pharmaceutical composition of the present invention is usually prepared as a parenteral formulation such as an injection, suspension, or intravenous infusion. Examples of pharmaceutically acceptable carriers include solvents, bases, diluents, excipients, analgesics, buffers, preservatives, stabilizers, suspensions, isotonic agents, surfactants, and solubilizers.

[0072] The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to various conditions such as the patient's weight, age, sex, and symptoms. The pharmaceutical composition of the present invention is applicable to mammals, including humans. [Examples]

[0073] Examples are given below to further explain the present invention. However, the present invention is not limited in any way to these examples.

[0074] [Example 1] CD34 + CD7 + Investigation of methods for producing T progenitor cells As a cell population containing hematopoietic endothelial cells (HECs), we used a suspension cell population differentiated from iPS cells (Ff-I01s04 strain: derived from peripheral blood mononuclear cells of healthy individuals) provided by the Center for iPS Cell Research and Application (IPSC) of Kyoto University, according to known methods (e.g., the method described in Cell Reports 2(2012)1722-1735 and International Publication No. 2017 / 221975).

[0075] Specifically, the Ff-I01s04 strain was placed in a 6-well plate treated with ultra-low adhesion in 1 × 10⁻¹⁶ layers. 6 Cells were seeded in cells / well (Day 0), and 50 ng / ml BMP4, 50 ng / ml bFGF, 50 ng / ml VEGF, and 2 μM SB431542 were added to 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 2-phosphate), and the cells were cultured for 4 days under hypoxic conditions (5% O2) (Day 4). Subsequently, the cells were cultured for another 4 days in medium supplemented with 50 ng / ml bFGF, 50 ng / ml VEGF, and 50 ng / ml SCF (Day 8) to obtain a cell population containing HEC. The above suspension cell population containing HEC was stained using the following antibody set.

[0076] [Table 1]

[0077] Hemogenic endothelial cells (HECs) induced from iPS cells were sorted using BD FACS AriaFusion (Figure 1), and seeded at 5,000 cells per well of a 24-well plate onto plates coated with RetroNectin (RN; Takara Bio Inc.) and Fc-tagged recombinant human delta-like 4 (DLL4-Fc; Sinobiological). The plates were prepared under concentration conditions of 5 μg / ml RN and 5 μg / ml DLL4-Fc. StemSpan SFEM II (StemCell Technologies) was used as the basal medium for culture. Furthermore, 300 ng / ml recombinant human (rh) stem cell factor (SCF, R&D Systems), 300 ng / ml rh Fms-like tyrosine kinase 3 ligand (Flt3L, Peprotech), 100 ng / ml rh thrombopoietin (TPO, Peprotech), 100 ng / ml rh interleukin 7 (IL-7, Peprotech), 100 ng / ml rh interleukin 6 (IL-6, Peprotech), 10 ng / ml rh interleukin 3 (IL-3, Peprotech) and 1 nM StemReginin 1 (SR1, Calbiochem) were added, and the cells were cultured for 11 days. On day 4 after the start of culture, 500 μl of the aforementioned medium was added. In addition, a half-volume medium exchange was performed on day 7 after the start of culture. On day 11 after the start of culture, cells were collected by pipetting, the number of cells was counted, then antibody staining was performed using the following antibody set, and CD34 was analyzed with a flow cytometer (FACS Aria Fusion, BD Bioscience) + / CD7 + It was confirmed that T progenitor cells (proT) were induced (Figure 2). The cells proliferated approximately 150-fold over 10 days of culture, of which about 25% were proT cells.

[0078] [Table 2]

[0079] [Example 2] Evaluation of the thymic engraftment ability of T progenitor cells neonate NOD.Cg-Prkdc at 2 days old scid Il2rg tm1Wjl In Example 1, 5 × 10⁶ mice (Charles River Japan Co., Ltd.) were used to induce the development of iPS cells. 5 A cell population containing T progenitor cells was suspended in 30 μl of administration solution (10 μl D-PBS(-), 10 μl 250 μg / ml anti-human mouse IL-7 antibody (M25, BioXCell), and 10 μl 50 μg / ml rhIL-7) and transplanted into the liver. Four weeks after administration, the thymus was harvested and evaluated by flow cytometry for the presence of human CD45-positive blood cells.

[0080] In the treatment group, human CD45 + CD3 + αβTCR + CD4 + CD8a + CD4 + CD8 + Double-positive cells were identified.

[0081] [Example 3] Evaluation of the importance of SR1 and IL-3 in T progenitor cell production iPS cell-derived hematopoietic endothelial cells (HECs) prepared in the same manner as in Example 1 were separated using BD FACS AriaFusion, resuspended in StemSpan SFEM II medium, and seeded in culture vessels (6-well plates) coated with RN and DLL4-Fc (225,000 cells / 6 wells). Three conditions were established for the addition of various factors: a control condition (containing 300 ng / ml recombinant human (rh) stem cell factor (R&D systems), 300 ng / ml rh Fms-like tyrosine kinase 3 ligand (peprotech), 100 ng / ml rh thrombopoietin (peprotech), 100 ng / ml rh interleukin 7 (peprotech), 100 ng / ml rh interleukin 6 (peprotech), 10 ng / ml rh interleukin 3 (peprotech), and 1 nM StemReginin 1 (SR1, Calbiochem)); a control condition (control condition) with interleukin 3 removed from the control condition; and a control condition (control condition) with SR1 removed from the control condition. Culture was carried out for 12 days. Midway through the culture, 500 μl of the aforementioned culture medium was added on the fourth day after the start of the culture. Furthermore, a half-volume culture medium change was performed on day 7 after the start of culture. On day 12 after the start of culture, cells were collected by pipetting, the number of cells was counted, and then the cells were stained with antibodies as described in Table 2 and measured using BD FACS AriaFusion. As a result, it was confirmed that cell proliferation decreased when SR1 or IL-3 was removed from the additives (Figure 3). In addition, flow cytometry analysis confirmed that the addition of SR1 also plays an important role in maintaining CD34 expression levels (Figure 4).

Claims

1. A method for producing T progenitor cells, (1) A method comprising the step of culturing CD34+ / CD43- / CD184- / CD73- cells in a medium containing an aromatic hydrocarbon receptor antagonist and interleukin-3 (IL-3), wherein the T precursor cells are derived from iPS cells.

2. The method according to claim 1, wherein the culture medium further comprises at least one selected from the group consisting of fibronectin, fibronectin fragments, and Delta-like 4 (DLL4).

3. The method according to claim 1, wherein the culture medium further comprises fibronectin and / or a fragment thereof, and DLL4.

4. The method according to any one of claims 1 to 3, wherein the aromatic hydrocarbon acceptor antagonist is StemRegenin-1 (SR1).

5. The method according to any one of claims 1 to 4, wherein the culture is performed in the absence of feeder cells.

6. A medium for T cell precursor differentiation for culturing CD34+ / CD43- / CD184- / CD73- cells, comprising an aromatic hydrocarbon receptor antagonist and IL-3, wherein the T cell precursor cells are derived from iPS cells.

7. The culture medium according to claim 6, further comprising at least one selected from the group consisting of fibronectin, fibronectin fragments, and DLL4.

8. The culture medium according to claim 6, further comprising fibronectin and / or a fragment thereof, and DLL4.

9. The culture medium according to any one of claims 6 to 8, wherein the aromatic hydrocarbon receptor antagonist is SR1.

10. A T progenitor cell inducer for inducing T progenitor cells from CD34+ / CD43- / CD184- / CD73- cells, comprising an aromatic hydrocarbon receptor antagonist and IL-3, wherein the T progenitor cells are derived from iPS cells.

11. The agent according to claim 10, further comprising at least one selected from the group consisting of fibronectin, fibronectin fragments, and DLL4.

12. The agent according to claim 10, further comprising fibronectin and / or a fragment thereof, and DLL4.

13. The agent according to any one of claims 10 to 12, wherein the aromatic hydrocarbon receptor antagonist is SR1.

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