WO2023053994A1 - 幹細胞の製造方法 - Google Patents

幹細胞の製造方法 Download PDF

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WO2023053994A1
WO2023053994A1 PCT/JP2022/034609 JP2022034609W WO2023053994A1 WO 2023053994 A1 WO2023053994 A1 WO 2023053994A1 JP 2022034609 W JP2022034609 W JP 2022034609W WO 2023053994 A1 WO2023053994 A1 WO 2023053994A1
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cells
stem cells
blood
producing
blood cells
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French (fr)
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剛士 田邊
健太 須藤
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I Peace Inc
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I Peace Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to cell technology, and to a method for producing stem cells.
  • 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 brought about by V(D)J gene rearrangements. The VJ gene rearrangement is shown in FIG.
  • DN1 double negative 1 cells, which are progenitor cells of T cells, proliferate in response to interleukin-7 (IL-7) and c-kit ligand (KL), which are strongly expressed in the cortical subcapsular region of the thymus.
  • IL-7 interleukin-7
  • KL c-kit ligand
  • a TCR comprises a variable (V) region and a constant (C) region.
  • V regions are highly diverse and form binding sites with antigens.
  • the V region gene exists in the germline DNA divided into the V gene, D (diversity) gene, and J (joining) gene, and in the process of differentiation into T cells, these become V-(D)-J. It is rearranged in one string on the chromosome and becomes a phenotypic gene.
  • J genes include JP1 gene, JP gene, J1 gene, JP2 gene, and J2 gene.
  • TCR ⁇ type T cells TCR consists of ⁇ and ⁇ chains.
  • TCR ⁇ type T cells TCR consists of ⁇ chain and ⁇ chain. The branching decision to become TCR ⁇ -type T cells or TCR ⁇ -type T cells is controlled by the silencer regions present in the TCR ⁇ locus and the TCR ⁇ locus.
  • TCR ⁇ type T cells are fewer than TCR ⁇ type T cells, but they occupy the majority in the epithelial intercellular lymphocyte population in the intestinal mucosa.
  • TCR ⁇ -type T cells sense various stresses that damage cells and induce immune responses.
  • TCR ⁇ type T cells are said to sense changes in properties associated with canceration of cells in addition to external stresses such as bacterial and viral infections.
  • TCR ⁇ type T cells proliferate and activate after recognizing intermediate products in mevalonate metabolism in the cholesterol synthesis pathway of antigen-presenting cells (APC) and isopentenyl pyrophosphate (IPP) as antigens. Therefore, cancer immunotherapy is performed in which a patient's TCR ⁇ T cells are activated outside the body and returned to the body.
  • APC antigen-presenting cells
  • IPP isopentenyl pyrophosphate
  • Patent Document 1 it has been proposed to induce iPS cells with reconstituted ⁇ -TCR genes by reprogramming blood cells stimulated with zoledronic acid (see Patent Document 1, for example).
  • Patent Document 1 it has been reported that iPS cells induced by this method do not reconstitute the ⁇ -TCR gene having the J1/J2 gene (see, for example, Non-Patent Document 1).
  • One of the objects of the present invention is to provide a method for efficiently inducing stem cells having a reconstituted ⁇ -TCR gene.
  • a method for producing stem cells according to an aspect of the present invention comprises applying phosphorylation to blood cells; deriving stem cells from blood cells.
  • the phosphate may be an intermediate or final product of the non-mevalonate pathway.
  • the phosphate may be (E)-4-hydroxy-3-methyl-2-butenyl diphosphate.
  • the above method for producing stem cells may further include applying interleukin to blood cells.
  • the interleukin may be at least one selected from the group consisting of IL-2, IL-15, and IL-23.
  • the blood cells may be mononuclear cells.
  • the stem cells may be iPS cells.
  • an inducer RNA may be introduced into blood cells in inducing stem cells from blood cells.
  • a Sendai virus vector may be used in inducing stem cells from blood cells.
  • a stealth RNA vector may be used in inducing stem cells from blood cells.
  • the stem cells may comprise a ⁇ -TCR rearrangement gene.
  • a method for producing blood cells according to an aspect of the present invention includes preparing stem cells produced by the method for producing stem cells described above, and inducing blood cells from the stem cells.
  • the blood cells may be ⁇ T cells.
  • cell clusters of stem cells may be seeded on feeder cells.
  • the feeder cells may be interstitial cells (stromal cells).
  • Stromal cells may be derived from bone marrow.
  • the stromal cells may be OP9 cells.
  • FIG. 4 is a schematic diagram showing VJ gene rearrangement. 4 is a graph showing the number of colonies of induced stem cells according to Example 1 and Comparative Example 1.
  • FIG. 1 is a photograph of induced stem cells according to Example 1.
  • FIG. 1 is a photograph of induced stem cells according to Example 1.
  • FIG. 1 is a photograph showing the result of PCR analysis of genomes of induced stem cells according to Example 1 and Comparative Example 1.
  • FIG. 1 is a photograph of immunostained cells according to Example 1.
  • FIG. 1 is a photograph of immunostained cells according to Example 1.
  • FIG. 1 is a photograph of immunostained cells according to Example 1.
  • FIG. 1 is a photograph of immunostained cells according to Example 1.
  • FIG. 1 is a dot plot by a flow cytometer showing the results of Example 1.
  • FIG. 4 is a photograph of cells according to Example 2.
  • FIG. 4 is a photograph of cells according to Example 2.
  • FIG. 4 is a photograph of cells according to Example 2.
  • a method for producing stem cells includes applying phosphorylation to blood cells and inducing stem cells from the blood cells.
  • Stem cells are, for example, induced pluripotent stem cells (iPS cells).
  • Blood cells may be human-derived or non-human animal-derived. Blood cells are separated from the blood. Blood includes, but is not limited to, peripheral blood and umbilical cord blood. Blood may be collected from an adult or from a minor. Anticoagulants such as ethylenediaminetetraacetic acid (EDTA), heparin, and biological standard blood storage solution A (ACD-A) solution may be used during blood collection.
  • EDTA ethylenediaminetetraacetic acid
  • ACD-A biological standard blood storage solution A
  • Blood cells are, for example, nucleated cells such as monocytes, neutrophils, eosinophils, basophils, and lymphocytes, and do not include red blood cells, 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, ⁇ T cells or ⁇ T cells. Blood cells need not be ⁇ T cells.
  • the phosphate may be an intermediate or final product of the non-mevalonate pathway.
  • the non-mevalonate pathway is a synthetic pathway for isopentenyl pyrophosphate (IPP) that does not go through mevalonate.
  • IPP isopentenyl pyrophosphate
  • Examples of non-mevalonate pathway intermediates 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-cyclo pyrophosphate (MEcPP), and (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMB-PP).
  • DOXP 1-de
  • the phosphorylation may be added to the medium for culturing the blood cells.
  • concentration of phosphorylation in the medium is, for example, 1 ⁇ mol/L or more and 50 ⁇ mol/L or less, 3 ⁇ mol/L or more and 20 ⁇ mol/L or less, or 5 ⁇ mol/L or more and 12 ⁇ mol/L or less.
  • the blood cells may be cultured for 1 day or more, 2 days or more, or 3 days or more in the medium supplemented with phosphorylation.
  • Blood cells may be cultured in medium supplemented with phosphate for 14 days or less, 10 days or more, or 7 days or more.
  • Interleukin may also be applied to blood cells.
  • Examples of interleukins include IL-2, IL-15, and IL-23.
  • interleukin When applying interleukin to blood cells, interleukin may be added to the medium for culturing blood cells.
  • concentration of interleukin in the medium is, for example, 5 ng/mL or more and 100 ng/mL or less, 10 ng/mL or more and 90 ng/mL or less, or 15 ng/mL or more and 80 ng/mL or less.
  • Blood cells may be cultured for 1 or more days, 2 or more days, or 3 or more days in a medium supplemented with interleukin.
  • the blood cells may be cultured in medium supplemented with interleukin for 14 days or less, 10 days or more, or 7 days or more.
  • Phosphate and interleukin may be applied simultaneously to blood cells.
  • Phosphate may be applied after applying interleukin to blood cells.
  • Examples of media for culturing blood cells include, but are not limited to, RPMI1640 medium, minimum essential medium ( ⁇ -MEM), Dulbecco's Modified Eagle Medium (DMEM), and F12 medium.
  • an inducer is introduced into the blood cells to which at least phosphorylation has been applied to induce stem cells from the blood cells.
  • induction refers to reprogramming, reprogramming, transformation, cell fate reprogramming, and the like.
  • the inducer introduced into blood cells may be RNA.
  • RNA may be mRNA.
  • Inducers include, for example, OCT3/4, SOX2, KLF4, and c-MYC.
  • OCT3/4 modified M 3 O may be used.
  • the inducer is 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 may be at least one selected from RNAs for these inducers are available from TriLink.
  • Inducer RNAs are pseudouridine ( ⁇ ), 5-methyluridine (5meU), N1-methylpseudouridine (me1 ⁇ ), 5-methoxyuridine (5moU), 5-hydroxymethyluridine (5hmU), 5-formyluridine.
  • 5fU 5-carboxymethyl ester uridine (5camU), thienoguanosine (thG), N4-methylcytidine (me4C), 5-methylcytidine (m5C), 5-methoxytidine (5moC), 5-hydroxymethylcytidine (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) may be modified with at least one selected from the group consisting of .
  • the inducer RNA may be polyadenylated.
  • RNA may be prepared by polyadenylation of in vitro transcribed (IVT) RNA.
  • IVT in vitro transcribed
  • RNA may be polyadenylated during IVT by using a DNA template encoding poly(A) ends.
  • RNA may be capped. To maximize the efficiency of expression in cells, it is preferred that most RNA molecules contain a cap.
  • the RNA may have a 5'cap[m7G(5')ppp(5')G] structure.
  • the sequence is a sequence that stabilizes RNA and promotes transcription. 5'triphosphate may be removed from RNA having 5'triphosphate by dephosphorylation treatment.
  • RNA may have [3'O-Me-m7G(5')ppp(5')G] as Anti-Reverse Cap Analog (ARCA).
  • ARCA is a sequence that is inserted before the start of transcription, doubling the efficiency of the RNA being transcribed.
  • the RNA may have a PolyA tail.
  • the RNA of the inducer may be a replicative RNA having self-proliferation ability.
  • Replicative RNA is RNA having the ability to self-replicate, and unlike normal RNA, it also has the ability to express proteins necessary for RNA replication.
  • the replicative RNA is derived from the Venezuelan equine encephalitis (VEE) virus, an alphavirus.
  • VEE Venezuelan equine encephalitis
  • Replicative RNA sequences include alphavirus replicon RNA, Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, and Western It may comprise a sequence obtained from an alphavirus selected from the group consisting of equine encephalitis virus (WEE).
  • EEE Eastern Equine Encephalitis virus
  • VEE Venezuelan Equine Encephalitis virus
  • Everglades virus a sequence obtained from an alphavirus selected from the group consisting of equine encephalitis virus (WEE).
  • replicative RNA includes Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, and 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. may contain a sequence obtained from
  • Replicative RNA for example, 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) - (VEE 3'UTR and poly A tail) - (optionally selectable marker) - Contains the promoter.
  • RF1-4 described above are factors that induce dedifferentiation of somatic cells into pluripotent cells.
  • RF2-3, RF3-4, and RF4 above are optional.
  • RF1-4 are 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, It may be selected from the group consisting of NR5A2, TBX3, MBD3sh, TH2A, and TH2B.
  • the method of introducing the inducer into blood cells is arbitrary.
  • vectors may be used to introduce inducers into blood cells.
  • the inducer may be introduced into blood cells by lipofection.
  • Sendai virus can be used as a vector.
  • Sendai virus is a virus whose genome is RNA and belongs to the family Paramyxoviridae of the order Mononegavirales.
  • the Sendai virus has an RNA genome and an envelope consisting of a lipid bilayer membrane encapsulating the RNA.
  • CytoTune (registered trademark, Invitrogen) can be used as a Sendai virus loaded with inducer RNA.
  • the Sendai virus vector may be a Sendai virus vector with improved persistence of infection.
  • a Sendai virus vector with improved persistence of infection is also called a stealth RNA vector.
  • SRV iPSC-1 Vector, SRV iPSC-2 Vector, SRV iPSC-3 Vector, and SRV iPSC-4 Vector (registered trademark, Tokiwa Bio Co., Ltd.) can be used as stealth RNA vectors. Details of the stealth RNA vector are described in Japanese Patent No. 4478788, Japanese Patent No. 4936482, Japanese Patent No. 5633075, and Japanese Patent No. 5963309.
  • the index of Sendai virus titer includes the multiplicity of infection (MOI).
  • the MOI of Sendai virus is, for example, 0.1 to 100.0, or 1.0 to 50.0.
  • An inducer may be introduced into blood cells that have been adherently cultured, or an inducer may be introduced into blood cells that have been suspension-cultured in a gel medium.
  • 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).
  • a basement membrane matrix such as Matrigel (Corning), CELLstart (registered trademark, ThermoFisher), or Laminin511 (iMatrix-511, nippi).
  • a stem cell medium such as a human ES/iPS medium such as Stemfit (Ajinomoto) can be used.
  • the stem cell medium is not limited to this, and various stem cell medium can be used.
  • Primate ES Cell Medium, mTeSR1, and TeSR2 (STEM CELL Technologies) may be used.
  • a stem cell culture medium is placed, for example, in a dish, well, tube, or the like.
  • the gel medium does not contain growth factors such as basic fibroblast growth factor (bFGF).
  • the gel medium contains growth factors such as bFGF at low concentrations of 400 ⁇ g/L or less, 40 ⁇ g/L or less, or 10 ⁇ g/L or less.
  • the gel medium does not contain TGF- ⁇ , or contains TGF- ⁇ at a low concentration of 600 ng/L or less, 300 ng/L or less, or 100 ng/L or less.
  • the gel medium does not have to be stirred. Also, the gel medium may not contain feeder cells.
  • the gel medium may contain at least one substance selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin.
  • the cells After introducing the inducer into the blood cells, the cells may be initialized in a liquid medium other than the gel medium, or the cells may be initialized in the gel medium.
  • the cells introduced with the inducer are collected, and at least one passaging is performed by seeding at least a portion of the collected and mixed cells in a culture medium.
  • clones of inducer-introduced cells may be mixed.
  • different clones of inducer-introduced cells may be mixed.
  • the cells into which the inducer has been introduced may be recovered, and at least a portion of the recovered and mixed cells may be seeded in a culture medium for subculturing, which may be performed multiple times.
  • the inducer-introduced cells may be harvested, and at least a portion of the harvested mixed cells may be seeded in culture medium and passaged until stem cells are established. In addition, all of the collected mixed cells may be seeded in the medium.
  • recovering the inducer-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passage means, for example, that the inducer-introduced cells are subjected to gene expression state It means to pass without distinction.
  • cells introduced with an inducer may be seeded in the same culture vessel without being differentiated by gene expression state.
  • recovering the inducer-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passaging means, for example, that the inducer-introduced cells are subjected to the degree of reprogramming. It means to pass without distinction.
  • at passaging cells introduced with an inducer may be seeded in the same culture vessel without being differentiated by the degree of reprogramming.
  • recovering the inducer-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passage means, for example, distinguishing the inducer-introduced cells by morphology It means to pass without For example, at passaging, the inducer-introduced cells may be seeded in the same culture vessel without being differentiated by morphology.
  • recovering the inducer-introduced cells and seeding at least a portion of the recovered and mixed cells in a culture medium for passage means, for example, size-discriminating the inducer-introduced cells. It means to pass without For example, at passaging, the inducer-introduced cells may be seeded in the same culture vessel without size discrimination.
  • recovering the inducer-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passage means passaging without cloning the inducer-introduced cells. It means to For example, when passage without cloning, it is not necessary to pick up colonies formed by cells into which the inducer has been introduced. For example, when passaging without cloning, multiple colonies formed by cells into which an inducer has been introduced may not be separated from each other. For example, during passaging, cells forming different colonies may be mixed and seeded in the same culture vessel. Also, for example, when passage without cloning, it is not necessary to clone a single colony formed by cells into which an inducer has been introduced. For example, at passaging colonies may be mixed and seeded in the same culture vessel.
  • the adherently cultured cells may be collected, and at least a portion of the collected and mixed cells may be seeded in a medium for passage.
  • cells may be detached from a culture vessel and at least a portion of the detached and mixed cells may be seeded into the same culture vessel.
  • the cells may be detached from the incubator with a detachment solution, and the detached and mixed whole cells may be passaged.
  • non-colony forming cells may be passaged.
  • the cells When passaging the inducer-introduced cells, the cells may be seeded in a medium or incubator at a low concentration.
  • the low concentration is, for example, 1 cell/cm 2 or more, 0.25 ⁇ 10 4 cells/cm 2 or less, 1.25 ⁇ 10 3 cells/cm 2 or less, 0.25 ⁇ 10 3 cells/cm 2 or less. cm 2 or less, 0.25 ⁇ 10 2 cells/cm 2 or less, or 0.25 ⁇ 10 1 cells/cm 2 or less.
  • low concentration means that 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. It is a concentration at which 11 or more cells do not come into contact with each other.
  • the state in which the entire bottom surface of the cell container is covered with cells is 100% confluent, and low concentration means 5% or less confluent, 4% or less confluent, 3% or less confluent, 2% or less confluent, 1% or less confluent, 0 0.5% confluent, 0.1% confluent, 0.05% confluent, or 0.01% confluent.
  • the low concentration is, for example, a concentration at which single cells do not contact each other in seeded cells.
  • wells of a well plate may be seeded with single cells.
  • a well plate may be a 12-well plate or a 96-well plate.
  • the proportion of cells in which Sendai virus remains is, for example, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0%.
  • Cells into which an inducer has been introduced may be cultured and passaged in a closed incubator.
  • a closed incubator does not exchange, for example, gases, viruses, microorganisms and impurities with the outside.
  • the cells into which the inducer has been introduced may be expanded by two-dimensional culture, or expanded by three-dimensional culture.
  • the whole adherent cultured cells may be cryopreserved as stem cells.
  • whole cells detached from the incubator with a detachment solution may be cryopreserved as stem cells.
  • the whole cells in suspension culture may be cryopreserved as stem cells.
  • Induced stem cells can express undifferentiated cell markers such as Nanog, OCT4, and SOX2. Induced stem cells can express TERT. Induced stem cells may exhibit temoutherase activity.
  • stem cells have been induced from blood cells can be confirmed, for example, from the morphology of the cells.
  • induced stem cells can form flat-shouldered colonies similar to ES cells and express alkaline phosphatase.
  • whether or not stem cells were induced from blood cells can be determined by a cytoflow meter from cell surface markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA5, which indicate undifferentiation. This may be done by analyzing whether at least one selected surface marker is positive.
  • TRA-1-60 is an iPS/ES cell specific antigen and is not detected in somatic cells. Since iPS cells can be generated only from the TRA-1-60 positive fraction, the TRA-1-60 positive cells are considered to be iPS cells.
  • Induced stem cells for example, have a ⁇ -TCR rearrangement gene.
  • a ⁇ -TCR rearranged gene is a TCR-encoding gene in which the TCR ⁇ region and the TCR ⁇ region have been rearranged.
  • the TCR ⁇ region includes V ⁇ -J ⁇ .
  • the TCR delta region includes V delta - D delta - J delta.
  • Induced stem cells are equipped with the ⁇ -TCR rearrangement genes, eg with the J1/J2 genes.
  • a method for producing blood cells includes preparing stem cells produced by the method for producing stem cells described above, and inducing blood cells from the stem cells.
  • the method of inducing blood cells from stem cells is not particularly limited.
  • the 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 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 cultured for 2 days in a medium containing growth factors such as VEGF, interleukins such as SCF, IL-3 and IL-6, cytokines such as Flt3L, and erythropoietin (EPO).
  • the cells are cultured in a medium containing SCF, interleukin such as IL-6, and EPO. This induces blood cells.
  • stem cells may be seeded on stromal cells and blood cells may be induced from the stem cells.
  • Stromal cells may be derived from bone marrow.
  • the stromal cells may be OP9 cells.
  • OP9 cells do not produce macrophage-stimulating factor (M-CSF) and function to support the differentiation of stem cells into blood cells.
  • M-CSF macrophage-stimulating factor
  • a stem cell colony is divided into a plurality of cell clumps, and the stem cell clumps are seeded on OP9 cells as feeder cells.
  • Blood cells are thereby induced from the stem cells. Blood cells that are induced are, for example, positive for CD34 and CD43.
  • the induced blood cells may be ⁇ type T cells.
  • Example 1 10 nmol/L (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP, Sigma-Aldrich®), 10% fetal bovine serum (Life Technologies), 1.0 ⁇ 10 ⁇ RPMI (Roswell Park Memorial Institute) 1640 medium (Gibco) containing 5 mol/L 2-mercaptoethanol (Nacalai Tesque), 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin (Life Technologies) as HMBPP-containing medium prepared.
  • HMBPP E-4-hydroxy-3-methyl-2-butenyl diphosphate
  • Human peripheral blood mononuclear cells were added to the medium, and the medium containing about 1 ⁇ 10 6 mononuclear cells was placed in a 24-well plate (Day 1). Medium was supplemented with 1 ⁇ L of 20 ⁇ g/mL IL-2 daily. Medium with cells was harvested from the plates on day 3, the medium was centrifuged and the supernatant removed. After that, 2 mL of fresh HMBPP-containing medium was added to the cells and 1 mL was added to 2 wells of a 24-well plate.
  • Fresh HMBPP-containing medium was added to the medium containing the cells collected from the 96-well plate on day 7 and placed in a 6-well plate. On the 8th, 10th, and 12th days, a stem cell medium (Stem Fit, Ajinomoto) was added, and then the medium was replaced with the stem cell medium.
  • a stem cell medium Ste Fit, Ajinomoto
  • FIG. 2 A photograph of the established iPS cells is shown in FIG.
  • genomic DNA was extracted from the iPS cells and analyzed by PCR and electrophoresis to determine whether or not they contained the reconstructed V ⁇ 9 gene.
  • Genomic DNA of ⁇ T cells was analyzed as a positive control, and the genome of iPS cells without the reconstructed V ⁇ 9 gene was similarly analyzed as a negative control.
  • FIG. 4 the iPS cells established in Example 1 were confirmed to have rearranged V ⁇ 9 genes with J1/J2 genes.
  • the established iPS cells were immunostained with antibodies against LIN28 and OCT3/4, which are pluripotent stem cell markers, the cells were positive for LIN28 and OCT3/4, as shown in FIG. Furthermore, when the established iPS cells were analyzed with a flow cytometer, they were TRA-1-60 positive as shown in FIG.
  • Comparative example 1 iPS cells were induced in the same manner as in Example 1, except that 5 ⁇ L of zoledronic acid (Zol, Sigma-Aldrich) was used instead of HMBPP in the HMBPP-containing medium. As shown in FIG. 2, in Tests 1 and 3 of Comparative Example 1, iPS cell colonies were not formed. In Test 2 of the comparative example, colonies of iPS cells were formed, but significantly less than in Test 2 of Example 1. As shown in FIG. 4, it was confirmed that the iPS cells established in Comparative Example 1 did not have the rearranged V ⁇ 9 gene with the J1/J2 gene.
  • zoledronic acid Zol, Sigma-Aldrich
  • Example 2 The iPS cell colonies prepared in Example 1 were detached from the culture vessel with 0.25% trypsin and 1 mg/mL collagenase IV, and divided into multiple cell clusters by pipetting. A culture vessel in which OP9 cells and OP9/DLL1 cells were cultured as feeder cells was prepared. OP9 and OP9/DLL1 cells were cultured in ⁇ -MEM medium supplemented with 20% fetal bovine serum (FBS). A plurality of cell clusters each consisting of iPS cells were seeded on the feeder cells.
  • FBS fetal bovine serum
  • FIG. 7 shows photographs of the iPS cells 5 days, 9 days, and 14 days after seeding the iPS cells on the feeder cells.
  • the iPS cells were observed to differentiate into blood progenitor cells.
  • FIG. 8 shows the results of analyzing the cells on the 14th day by flow cytometry. The cells were confirmed to be positive for blood cell markers CD34 and CD43.

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WO2018143243A1 (ja) * 2017-02-03 2018-08-09 国立大学法人神戸大学 人工多能性幹細胞の作製方法
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