Stem cell production method

Abstract

The present disclosure provides a method for producing stem cells, said method comprising applying a phosphate to blood cells and inducing stem cells from the blood cells. The phosphate may be an intermediate or final product of the non-mevalonic acid pathway. The phosphate may be (E)-4-hydroxy-3-methyl-2-butenyl diphosphate.

Description

[Technical Field]

[0001] The present invention relates to cell technology and to a method for producing stem cells. [Background technology]

[0002] T cells derived from hematopoietic stem cells play an important role in immunity. T cells express a wide variety of T cell receptors (TCRs) on their surface. TCR diversity is achieved by V(D)J gene rearrangement. VJ gene rearrangement is shown in Figure 1.

[0003] DN1 (double negative 1) cells, precursors of T cells, proliferate in response to interleukin-7 (IL-7) and c-kit ligand (KL), which are highly expressed in the cortical subcapsular region of the thymus, and express CD25 (interleukin-2 receptor α chain) to become DN2 cells. DN2 cells gradually lose CD44 expression and become DN3 cells.

[0004] TCRs comprise a variable (V) region and a constant (C) region. The amino acid sequence of the V region is highly diverse and forms the antigen-binding site. The V region genes are separated into V genes, D (diversity) genes, and J (joining) genes in germline DNA, and during the process of differentiation into T cells, these are reconstituted into a single V-(D)-J sequence on the chromosome to become an expressed gene. The J genes include the JP1 gene, JP gene, J1 gene, JP2 gene, and J2 gene.

[0005] DN3 cells, which have undergone V(D)J gene rearrangement at the TCRβ and TCRγ loci, become either TCRαβ T cells or TCRγδ T cells. In TCRαβ T cells, the TCR consists of α and β chains. In TCRγδ T cells, the TCR consists of γ and δ chains. The branching decision to become TCRαβ T cells or TCRγδ T cells is controlled by silencer regions present in the TCRγ and TCRα loci.

[0006] Although TCRγδ T cells are fewer in number than TCRαβ T cells, they account for the majority of the intraepithelial lymphocyte population in the intestinal mucosa. TCRγδ T cells sense various stresses that damage cells and induce immune responses. TCRγδ T cells are said to sense external stresses such as bacterial and viral infections, as well as changes in the properties of cells associated with canceration.

[0007] TCRγδ T cells proliferate and become activated after recognizing isopentenyl pyrophosphate (IPP), an intermediate product of mevalonate metabolism in the cholesterol synthesis pathway of antigen-presenting cells (APCs), as an antigen. Therefore, cancer immunotherapy involves activating a patient's TCRγδ T cells ex vivo and then returning them to the body. However, because only 1-5% of TCRγδ T cells exist in peripheral blood, it is difficult to obtain sufficient TCRγδ T cells even with blood sampling.

[0008] For this reason, it has been proposed to induce iPS cells with rearranged γδ-TCR genes by reprogramming blood cells stimulated with zoledronic acid (see, for example, Patent Document 1). However, it has been reported that iPS cells induced by this method do not rearrange γδ-TCR genes with J1 / J2 genes (see, for example, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2018 / 143243 [Non-patent literature]

[0010] [Non-Patent Document 1] DAISUKE WATANABE et al., “The Generation of Human cdT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture,” STEM CELLS TRANSLATIONAL MEDICINE, 2018;7:34-44 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a method for efficiently inducing stem cells carrying rearranged γδ-TCR genes. [Means for solving the problem]

[0012] A method for producing stem cells according to an embodiment of the present invention includes applying a phosphorylated compound to blood cells; and deriving stem cells from blood cells.

[0013] In the above-mentioned method for producing stem cells, the phosphorylated product may be an intermediate product or an end product of the non-mevalonate pathway.

[0014] In the above-mentioned method for producing stem cells, the phosphorylated product may be (E)-4-hydroxy-3-methyl-2-butenyl diphosphate.

[0015] The above-mentioned method for producing stem cells may further comprise applying interleukin to the blood cells.

[0016] In the above-mentioned method for producing stem cells, the interleukin may be at least one selected from the group consisting of IL-2, IL-15, and IL-23.

[0017] In the above-mentioned method for producing stem cells, the blood cells may be mononuclear cells.

[0018] In the above-mentioned method for producing stem cells, the stem cells may be iPS cells.

[0019] In the above-mentioned method for producing stem cells, in inducing stem cells from blood cells, inducer RNA may be introduced into the blood cells.

[0020] In the above-mentioned method for producing stem cells, a Sendai virus vector may be used to induce stem cells from blood cells.

[0021] In the above-mentioned method for producing stem cells, a stealth RNA vector may be used in inducing stem cells from blood cells.

[0022] In the above-mentioned method for producing stem cells, the stem cells may comprise a γδ-TCR rearrangement gene.

[0023] A method for producing blood cells according to an aspect of the present invention includes preparing stem cells produced by the above-described method for producing stem cells, and inducing blood cells from the stem cells.

[0024] In the above-mentioned method for producing blood cells, the blood cells may be γδ T cells.

[0025] In the above-mentioned method for producing blood cells, when inducing blood cells from stem cells, cell clusters of stem cells may be seeded on feeder cells.

[0026] In the above-mentioned method for producing blood cells, the feeder cells may be stromal cells. The stromal cells may be derived from bone marrow. The stromal cells may be OP9 cells. [Effects of the Invention]

[0027] According to the present invention, it is possible to provide a method for efficiently inducing stem cells having rearranged γδ-TCR genes. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram showing VJ gene rearrangement. [Figure 2] 1 is a graph showing the number of colonies of stem cells induced according to Example 1 and Comparative Example 1. [Figure 3A] 1 is a photograph of induced stem cells according to Example 1. [Figure 3B] 1 is a photograph of induced stem cells according to Example 1. [Figure 4] 1 shows photographs showing the results of PCR analysis of the genomes of stem cells induced in Example 1 and Comparative Example 1. [Figure 5A] 1 is a photograph of immunostained cells according to Example 1. [Figure 5B] 1 is a photograph of immunostained cells according to Example 1. [Figure 5C] 1 is a photograph of immunostained cells according to Example 1. [Figure 5D] 1 is a photograph of immunostained cells according to Example 1. [Figure 6] 1 is a dot plot obtained by a flow cytometer showing the results of Example 1. [Figure 7A] 1 is a photograph of cells according to Example 2. [Figure 7B] 1 is a photograph of cells according to Example 2. [Figure 7C] 1 is a photograph of cells according to Example 2. [Figure 8] 1 is a dot plot obtained by a flow cytometer showing the results of Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0029] Hereinafter, embodiments of the present invention will be described in detail. Note that the following embodiments are merely examples for embodying the technical concept of the present invention, and the technical concept of the present invention is not limited to the combination of components described below. Various modifications can be made to the technical concept of the present invention within the scope of the claims.

[0030] A method for producing stem cells according to an embodiment includes applying a phosphorylation agent to blood cells and inducing stem cells from the blood cells, such as induced pluripotent stem cells (iPS cells).

[0031] The blood cells may be derived from a human or non-human animal. The blood cells are separated from blood, such as, but not limited to, peripheral blood and umbilical cord blood. The blood may be collected from an adult or a minor. Anticoagulants such as ethylenediaminetetraacetic acid (EDTA), heparin, and ACD-A (Biologically Certified Blood Preservative Solution) may be used during collection.

[0032] Blood cells are nucleated cells such as monocytes, neutrophils, eosinophils, basophils, and lymphocytes, and do not include erythrocytes, granulocytes, and platelets. Blood cells may be, for example, endothelial progenitor cells, blood stem / progenitor cells, T cells, or B cells. T cells may be, for example, αβ T cells or γδ T cells. Blood cells do not have to be γδ T cells.

[0033] The phosphate may be an intermediate or end product of the non-mevalonate pathway. The non-mevalonate pathway is a synthetic pathway for isopentenyl pyrophosphate (IPP) that does not involve mevalonate. Examples of intermediate products of the non-mevalonate pathway include 1-deoxy-D-xylulose 5-phosphate (DOXP), 2-C-methyl-D-erythritol 4-phosphate (MEP), 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME), 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP), 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP), and (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMB-PP).

[0034] When applying phosphate to blood cells, the phosphate may be added to the culture medium in which the blood cells are cultured. The concentration of the phosphate in the culture medium is, for example, 1 μmol / L to 50 μmol / L, 3 μmol / L to 20 μmol / L, or 5 μmol / L to 12 μmol / L. The blood cells may be cultured in the phosphate-added culture medium for 1 day or more, 2 days or more, or 3 days or more. The blood cells may be cultured in the phosphate-added culture medium for 14 days or less, 10 days or more, or 7 days or more.

[0035] The blood cells may further be administered interleukins, examples of which include IL-2, IL-15, and IL-23.

[0036] When applying interleukin to blood cells, interleukin may be added to the culture medium in which the blood cells are cultured. The concentration of interleukin in the culture medium is, for example, 5 ng / mL to 100 ng / mL, 10 ng / mL to 90 ng / mL, or 15 ng / mL to 80 ng / mL. Blood cells may be cultured in the culture medium containing interleukin for 1 day or more, 2 days or more, or 3 days or more. Blood cells may be cultured in the culture medium containing interleukin for 14 days or less, 10 days or more, or 7 days or more.

[0037] The phosphate may be applied to the blood cells followed by application of the interleukin. The phosphate and the interleukin may be applied to the blood cells simultaneously. The interleukin may be applied to the blood cells followed by application of the phosphate.

[0038] Examples of media for culturing blood cells include, but are not limited to, RPMI1640 medium, minimal essential medium (α-MEM), Dulbecco's modified Eagle's medium (DMEM), and F12 medium.

[0039] Next, an inducer is introduced into the phosphorylated blood cells to induce stem cells from the blood cells. In inducing stem cells, induction refers to reprogramming, reprogramming, transformation, cell fate reprogramming, etc.

[0040] The inducer introduced into the blood cells may be RNA. The RNA may be mRNA. Examples of the inducer include OCT3 / 4, SOX2, KLF4, and c-MYC. M3O, an improved version of OCT3 / 4, may also be used as the inducer. The inducer may also be at least one selected from the group consisting of LIN28A, FOXH1, LIN28B, GLIS1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, TH2B, and p53DD. RNAs of these inducers are available from TriLink. Note that although gene symbols are listed here in human form, capitalization is not intended to limit the species. For example, even if a gene is written in all capital letters, it does not exclude the inclusion of a mouse or rat gene. However, in the examples, gene symbols are written in accordance with the species actually used.

[0041] The inducer RNAs were pseudouridine (Ψ), 5-methyluridine (5meU), N1-methylpseudouridine (melΨ), 5-methoxyuridine (5moU), 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), 5-carboxymethylesteruridine (5camU), thienoguanosine (thG), N4-methylcytidine (me4C), 5-methylcytidine (m5C), 5-methyoxycytidine (5moC), 5-hydroxymethyluridine (5camU), thienoguanosine (thG), N4-methylcytidine (me4C), 5-methylcytidine (m5C), 5-methyloxycytidine (5moC), 5-hydroxymethyluridine (5camU), thienoguanosine (thG ... It may be modified with at least one selected from the group consisting of 5-methylcytidine (5hmC), 5-hydroxycytidine (5hoC), 5-formcytidine (5fC), 5-carboxycytidine (5caC), N6-methyl-2-aminoadenosine (m6DAP), diaminopurine (DAP), 5-methyluridine (m5U), 2'-O-methyluridine (Um or m2'-OU), 2-thiouridine (s2U), and N6-methyladenosine (m6A).

[0042] The inducer RNA may be polyadenylated.

[0043] Inducer RNA may be prepared by polyadenylation of in vitro transcribed (IVT) RNA. RNA may be polyadenylated during IVT by using a DNA template encoding a poly(A) tail. RNA may be capped. To maximize expression efficiency in cells, it is preferable that most RNA molecules contain a cap. RNA may have a 5' cap [m7G(5')ppp(5')G] structure. This sequence stabilizes RNA and promotes transcription. From RNA with a 5' triphosphate, the 5' triphosphate may be removed by dephosphorylation. RNA may have an anti-reverse cap analog (ARCA) [3'O-Me-m7G(5')ppp(5')G]. ARCA is a sequence inserted before the transcription start site, doubling the efficiency of transcribed RNA. RNA may have a polyA tail.

[0044] Furthermore, the inducer RNA may be replicative RNA, which has the ability to self-replicate. Replicative RNA is RNA that has the ability to self-replicate, and unlike normal RNA, it also has the ability to express proteins necessary for RNA replication. Replicative RNA is derived from the Venezuelan equine encephalitis (VEE) virus, a type of alphavirus. By transfecting replicative RNA into cells, it is possible to cause the cells to express RNA that continues to produce inducers, thereby eliminating the need to introduce RNA into cells multiple times.

[0045] The replicative RNA sequence may comprise a sequence obtained from an alphavirus selected from the group consisting of alphavirus replicon RNA, Eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, and Western equine encephalitis virus (WEE).

[0046] The replicative RNA may also comprise sequences from an alphavirus selected from the group consisting of Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.

[0047] For example, the replicative RNA contains, from 5' to 3', (VEE RNA replicase)-(promoter)-(RF1)-(self-cleaving peptide)-(RF2)-(self-cleaving peptide)-(RF3)-(IRES or core promoter)-(RF4)-(IRES or any promoter)-(optional selectable marker)-(VEE 3'UTR and polyA tail)-(optional selectable marker)-promoter. RF1-4 above are factors that induce dedifferentiation of somatic cells into pluripotent cells. RF2-3, RF3-4, and RF4 above are optional. The RF1-4 may be selected from the group consisting of OCT3 / 4, KLF4, SOX-2, c-MYC, LIN28A, LIN28B, GLIS1, FOXH1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, and TH2B.

[0048] The method for introducing the inducer into the blood cells is arbitrary. For example, the inducer may be introduced into the blood cells using a vector. The inducer may be introduced into the blood cells by lipofection.

[0049] Sendai virus (Sev) can be used as a vector. Sendai virus is a virus that belongs to the Paramyxoviridae family of the Mononegavirales order and has an RNA genome. Sendai virus has an RNA genome and an envelope made of a lipid bilayer membrane that encapsulates the RNA.

[0050] CytoTune (registered trademark, Invitrogen) can be used as a Sendai virus carrying an inducer RNA. The Sendai virus vector may be a Sendai virus vector with improved infection persistence. Sendai virus vectors with improved infection persistence are also called stealth RNA vectors. Examples of stealth RNA vectors that can be used include SRV iPSC-1 Vector, SRV iPSC-2 Vector, SRV iPSC-3 Vector, and SRV iPSC-4 Vector (registered trademark, Tokiwa Bio Co., Ltd.). Details of stealth RNA vectors are described in Japanese Patent Nos. 4478788, 4936482, 5633075, and 5963309.

[0051] An index of the titer of Sendai virus is the multiplicity of infection (MOI). The MOI of Sendai virus is, for example, 0.1 to 100.0, or 1.0 to 50.0.

[0052] The inducer may be introduced into blood cells that are being cultured in an adherent manner, or into blood cells that are being cultured in a suspension manner in a gel medium.

[0053] Blood cells into which an inducer is introduced may be cultured feeder-free using a basement membrane matrix such as Matrigel (Corning), CELLstart (registered trademark, ThermoFisher), or Laminin511 (iMatrix-511, nippi).

[0054] As a medium for culturing blood cells into which an inducing factor is introduced, for example, a stem cell medium such as a human ES / iPS medium such as Stemfit (Ajinomoto) can be used.

[0055] However, the stem cell medium is not limited to this, and various stem cell media can be used. For example, Primate ES Cell Medium, mTeSR1, and TeSR2 (STEMCELL Technologies) may be used. The stem cell medium is placed in, for example, a dish, a well, or a tube.

[0056] The gel medium does not contain a growth factor such as basic fibroblast growth factor (bFGF), or alternatively, the gel medium contains a growth factor such as bFGF at a low concentration of 400 μg / L or less, 40 μg / L or less, or 10 μg / L or less.

[0057] The gel medium does not contain TGF-β or contains TGF-β at a low concentration of 600 ng / L or less, 300 ng / L or less, or 100 ng / L or less.

[0058] The gel medium does not have to be stirred, and the gel medium does not have to contain feeder cells.

[0059] The gel medium may contain at least one substance selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin.

[0060] After introducing an inducer into blood cells, the cells may be reprogrammed in a liquid medium other than a gel medium, or in a gel medium.

[0061] After introducing an inducer into blood cells and culturing the cells, the inducer-introduced cells may be recovered, and at least a portion of the recovered mixed cells may be seeded into a medium, followed by at least one passaging. In the passaging, clones of the inducer-introduced cells may be mixed. In the passaging, different clones of the inducer-introduced cells may be mixed. Thereafter, the inducer-introduced cells may be recovered, and at least a portion of the recovered mixed cells may be seeded into a medium and passaged multiple times. Until stem cells are established, the inducer-introduced cells may be recovered, and at least a portion of the recovered mixed cells may be seeded into a medium and passaged. Note that all of the recovered mixed cells may be seeded into a medium.

[0062] Here, recovering cells into which an inducer has been introduced and seeding at least a portion of the recovered and mixed cells into a medium for passaging refers to, for example, passaging the cells into which an inducer has been introduced without distinguishing them based on their gene expression state. For example, during passaging, the cells into which an inducer has been introduced may be seeded into the same culture vessel without distinguishing them based on their gene expression state. Alternatively, recovering cells into which an inducer has been introduced and seeding at least a portion of the recovered and mixed cells into a medium for passaging refers to, for example, passaging the cells into which an inducer has been introduced without distinguishing them based on the degree of reprogramming. For example, during passaging, the cells into which an inducer has been introduced may be seeded into the same culture vessel without distinguishing them based on the degree of reprogramming.

[0063] Alternatively, recovering cells into which an inducer has been introduced and seeding at least a portion of the recovered and mixed cells into a medium for passage means, for example, passaging the cells into which an inducer has been introduced without distinguishing them by morphology. For example, during passaging, the cells into which an inducer has been introduced may be seeded into the same culture vessel without distinguishing them by morphology. Alternatively, recovering cells into which an inducer has been introduced and seeding at least a portion of the recovered and mixed cells into a medium for passage means, for example, passaging the cells into which an inducer has been introduced without distinguishing them by size. For example, during passaging, the cells into which an inducer has been introduced may be seeded into the same culture vessel without distinguishing them by size.

[0064] Alternatively, recovering cells into which an inducer has been introduced and seeding at least a portion of the recovered mixed cells into a medium for passage refers to passaging cells into which an inducer has been introduced without cloning. For example, when passaging without cloning, it is not necessary to pick up colonies formed by cells into which an inducer has been introduced. For example, when passaging without cloning, it is not necessary to separate multiple colonies formed by cells into which an inducer has been introduced. For example, during passaging, cells that have formed multiple different colonies may be mixed and seeded in the same culture vessel. Also, for example, when passaging without cloning, it is not necessary to clone a single colony formed by cells into which an inducer has been introduced. For example, during passaging, colonies may be mixed and seeded in the same culture vessel.

[0065] For example, when cells into which an inducer has been introduced are cultured in adhesion, the cells in adhesion culture may be recovered, and at least a portion of the recovered and mixed cells may be seeded in a medium and passaged. For example, when passaged, the cells may be detached from the culture vessel, and at least a portion of the detached and mixed cells may be seeded in the same culture vessel. For example, the cells may be detached from the culture vessel with a detachment solution, and the detached and mixed cells may be passaged as a whole. For example, cells that have not formed colonies may be passaged. When cells into which an inducer has been introduced are cultured in suspension, the suspension cultured cells may be passaged as a whole.

[0066] When the cells into which the inducer has been introduced are subcultured, the cells may be seeded in a medium or culture vessel at a low density. Here, a low density refers to, for example, 1 cell / cm. 2 or more, 0.25 x 10 4 cells / cm 2 Below, 1.25 x 10 3 cells / cm 2 Below, 0.25 x 10 3 cells / cm 2 Below, 0.25 x 10 2 cells / cm 2 or less, or 0.25 x 10 1 cells / cm 2 or less. Alternatively, a low concentration refers to a concentration at which 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less cells can contact each other, but 11 or more cells do not contact each other. Note that there may be multiple cell clumps in which 10 or less cells are in contact with each other. Alternatively, a state in which the entire bottom surface of the cell container is covered with cells is considered to be 100% confluent, and a low concentration refers to 5% or less confluent, 4% or less confluent, 3% or less confluent, 2% or less confluent, 1% or less confluent, 0.5% or less confluent, 0.1% or less confluent, 0.05% or less confluent, or 0.01% or less confluent. Alternatively, a low concentration refers to a concentration at which single cells do not contact each other among the seeded cells. For example, single cells may be seeded into the wells of a well plate. The well plate may be a 12-well plate or a 96-well plate. According to the findings of the present inventors, when cells into which an inducer has been introduced are passaged, seeding the cells in a medium at a low concentration makes it possible to suppress the persistence of Sendai virus in stem cells induced from the cells. The percentage of cells in which Sendai virus persists among the induced stem cells is, for example, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0%.

[0067] The cells into which the inducer has been introduced may be cultured and passaged in a closed culture vessel. A closed culture vessel does not allow, for example, the exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The cells into which the inducer has been introduced may be expanded in two-dimensional culture or three-dimensional culture.

[0068] After the cells into which the inducer has been introduced are induced to become stem cells and the stem cells are established, the whole cells in the adherent culture may be cryopreserved as stem cells. For example, the whole cells detached from the culture vessel with a detachment solution may be cryopreserved as stem cells. Furthermore, after the cells into which the inducer has been introduced are induced to become stem cells, the whole cells in the suspension culture may be cryopreserved as stem cells.

[0069] The induced stem cells may express undifferentiated cell markers such as Nanog, OCT4, and SOX2. The induced stem cells may express TERT. The induced stem cells may exhibit catabolism activity.

[0070] Furthermore, whether stem cells have been induced from blood cells can be confirmed, for example, by cell morphology. For example, induced stem cells may form flat colonies similar to ES cells and express alkaline phosphatase. Alternatively, whether stem cells have been induced from blood cells may be determined by analyzing using a cytometer whether the cells are positive for at least one surface marker selected from TRA-1-60, TRA-1-81, SSEA-1, and SSEA5, which are cell surface markers indicating undifferentiation. TRA-1-60 is an antigen specific to iPS / ES cells and is not detected in somatic cells. Since iPS cells can be generated only from the TRA-1-60-positive fraction, TRA-1-60-positive cells are considered to be iPS cells.

[0071] The induced stem cells have, for example, a γδ-TCR rearrangement gene. A γδ-TCR rearrangement gene is a gene encoding a TCR in which a rearrangement of the TCRγ region and the TCRδ region has occurred. The TCRγ region contains Vγ-Jγ. The TCRδ region contains Vδ-Dδ-Jδ. The induced stem cells have, for example, a γδ-TCR rearrangement gene having J1 / J2 genes.

[0072] A method for producing blood cells according to an embodiment includes preparing stem cells produced by the above-described method for producing stem cells, and inducing blood cells from the stem cells.

[0073] The method for inducing blood cells from stem cells is not particularly limited. For example, prepared cells are cultured for 4 days in a medium containing a GSK3 inhibitor such as CHIR99021, a bone morphogenetic protein such as BMP-4, and a growth factor such as VEGF. The cells are then cultured for 2 days in a medium containing an ALK5 inhibitor such as SB431542, growth factors such as VEGF and bFGF, and stem cell factor (SCF). The cells are then cultured for 2 days in a medium containing a growth factor such as VEGF, SCF, interleukins such as IL-3 and IL-6, cytokines such as Flt3L, and erythropoietin (EPO). The cells are then cultured in a medium containing SCF, interleukins such as IL-6, and EPO. This induces blood cells.

[0074] Alternatively, blood cells can be induced from stem cells by seeding them on stromal cells (stromal cells). The stromal cells may be derived from bone marrow. The stromal cells may be OP9 cells. OP9 cells do not produce macrophage colony-stimulating factor (M-CSF) and have the function of supporting the differentiation of stem cells into blood cells. For example, a stem cell colony is divided into multiple cell clusters, and the stem cell clusters are seeded on OP9 cells as feeder cells. This allows blood cells to be induced from stem cells. The induced blood cells are, for example, positive for CD34 and CD43.

[0075] The induced blood cells may be γδ T cells.

[0076] Example 1 10 nmol / L (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP, Sigma-Aldrich, registered trademark), 10% fetal bovine serum (Life Technologies), 1.0 × 10 -5 RPMI (Roswell Park Memorial Institute) 1640 medium (Gibco) containing 100 mol / L 2-mercaptoethanol (Nacalai Tesque), 100 U / mL penicillin, and 100 μg / mL streptomycin (Life Technologies) was prepared as an HMBPP-containing medium.

[0077] Approximately 1 × 10 human peripheral blood mononuclear cells were added to the medium. 6 The medium containing monocytes was placed in a 24-well plate (day 1). 1 μL of 20 μg / mL IL-2 was added to the medium every day. On day 3, the medium containing the cells was collected from the plate, centrifuged, and the supernatant was removed. Then, 2 mL of fresh HMBPP-containing medium was added to the cells, and 1 mL of the medium was placed in each of two wells of a 24-well plate.

[0078] On day 6, the medium containing the cells was collected from the plate, centrifuged, and the supernatant was removed. Then, HMBPP-containing medium was added to the cells, and 2 × 10 4 Culture medium containing monocytes was placed in a 96-well plate. KLF4, OCT3 / 4, SOX2, and c-MYC were transfected into the cells using CytoTune-iPS 2.0 (Thermo Fisher Scientific). The multiplicity of infection (MOI) was adjusted to 20–30.

[0079] On day 7, fresh HMBPP-containing medium was added to the medium containing cells recovered from the 96-well plate and placed in a 6-well plate. On days 8, 10, and 12, stem cell medium (Stem Fit, Ajinomoto) was added, and the medium was then replaced with stem cell medium.

[0080] As shown in Figure 2, it was confirmed that multiple colonies of iPS cells had formed on day 14. Photographs of the established iPS cells are shown in Figure 3. Genomic DNA was extracted from the iPS cells and analyzed by PCR and electrophoresis to determine whether they contained a rearranged Vγ9 gene. Genomic DNA from γδ T cells was used as a positive control, and the genome of iPS cells lacking the rearranged Vγ9 gene was used as a negative control. As a result, as shown in Figure 4, it was confirmed that the iPS cells established in Example 1 contained a rearranged Vγ9 gene containing the J1 / J2 gene.

[0081] The established iPS cells were also immunostained with antibodies against pluripotent stem cell markers LIN28 and OCT3 / 4, and the cells were found to be positive for LIN28 and OCT3 / 4, as shown in Figure 5. Furthermore, the established iPS cells were analyzed using a flow cytometer and were found to be positive for TRA-1-60, as shown in Figure 6.

[0082] (Comparative Example 1) iPS cells were induced in the same manner as in Example 1, except that the HMBPP in the HMBPP-containing medium was replaced with 5 μL of zoledronic acid (Zol, Sigma-Aldrich). As shown in Figure 2, no iPS cell colonies were formed in Tests 1 and 3 of Comparative Example 1. In Test 2 of Comparative Example, iPS cell colonies were formed, but the number was significantly smaller than in Test 2 of Example 1. As shown in Figure 4, it was confirmed that the iPS cells established in Comparative Example 1 did not have a rearranged Vγ9 gene carrying the J1 / J2 gene.

[0083] Example 2 The iPS cell colonies prepared in Example 1 were detached from the culture vessel using 0.25% trypsin and 1 mg / mL collagenase IV and divided into multiple cell clumps by pipetting. A culture vessel in which OP9 cells and OP9 / DLL1 cells were cultured as feeder cells was prepared. OP9 cells and OP9 / DLL1 cells were cultured in α-MEM medium supplemented with 20% fetal bovine serum (FBS). Multiple cell clumps composed of iPS cells were seeded onto the feeder cells.

[0084] Figure 7 shows photographs of iPS cells on days 5, 9, and 14 after seeding on feeder cells. The process of differentiation of iPS cells into blood progenitor cells was observed. Figure 8 shows the results of flow cytometry analysis of cells on day 14. The cells were confirmed to be positive for CD34 and CD43, which are blood cell markers.

Claims

1. applying (E)-4-hydroxy-3-methyl-2-butenyl diphosphate to blood cells; deriving stem cells from said blood cells; A method for producing stem cells having a γδ-TCR rearrangement gene carrying the J1 / J2 gene, comprising:

2. The method for producing stem cells according to claim 1 , further comprising administering an interleukin to the blood cells.

3. The method for producing stem cells according to claim 2, wherein the interleukin is at least one selected from the group consisting of IL-2, IL-15, and IL-23.

4. The method for producing stem cells according to claim 1 , wherein the blood cells are mononuclear cells.

5. The method for producing stem cells according to claim 1 , wherein the stem cells are iPS cells.

6. The method for producing stem cells according to claim 1 , wherein in inducing the stem cells from the blood cells, an inducer RNA is introduced into the blood cells.

7. The method for producing stem cells according to claim 1 , wherein a Sendai virus vector is used in inducing the stem cells from the blood cells.

8. The method for producing stem cells according to claim 1, wherein the stem cells comprise a γδ-TCR rearrangement gene.

9. providing stem cells having a γδ-TCR rearrangement gene carrying the J1 / J2 gene, which are produced by the method for producing stem cells according to claim 1; deriving blood cells from the stem cells; A method for producing blood cells, comprising:

10. The method for producing blood cells according to claim 9 , wherein the blood cells are γδ T cells.

11. The method for producing blood cells according to claim 9 , wherein in inducing the blood cells from the stem cells, cell clusters of the stem cells are seeded on feeder cells.

12. The method for producing blood cells according to claim 11 , wherein the feeder cells are stromal cells.

Images