WO2021090594A1 - Neutrophil progenitor cells and method for producing same - Google Patents

Neutrophil progenitor cells and method for producing same Download PDF

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WO2021090594A1
WO2021090594A1 PCT/JP2020/036094 JP2020036094W WO2021090594A1 WO 2021090594 A1 WO2021090594 A1 WO 2021090594A1 JP 2020036094 W JP2020036094 W JP 2020036094W WO 2021090594 A1 WO2021090594 A1 WO 2021090594A1
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gene
cells
neutrophil
cell
expression
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Japanese (ja)
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将 宮内
峰夫 黒川
雄介 伊藤
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国立大学法人 東京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4648—Bacterial antigens
    • A61K39/464821—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • Cancer patients suffer from bone marrow damage and a decrease in white blood cells, especially neutrophils, due to cancer treatment with anticancer drugs and / or radiation therapy and pretreatment for bone marrow transplantation. Loss of neutrophils in cancer patients can severely damage the immune system, causing fever and rapidly fatal infections. Antibacterial agents, steroids, and antipyretics are administered to patients with fever due to neutropenia, and granulocyte colony-stimulating factors, bone marrow growth factors, etc. are administered for the purpose of proliferating neutrophils. To. However, in subjects whose neutrophils are already depleted, the reactivity of neutrophil proliferation is poor and it may not be possible to generate sufficient neutrophils.
  • GTX Granulocyte Transfusion Therapy
  • Non-Patent Document 5 Stem Cells (2016), vol.34, p.1513-1526.
  • Non-Patent Document 6 Stem Cells Translational Medicine (2019) vol. 8, 557-567). Further, a method for obtaining neutrophils by introducing ETV2 mM mRNA to induce bone marrow endothelial progenitor cells and differentiating them is also disclosed.
  • Non-Patent Document 7 Stem Cell Reports vol 13, 1099-1110).
  • the present inventors improved proliferativeness by forcibly expressing at least one gene belonging to the BCL2 family in stem cell-derived hematopoietic progenitor cells in which the c-Myc gene and the BMI1 gene were forcibly expressed.
  • neutrophil progenitor cells can be obtained. It was also found that the neutrophil progenitor cells thus obtained rapidly differentiate into neutrophil-like cells by suppressing or stopping the expression of the forcibly expressed gene. Furthermore, it was confirmed that the neutrophil-like cells thus obtained can exert a function as neutrophils. Therefore, the present invention relates to the following:
  • [1] has the ability to grow beyond 12 weeks after induction of differentiation from stem cells, 10 13 or more is capable of growing to cell number, with the ability to differentiate into neutrophil-like cells, stem cell-derived neutrophil Progenitor cells.
  • the neutrophil progenitor cell according to item 1 wherein the stem cell is an induced pluripotent stem cell or a hematopoietic stem cell.
  • the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family are forcibly expressed.
  • Neutrophil progenitor cells [14] The neutrophil progenitor cell according to any one of items 1 to 13, wherein the neutrophil-like cell has migration properties equivalent to those of human isolated neutrophils when measured by a migration assay. ..
  • At least one of the neutrophil-like cells selected from the group consisting of LFA-1 integrin (CD11a / CD18), integrin ⁇ -M (CD11b), CXCR1, FPR1, and L-selectin (CD62L).
  • the neutrophil progenitor cell according to any one of items 1 to 14, which expresses an adhesion factor.
  • Stem cell-derived neutrophils comprising an expression vector capable of expressing DNA encoding at least one gene selected from the group consisting of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family. Like cells.
  • the neutrophil precursor cells after induction of differentiation from stem cells, have the ability to grow beyond 12 weeks, it can be grown to 10 13 or more in the number of cells, one of the items 21-25 one The manufacturing method described in the section.
  • the production method according to item 27, wherein at least one gene belonging to the BCL2 family is BCL-XL.
  • the production method according to item 28 which comprises the step of culturing in the absence of feeder cells.
  • Neutrophil progenitor cells produced by the production method according to any one of items 21 to 29.
  • a method of treating a subject suffering from a disease caused by neutropenia, neutrophil dysfunction, or immunodeficiency comprising the step of administering the neutrophil-like cell according to any one of items 16 to 21 and 31 to the subject.
  • a research composition comprising the neutrophil-like cell according to any one of items 16 to 21 and 31.
  • the neutrophil according to any one of items 16 to 21, and 31 for use in the treatment or prevention of diseases caused by neutropenia, neutrophil dysfunction, or immunodeficiency. Neutrophils.
  • the present invention provides highly proliferative neutrophil progenitor cells.
  • a sufficient number of neutrophil-like cells can be provided from neutrophil progenitor cells for treatment, experiments, etc., and such neutrophil-like cells have the same functions and properties as living neutrophils. Can be used for treatment and experiments.
  • FIG. 1A shows an experimental design for cytokine addition schedule and gene transfer timing. After the introduction of the c-Myc and BMI1 genes, doxycycline is added until differentiation into NeuCs-XL is initiated, during which time the introduced genes are forcibly expressed. When initiating differentiation, the medium is replaced with a doxycycline-free medium, which suppresses or arrests the expression of the transgene.
  • FIG. 1B shows an example of an alternative cytokine addition schedule.
  • FIG. 2A shows the number of cells when BCL2A1, BCL-XL, and MCL1 were introduced as BCL2 family genes after culturing NeuPs into which c-Myc and BMI1 were introduced for up to 10 weeks in the presence of feeder cells. Show change.
  • FIG. 1A shows an experimental design for cytokine addition schedule and gene transfer timing. After the introduction of the c-Myc and BMI1 genes, doxycycline is added until differentiation into NeuCs-XL is initiated, during which time the
  • FIG. 2B shows the case where NeuPs into which c-Myc and BMI1 have been transgenic are cultured in the presence of feeder cells for up to 10 weeks, then BCL2A1, BCL-XL, and MCL1 are introduced and cultured in the absence of feeder cells. It shows the change in the number of cells in.
  • FIG. 2C shows a longer-term proliferative property when BCL-XL is expressed as a BCL2 family gene 10 to 17 days after the gene transfer of c-Myc and BMI1.
  • FIG. 2D shows a proliferation test of NeuPs derived from hematopoietic stem progenitor cells of cord blood into which BCL-XL was transgenic 10 to 17 days after transfection of c-Myc and BMI1.
  • FIG. 2E shows changes in the number of cells of NeuPs derived from hematopoietic stem progenitor cells of cord blood when cultured in the absence of doxycycline to induce differentiation.
  • FIG. 2F shows NeuPs derived from hematopoietic stem progenitor cells of cord blood, NeuCs obtained by culturing in the absence of doxycycline, cord blood CD34-positive cells, granulocytic surface antigen CD33, and erythrocytic surface. The results of analysis of the antigen CD235A by flow cytometry are shown.
  • FIG. 3A shows intracellular neutrophil precursor transgenes before replacement with doxycycline-free medium (Dox-on), 2 days after replacement (Dox-off day2), and 4th day (Dox-off day4). Shows changes in protein content.
  • FIG. 3B shows changes in the expression of cell surface markers of neutrophil precursors before and after replacement with a doxycycline-free medium (Dox-on) and after replacement (Dox-off day 4).
  • FIG. 3C shows changes in cell and nucleus morphology before replacement with doxycycline-free medium (Dox-on), after replacement (Dox-off day4), and after replacement with LPS (Dox-off day4 + LPS 300 ng / ml). .. FIG. FIG.
  • FIG. 4A shows the change in the gene expression profile when differentiating from NeuPs-XL to NeuCs-XL, and the gene ontology function enrichment analysis is performed to show the most prominent top 30 GO categories.
  • FIG. 4B shows the changes in the gene expression profile before and after LPS stimulation in NeuCs-XL, and the gene ontology function enrichment analysis is performed to show the most prominent top 30 GO categories.
  • FIG. 4C shows the results of principal component analysis of changes in gene expression profiles between NeuPs-XL, LPS-unstimulated NeuCs-XL, and LPS-stimulated NeuCs-XL.
  • FIG. 4D shows gene set enrichment analysis of the NF ⁇ B pathway before and after LPS stimulation in NeuCs-XL.
  • FIG. 5 shows the genes for inflammatory cytokines (IL-1A, IL-1B, CXCL2, IL-8) in NeuCs-XL without LPS stimulation ((LPS (-)) or with stimulation ((LPS (+))). The result of measuring the expression change by quantitative PCR is shown.
  • FIG. 6 shows the intracellular NF ⁇ B and MAPK pathway factors (NF ⁇ B, I ⁇ B ⁇ , p38) before LPS stimulation ((LPS (-)) and 15 minutes, 30 minutes, and 60 minutes after stimulation in NeuCs-XL. Changes in the amount of ERK) protein and the amount of phosphorylated protein (p-) are shown by Western blot.
  • FIG. 1 shows the genes for inflammatory cytokines (IL-1A, IL-1B, CXCL2, IL-8 in NeuCs-XL without LPS stimulation ((LPS (-)) or with stimulation ((LPS (+))). The result of measuring the expression change by quantitative PCR is shown.
  • FIG. 6 shows the
  • FIG. 8 shows the results of a migration assay performed on NeuCs-XL. Dose-dependent FBS when saline (PBS), fetal bovine serum (FBS 0.1%), fetal bovine serum (FBS 1%), or fetal bovine serum (FBS 10%) is added to the lower chamber. It is shown that the migratory activity is increased.
  • PBS saline
  • FBS 0.1%) fetal bovine serum
  • FBS 1% fetal bovine serum
  • FBS 10% fetal bovine serum
  • FIGS. 10D to 10F show graphs quantifying the phagocytic action of NeuPs-XL and NeuCs-XL (D: Staphylococcus aureus (S. aureus).
  • FIG. 11A shows the results of mixing NeuPs-XL, NeuCs-XL, or human peripheral blood neutrophils with FITC-labeled and heat-inactivated E. coli suspension, and detecting E. coli-phagocytotic cells by flow cytometry. Is shown.
  • FIG. 11B shows a graph in which the flow cytometry results of the phagocytosis assay of FIG. 11A are quantified and compared.
  • FIG. 11A shows the results of mixing NeuPs-XL, NeuCs-XL, or human peripheral blood neutrophils with FITC-labeled and heat-inactivated E. coli suspension, and detecting E. coli-phagocytotic cells by flow cytometry. Is shown.
  • FIG. 11B shows a graph in which the flow cytometry results of the phagocytosis assay of FIG. 11A are quantified and compared.
  • FIG. 12A shows the results of an oxidative burst assay in NeuPs-XL, NeuCs-XL, or human peripheral blood neutrophils, showing the fluorescence of dihydrorhodamine in the presence or absence of PMA.
  • FIG. 12B shows a graph comparing the assay results of FIG. 12A quantified.
  • FIG. 13A shows an experimental design of in vivo imaging after low dose (2.0 ⁇ 10 5) administration of NeuCs. In this experimental design, Luc2-expressing NeuCs-XL is administered to NSG mice irradiated with radiation (2.0 Gy) after LPS stimulation.
  • FIG. 13B is a graph showing an increase in white blood cell count in NSG mice (LPS) intraperitoneally administered with LPS.
  • FIG. 13C shows an intraperitoneal injection of 2.0 ⁇ 10 5 luc2-expressing NeuCs-XL in NSG mice without LPS stimulation (LPS ( ⁇ )) and with LPS stimulation (LPSi.p.) 1 hour later.
  • LPS LPS stimulation
  • FIG. 14A shows an experimental design of in vivo imaging after administration of high doses (1.0 ⁇ 10 7 cells (corresponding to 5.0 ⁇ 10 8 cells / kg)) of NeuCs.
  • FIG. 14B shows the results of in vivo imaging taken 1 hour and 48 hours after administration of NeuCs.
  • FIG. 14A shows an experimental design of in vivo imaging after administration of high doses (1.0 ⁇ 10 7 cells (corresponding to 5.0 ⁇ 10 8 cells / kg)) of NeuCs.
  • FIG. 14B shows the results of in vivo imaging taken 1 hour and 48 hours after administration of NeuCs.
  • FIG. 14C shows the results of flow cytometry analysis of mouse CD45-positive cells and human CD45-positive cells in mouse blood collected 1 hour and 48 hours after administration of NeuCs, respectively.
  • FIG. 14D shows the number of human CD45 + cells in peripheral blood (PB) collected 1 hour and 48 hours after administration of NeuCs.
  • FIG. 15A shows an experimental design of in vivo imaging after NeuCs administration in a biofilm chronic infection model.
  • FIG. 15B shows a photograph of a biofilm (bacterial adherent catheter segment) implanted subcutaneously.
  • FIG. 15C shows the results of in vivo imaging in which 1 ⁇ 10 7 luc2-expressing NeuCs-XL were intravenously injected into mice 7 days after biofilm transplantation and imaged 5 minutes, 30 minutes, 60 minutes, and 120 minutes later. Is shown.
  • FIG. 16A shows an experimental design for a survival test after administration of NeuCs-XL in an acute lethal peritonitis model.
  • FIG. 16B shows the results of a survival test after administration of NeuCs-XL in a model of acute lethal peritonitis.
  • FIG. 16C is a graph showing the number of bacteria in the abdominal cavity after administration of NeuCs-XL in an acute lethal peritonitis model.
  • neutrophil progenitor cells are cells having self-proliferation and differentiation into neutrophils or neutrophil-like cells.
  • neutrophil progenitor cells are produced from stem cells, particularly induced pluripotent stem cells, embryonic stem cells, etc. via hematopoietic progenitor cells, or from living body-derived hematopoietic stem cells or hematopoietic progenitor cells.
  • differentiation is induced by culturing stem cells such as induced pluripotent stem cells in the presence of vascular endothelial growth factor (VEGF) to obtain hematopoietic progenitor cells (HPC).
  • stem cells such as induced pluripotent stem cells in the presence of vascular endothelial growth factor (VEGF) to obtain hematopoietic progenitor cells (HPC).
  • VEGF vascular endothelial growth factor
  • neutrophil progenitor cells can be obtained by forcibly expressing the c-Myc gene and the BMI1 gene in HPC.
  • neutrophil progenitor cells (NeuPs) can be obtained by forcibly expressing the c-Myc gene and the BMI1 gene in living body-derived hematopoietic stem cells and / or hematopoietic progenitor cells (hematopoietic progenitor cells). ..
  • NeuPs can grow up to 12 weeks, and up to 10 12 can grow by growing up to 12 weeks.
  • BCL2-family gene in NeuPs it is possible to proliferate over 12 weeks, preferably over 13 weeks, more preferably over 15 weeks, and even more preferably over 17 weeks.
  • Sphere progenitor cells were obtained.
  • the number of neutrophil progenitor cells for which the BCL-family gene was forcibly expressed was 10 13 or more, 10 15 or more, preferably 10 18 or more, 10 20 or more, 10 23 or more, and more preferably 10. It is possible to grow up to 25 cells.
  • neutrophil progenitor cells rapidly differentiate into neutrophil-like cells (NeuCs) by stopping or suppressing the expression of the forcibly expressed gene.
  • NeuPs-XL neutrophil progenitor cells obtained by forcibly expressing BCL-XL
  • neutrophil-like cells obtained by further differentiation are particularly designated as NeuPs-XL. Let it be NeuCs-XL.
  • neutrophil-like cells refer to cells that differentiate from hematopoietic progenitor cells or neutrophil progenitor cells and exert some or all of the functions and properties of neutrophils.
  • the hematopoietic progenitor cells are cultured in the presence of G-CSF to differentiate into neutrophil-like cells on days 9 to 14.
  • neutrophil progenitor cells in which the c-Myc gene and the BMI1 gene are forcibly expressed are differentiated into neutrophil-like cells within 4 days by stopping or suppressing the expression of these genes.
  • the function of neutrophils is to suppress infectious diseases. More specifically, neutrophils accumulate at the site of infection and have a bactericidal ability against bacteria and fungi. When the functions of neutrophils are further classified, neutrophil-like cells exert at least one function selected from the group consisting of adhesive ability, migration ability, phagocytosis ability, and bactericidal ability.
  • Neutrophil-like cells express cell markers similar to neutrophils isolated in vivo. Examples of such a cell marker include CD16b, CD66b and the like.
  • Induced pluripotent stem cells in the present invention refer to stem cells produced by reprogramming somatic cells.
  • the type of somatic cell used here is not particularly limited, and any somatic cell can be used.
  • somatic cells such as bone marrow-derived CD34 + cells, cord blood-derived cells, and cutaneous fibroblasts can be used to establish induced pluripotent stem cells.
  • somatic cells such as bone marrow-derived CD34 + cells, cord blood-derived cells, and cutaneous fibroblasts can be used to establish induced pluripotent stem cells.
  • the neutrophil-like cells produced by the method of the present invention are administered to a patient suffering from a disease related to neutropenia, neutrophil dysfunction, or immunodeficiency, the patient suffering from the disease is treated. It is preferable to use somatic cells isolated from themselves.
  • the following gene combinations can be used: (I) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40largeT gene (iv) ) Oct gene, Klf gene, Sox gene Particularly preferably, Oct3 / 4, KLF4, SOX2, c-Myc can be used as the reprogramming gene.
  • Hematopoietic stem cells are cells that are self-proliferating and differentiate into all blood cells. Hematopoietic stem cells differentiate into hematopoietic progenitor cells, especially lymphocytic progenitor cells and myeloid progenitor cells. Lymphocyte progenitor cells differentiate into T lymphocytes and B lymphocytes. Myeloid progenitor cells differentiate into granulocytes, monocytes, macrophages, erythrocytes, platelets and the like. Hematopoietic stem cells are present in the bone marrow in vivo and also in cord blood.
  • Hematopoietic stem cells can be isolated as CD34-positive cells from bone marrow or cord blood, for example.
  • Cells isolated as CD34-positive cells from bone marrow or umbilical cord blood contain both hematopoietic stem cells and hematopoietic progenitor cells, and thus can also be referred to as hematopoietic stem progenitor cells.
  • the present invention relates to stem cell-derived neutrophil progenitor cells, or living hematopoietic stem cells and / or neutrophil-like cells derived from hematopoietic progenitor cells.
  • Neutrophil progenitor cells are cells that are self-proliferating and capable of differentiating into neutrophil-like cells. More specifically, neutrophil progenitor cells of the present invention have the ability to grow beyond 12 weeks after induction of differentiation to hematopoietic progenitor cells, it can be grown to 10 13 or more in the number of cells.
  • gene expression can be controlled by using a gene expression vector designed to remove or disrupt the gene expression vector introduced under specific conditions.
  • the type of expression vector is not particularly limited, and may be a viral vector or a plasmid vector.
  • a viral vector can also be used in which the introduced gene is integrated into the chromosome of the cell.
  • the virus vector that can be used in the present invention include a retrovirus vector (including a lentivirus vector), an adenovirus vector, and an adeno-associated virus vector.
  • the gene introduced in the present invention may be loaded on one expression vector, or may be loaded separately on two or three expression vectors.
  • the stem cell-derived neutrophil progenitor cell according to the present invention or the neutrophil-like cell differentiated from the neutrophil progenitor cell, has a c-Myc gene, a BMI1 gene, and at least one gene belonging to the BCL2 family.
  • the neutrophil progenitor cell or neutrophil-like cell according to the present invention is selected from the group consisting of at least one gene belonging to the c-Myc gene, the BMI1 gene, and the BCL2 family, or at least 1, at least 2, or.
  • the BCL2 family gene is a gene family responsible for suppressing or inducing apoptosis, and BCL2, BAX, BAK, BCL2A1, BCL-XL, MCL1 and the like are known. At least one gene belonging to the BCL2 family gene can also be simply referred to as a BCL2 family gene.
  • BCL2 has been identified as a protein involved in the chromosomal translocation of follicular lymphoma.
  • the BCL2 family which has an apoptosis-promoting effect, is localized in the outer mitochondrial membrane and becomes a signal of the apoptosis cascade by increasing the permeability of the membrane.
  • the BCL2 family having an apoptosis-suppressing action is considered to inhibit the action of the BCL2 family having an apoptosis-promoting action.
  • the anti-apoptotic BCL2 family include BCL2A1 (NM_004049), BCL-XL (NM_001317919), and MCL1 (NM_021960), and when these genes are expressed in neutrophil progenitor cells, they exert an anti-apoptotic effect. It is considered.
  • the BMI1 gene (NM_005180) is one of the proteins that form the polycomb complex 1, and is also called the polycomb group ring finger protein (PCGF4) or ring finger 0 protein 51 (RNF51). It is known to regulate cell cycle inhibitory genes p16 and p19, and has been reported as an oncogene.
  • the invention also relates to a method for producing neutrophil progenitor cells. Specifically, the following steps: A step of culturing a hematopoietic progenitor cell derived from a stem cell or a hematopoietic stem cell and / or a hematopoietic progenitor cell derived from a living body under forced expression of the c-Myc gene and the BMI1 gene. Includes the step of culturing under forced expression of at least one gene belonging to the BCL2 family.
  • Stem cell-derived hematopoietic progenitor cells can be obtained by culturing stem cells, especially iPS cells, in the presence of VEGF to obtain hematopoietic progenitor cells. Therefore, the method for producing neutrophil progenitor cells may include a step of culturing stem cells in the presence of VEGF to obtain hematopoietic progenitor cells. More specifically, stem cells can be cultured in VEGF-added medium for 10 to 20 days, preferably 12 days to about 2 weeks, and CD34 + / CD43 + cells can be separated as hematopoietic progenitor cells. From the viewpoint of differentiating into neutrophil-like cells in the future, myelocyte hematopoietic progenitor cells are preferable.
  • the VEGF-added medium is a known blood cell culture medium to which VEGF has been added.
  • Living body-derived hematopoietic stem cells or hematopoietic progenitor cells mainly refer to umbilical cord blood or bone marrow-derived hematopoietic stem cells and / or hematopoietic progenitor cells. These cells can be obtained by isolation from cord blood or bone marrow as CD34-positive cells.
  • Hematopoietic stem cells and hematopoietic progenitor cells may be distinguished or may be referred to as "hematopoietic stem progenitor cells" without distinction.
  • hematopoietic stem progenitor cells By forcibly expressing the c-Myc gene and the BMI gene in hematopoietic stem cells and hematopoietic progenitor cells, both are differentiated into neutrophil progenitor cells (NeuPs).
  • NeuPs neutrophil progenitor cells
  • any culture medium well known in the art can be used.
  • Cytokines that promote blood cell differentiation can be added to the blood cell culture medium.
  • at least one cytokine selected from the group consisting of SCF, TPO, FLT3-L, and GCSF can be used on an appropriate schedule.
  • Cytokine addition schedules can be appropriately selected by those skilled in the art so as to maintain the high proliferative capacity of neutrophil progenitor cells. Examples of cytokine addition schedules are shown in FIGS. 1A and 1B.
  • the cells are co-cultured with feeder cells to maintain, proliferate and promote differentiation.
  • the feeder cell used any feeder cell known in the art can be used.
  • the step of forcibly expressing the c-Myc gene and the BMI1 gene in the hematopoietic progenitor cells and / or the hematopoietic stem cells is performed by introducing a vector for expressing the c-Myc and BMI1 genes.
  • c-Myc is infected with a lentivirus equipped with a gene expression vector in which the c-Myc and BMI1 genes are operably arranged under the control of the Tet-on / off system, and cultured in the presence of doxycycline.
  • the gene and the BMI1 gene can be forcibly expressed.
  • the neutrophil progenitor cells differentiated by forced expression of the c-Myc gene and BMI1 gene can be cultured up to 12 weeks after gene transfer, but by further forced expression of the BCL2 family gene, they can proliferate after 12 weeks. It has the ability to grow to a cell number of 10 13 or more, preferably 10 15 or more, preferably 10 18 or more, 10 20 or more, 10 23 or more, and more preferably 10 25.
  • the obtained neutrophil progenitor cells can proliferate independently of feeder cells.
  • Neutrophil progenitor cells capable of feeder cell-independent proliferation are preferable from the viewpoint of quality control.
  • neutrophil-like cells differentiated from neutrophil progenitor cells are administered for therapeutic purposes, it is more preferable from the viewpoint of avoiding contamination with feeder cells. Therefore, from the viewpoint of enabling feeder cell-independent proliferation, it is preferable to use BCL-XL as the BCL2 family gene in the present invention.
  • the method for producing neutrophil-like cells of the present invention specifically describes the following steps: A step of culturing a hematopoietic progenitor cell derived from a stem cell or a hematopoietic stem cell and / or a hematopoietic progenitor cell derived from a living body under forced expression of the c-Myc gene and the BMI1 gene.
  • the step of culturing under forced expression of at least one gene belonging to the BCL2 family It includes a step of suppressing the forced expression of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family and culturing.
  • the neutrophil-like cells obtained by the present invention exert some or all of the functions and properties of neutrophils.
  • the main function of neutrophils is to control infectious diseases, but the control of infectious diseases is by accumulating at the site of infectious disease and killing bacteria. More specifically, the functions involved in the suppression of neutrophil infections can be classified into adhesive ability, migratory ability, phagocytic ability, and bactericidal ability.
  • the neutrophil-like cells of the present invention can be identified by their migratory ability.
  • the migration ability can be determined by performing a migration assay using a migration plate having a chamber blocked by a pore membrane.
  • neutrophils isolated from blood preferably human isolated neutrophils, are comparable in size.
  • Cells having a migration assay can be neutrophil-like cells.
  • the neutrophil-like cells of the present invention can be identified by their phagocytic ability.
  • Phagocytosis can be determined by a phagocytosis assay. As an example, it is carried out by suspending bacteria and neutrophil-like cells and determining the number of cells phagocytosed by the neutrophil-like cells. Specifically, bacteria can be counted by staining with Gram stain or the like, or bacteria pre-fluorescently labeled can be used and counted based on fluorescence.
  • Isolated neutrophils preferably cells having the same phagocytic capacity as human isolated neutrophils, can be identified as neutrophil-like cells.
  • the neutrophil-like cells of the present invention can be identified by their bactericidal activity.
  • the bactericidal ability of neutrophil-like cells can be determined by an oxidative burst assay.
  • Isolated neutrophils preferably cells having similar bactericidal activity as human isolated neutrophils, can be identified as neutrophil-like cells.
  • the invention in another aspect of the invention, relates to a composition comprising neutrophil-like cells.
  • the compositions of the invention are used in Granulocyte Transfusion Therapy (GTX), which replaces neutrophils by blood transfusion, to treat or prevent diseases associated with neutropenia / dysfunction.
  • GTX Granulocyte Transfusion Therapy
  • Composition for use. Diseases related to neutropenia / dysfunction include neutropenia, neutropenic fever, and immunodeficiency.
  • the compositions of the present invention may be prophylactically administered to a patient undergoing cancer treatment with anti-cancer drug administration and / or radiation therapy.
  • the invention may relate to a therapeutic or prophylactic method comprising administering neutrophil-like cells.
  • Neutrophil-like cells are administered to subjects who have or are at risk of causing neutropenia / dysfunction.
  • Such subjects include, for example, patients undergoing cancer treatment with anticancer drug administration and / or radiotherapy, patients with bone marrow damage due to pre-transplantation of bone marrow transplantation, and the presence of cancer.
  • Examples include immunodeficiency patients with neutropenia and dysfunction due to hereditary diseases, and congenital immunodeficiency patients due to hereditary diseases.
  • mice BALB / c mice and NOD.Cg-Prkdc scid Il2rg tm1Wjl / Szj (NSG) mice were purchased from Japan SLC, Inc. and Charles River Laboratories Japan Co., Ltd., respectively. All mice used were 8-12 weeks old and all animal studies adhered to the University of Tokyo animal testing guidelines.
  • Bone marrow CD34 (+) cell-derived iPSC Bone marrow CD34 (+) cell-derived iPSCs were produced according to Stem Cell Reports 10, (2016).
  • Cord blood-derived iPSC (610B1) was provided by the Center for iPS Cell Research and Application (Kyoto University).
  • Mouse C3H10T1 / 2 cells were used as feeder cells.
  • lentiviral vector plasmids were used to produce constitutive or inducible protein-expressing lentiviruses, respectively: CSII-EF-MCS-IRES2-Venus (RIKEN BRC) or CSIV-TRE-RfA-UbC-KT ( RIKEN BRC).
  • CSII-EF-MCS-IRES2-Venus RIKEN BRC
  • CSIV-TRE-RfA-UbC-KT RIKEN BRC
  • Human BCL2A1, BCL-XL, MCL1, c-Myc, and BMI1 were each operably integrated into a lentiviral vector plasmid. All plasmids constructed were validated by Sanger sequencing.
  • Lentivirus HEK293T cells were transiently transfected with a lentiviral vector plasmid, MD2G packaging plasmid (Invitrogen) and PAX2 envelope plasmid (Invitrogen) in which the transgene was incorporated to obtain a lentivirus supernatant. After 48 hours, the virus supernatant was collected and used for infection. Cells transduced with the vector were selected and subjected to in vitro culture. When a doxycycline-inducible lentiviral vector was used, 1 ⁇ g / ml doxycycline (Dox) was added to induce transgene expression.
  • Dox doxycycline
  • Example 1 Establishment of neutrophil progenitor cells (NeuPs) (1) Induction of differentiation from pluripotent stem cells (iPSC) to hematopoietic progenitor cells (HPC) 1% insulin-transferase-selenium-ethanolamine (ITSX; Life) Technologies), 1% penicillin / streptomycin / glutamine (PSG; Life Technologies), 0.45 mmol / L monothioglycerol (Sigma-Aldrich), 50 mg / mL ascorbic acid (Sigma-Aldrich), and 15% highly filtered FBS ( IPSCs on C3H10T1 / 2 cells treated with mitomycin C in Iscob-modified Darbecco medium (Sigma-Aldrich) containing Thermo Fisher Scientific) and supplemented with 20 ng / mL human recombinant vascular endothelial growth factor (VEGF; Peprotech).
  • iPSC pluripotent stem cells
  • HPC hema
  • Clusters were transferred and co-cultured for hematopoietic differentiation.
  • the cell culture was subjected to 5% CO 2 at 37 ° C., and the medium was changed on the 4, 7, 10, 12, and 14 days.
  • the iPSC sac broken with a pipette tip was filtered through a cell strainer, and CD34 + / CD43 + HPC was isolated by fluorescence activated cell selection (FACS) to obtain iPSCs-derived HPCs.
  • FACS fluorescence activated cell selection
  • FACS was performed using FACSAria II and FACSAria III cell sorters (BD Biosciences). The data were analyzed by FlowJo (TreeStar, Ashland, OR, USA).
  • APC-binding anti-human CD34 (4H11; eBioscience) antibody and PE-binding anti-human CD43 (DTF1; Beckman Coulter) antibody were used to isolate iPSCs-derived HPCs.
  • G-CSF was used, and 100 ng / ml G-CSF was used after 6 days.
  • Lentivirus introduction of BCL-XL was carried out from the 10th day to the 17th day to obtain neutrophil progenitor cells (NeuPs).
  • the cytokine addition schedule is shown in FIG. 1A.
  • BCL2A1, BCL-XL, and MCL1 were introduced into NeuPs as BCL2 family genes.
  • a CSII-EF-MCS-IRES2-Venus lentiviral vector operably linked with the DNA of each BCL family gene was used. 3000 Venus (+) cells were seeded by FACS, containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), 15% highly filtered FBS (Thermo Fisher Scientific), 100 ng / ml G-CSF.
  • FIG. 2A shows changes in the number of cells when BCL2A1, BCL-XL, and MCL1 are introduced as BCL2 family genes. Then, after introducing the BCL2A1, BCL-XL, and MCL1 genes 70 days later, the change in the number of cells when cultured in the absence of feeder cells is shown in FIG. 2B.
  • FIG. 2B shows changes in the number of cells when cultured in the absence of feeder cells.
  • 2C shows a longer-term proliferative property when BCL-XL is expressed as a BCL2 family gene. After 16 weeks of culture, the number of cells reached 10 23, which was 10 12 times the number of neutrophils required for human transplantation.
  • Neutrophil progenitor cells obtained by expressing BCL-XL as the BCL2 family were designated as NeuPs-XL, and neutrophil-like cells obtained by differentiating them were designated as NeuCs-XL.
  • IMDM IMDM (Sigma-Aldrich) containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), and 15% highly filtered FBS (Thermo Fisher Scientific), 1 ⁇ g in a 37 ° C., 5% CO 2 atmosphere.
  • Recombinant human SCF (Peprotech), TPO (Peprotech), FLT3-L (Peprotech), and G-CSF (Peprotech) were added to the medium.
  • BCL-XL was gene-introduced into NeuPs 10 to 17 days after the initial introduction.
  • a CSII-EF-MCS-IRES2-Venus lentiviral vector operably linked with BCL-XL DNA was used for introduction.
  • 3000 Venus (+) cells were seeded by FACS, containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), 15% highly filtered FBS (Thermo Fisher Scientific), 100 ng / ml G-CSF.
  • IMDM Sigma-Aldrich
  • the cells were cultured on mitomycin C-treated OP9 feeder cells at 37 ° C.
  • Fig. 2D BCL-XL lentivirus was introduced, and after culturing for 14 days or more, it was replaced with a semi-solid medium (Methocult4434 classic) without docycyclin, and c-Myc, BMI1, and BCL-XL were silenced and induced to differentiate.
  • UCB-HSPC-NeuCs derived from hematopoietic stem progenitor cells derived from cord blood were obtained.
  • the change in cell number during the induction of differentiation is shown in FIG. 2E.
  • the expression of CD33 which is a granulocyte-based cell marker
  • CD235A which is an erythroblast-based cell marker
  • Example 2 Identification of the properties of NeuCs (1) Flow cytometry Cell surface markers were used for NeuPs-XL before silencing of c-Myc, BMI1, and BCL-XL, and NeuCs-XL 4 days after silencing. For analysis, flow cytometry was performed using APC-binding anti-human CD16b antibody (REA584; Miltenyi Biotec) and APC-binding anti-human CD66b antibody (G10F5; Biolegend) (Fig. 3B).
  • APC-binding anti-human CD16b antibody (REA584; Miltenyi Biotec)
  • APC-binding anti-human CD66b antibody G10F5; Biolegend
  • NeuPs-XL In NeuPs-XL (Dox-on), the expression of CD16b and CD66b was not observed, while in NeuCs (Dox-off), the expression of CD16b and CD66b was increased, and the expression was divided into neutrophil-like cells (NeuCs-XL). It was shown that it has become.
  • Western blot was performed using an anti-mouse antibody (Santa Cruz Biotechnology, sc-2357). The images were taken using an Immobilon (Millipore) and a LAS-4000 image analyzer (FUJIFILM, Tokyo, Japan) (Fig. 6). Western blotting showed that LPS stimulation rapidly activates another downstream pathway group associated with LPS stimulation, the NF ⁇ B and MAPK pathways, within 15 minutes (Fig. 6).
  • Example 3 Integrin Function Assay for NeuCs (1) Flow Cytometry for Adhesion Molecules To investigate changes in adhesion during differentiation from NeuPs-XL (Dox-on) to NeuCs-XL (Dox-off day 4) Flow for the adhesion molecules LFA-1 integrin (CD11a / CD18) and integrin ⁇ -M (CD11b), and the proteins of the chemokine and chemotractant receptors CXCR1 and FPR1 against NeuPs-XL and NeuCs-XL. Cytometry was performed.
  • LPS L-selectin
  • Oxidative Burst Assay To evaluate oxidative rupture associated with bactericidal function, erythrocytes in human peripheral blood (PB) were lysed and leukocytes were analyzed. 1.0 ⁇ 10 5 NeuPs, NeuPs-XL, NeuCs, NeuCs-XL or human leukocytes 0.5% bovine serum albumin (FUJIFILM), 1 unit / ml catalase (Santa) and 100 ⁇ M dihydrorhodamine 123 (DHR123) Suspended in HBSS (FUJIFILM) containing hydrochloride (FUJIFILM).
  • PB peripheral blood
  • FUJIFILM bovine serum albumin
  • DHR123 dihydrorhodamine 123
  • Example 4 In vivo Function Assay for NeuCs (1) In vivo Imaging of Luc2-Expressed NeuCs-XL (a) Establishment of Luc2-Expressed NeuPs-XL and Luc2-Expressed NeuCs-XL Using CSII-EF-MCS-IRES2-Venus, NeuPs -The luc2 lentivirus was introduced into XL. A single cell of NeuPs-XL expressing Venus was isolated by FACS, amplified and differentiated into NeuCs-XL. In vitro, luc2 expression in NeuCs-XL was confirmed by exposure to D-luciferin (VivoGIo TM Luciferin; Promega).

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Abstract

A purpose of the present invention is to produce neutrophil-like cells in numbers that can be used in granulocyte transfusion therapy. It became possible to vastly improve proliferation by further forcibly expressing at least one gene belonging to the BCL2 family in neutrophil progenitor cells in which an Myc-C gene and a BMI1 gene are forcibly expressed. As a result, neutrophil progenitor cells having proliferation ability for more than 12 weeks after induction of differentiation from stem cells and capable of proliferating to a cell count of 1013 or higher were produced, leading to the present invention. Therefore, the present invention relates to such neutrophil progenitor cells, and a method for producing the same. The present invention also relates to neutrophil-like cells induced to differentiate from proliferated neutrophil progenitor cells, and a method for producing the same.

Description

好中球前駆細胞及びその製造方法Neutrophil progenitor cells and their production method
　本発明は、幹細胞から分化誘導された好中球前駆細胞、及びその製造方法に関する。さらに、本発明は増殖された好中球前駆細胞から分化誘導された好中球様細胞、及びその製造方法にも関する。 The present invention relates to neutrophil progenitor cells induced to differentiate from stem cells and a method for producing the same. Furthermore, the present invention also relates to neutrophil-like cells induced to differentiate from proliferated neutrophil progenitor cells, and a method for producing the same.
　好中球は、細菌感染および真菌感染に対する身体の主な防御因子である。好中球数は他の血球数ほど安定せず、活動状態、不安、感染症、薬剤など多くの因子に依存して短期間のうちに大きく変化することがある。好中球が200/μL未満に減少すると、炎症反応が消失することがある。好中球減少症が存在すると、細菌や真菌感染に対する炎症反応が無力となる。 Neutrophils are the body's main defense against bacterial and fungal infections. Neutrophil counts are not as stable as other blood cell counts and can change significantly over a short period of time depending on many factors such as activity, anxiety, infections, and drugs. When neutrophils are reduced to less than 200 / μL, the inflammatory response may disappear. The presence of neutropenia neutralizes the inflammatory response to bacterial and fungal infections.
　がん患者は、抗がん剤投与および／または放射線治療によるがん治療や骨髄移植の移植前処理を受けることで骨髄にダメージを受け、白血球、特に好中球が減少する。がん患者で好中球が失われると免疫系が大きく損なわれ、発熱を呈するとともに、急速に死に至る感染症につながる恐れがある。好中球減少症による発熱を呈した患者に対しては、抗菌薬、ステロイド、解熱剤が投与され、また好中球を増殖する目的で、顆粒球コロニー形成刺激因子、骨髄増殖因子などが投与される。しかしながら、既に好中球が減少している対象では、好中球増殖の反応性は乏しく、十分な好中球を生成することができないことがある。そうした患者には、骨髄性前駆細胞の輸注が臨床的に利用可能ではあるが、ドナー不足の問題がある。また、好中球を輸血により補充する顆粒球輸血療法（Granulocyte Transfusion Therapy：ＧＴＸ）も行われている。ＧＴＸが治療効果を発揮するためには、１０9細胞／ｋｇという高用量で好中球を投与する必要があり、ＧＴＸが確立されてから半世紀が経過した現在も、輸血に適した好中球の提供が重要な課題となっている。末梢血の造血前駆細胞（ＨＰＣ）を増殖させて好中球に分化させる手法により、動員末梢血において４０００倍まで増殖させ、１０10細胞数まで増幅可能になった（非特許文献１：Biotechnology and Bioengineering vol. 104, No. 4, p. 832-840)。また、臍帯血由来の造血幹細胞（ＨＳＣ）をex vivoで増殖させることで、４９０００倍まで増殖させる技術も開発されている（非特許文献２：Plos One 12 (7): e0180832）。しかしながら、これらの手法ではＧＴＸの治療に十分な数の好中球が依然として得られていない。 Cancer patients suffer from bone marrow damage and a decrease in white blood cells, especially neutrophils, due to cancer treatment with anticancer drugs and / or radiation therapy and pretreatment for bone marrow transplantation. Loss of neutrophils in cancer patients can severely damage the immune system, causing fever and rapidly fatal infections. Antibacterial agents, steroids, and antipyretics are administered to patients with fever due to neutropenia, and granulocyte colony-stimulating factors, bone marrow growth factors, etc. are administered for the purpose of proliferating neutrophils. To. However, in subjects whose neutrophils are already depleted, the reactivity of neutrophil proliferation is poor and it may not be possible to generate sufficient neutrophils. Infusion of myeloid progenitor cells is clinically available for such patients, but there is a problem of donor shortage. In addition, Granulocyte Transfusion Therapy (GTX), in which neutrophils are replaced by blood transfusion, is also performed. For GTX exert a therapeutic effect, 109 need to administer high doses neutrophils that cells / kg, still, Medium good suitable for transfusion half a century since the GTX is established has elapsed Providing spheres has become an important issue. By a technique of proliferating peripheral blood hematopoietic progenitor cells (HPC) to differentiate into neutrophils, it has become possible to proliferate up to 4000 times in mobilized peripheral blood and amplify up to 10 10 cells (Non-Patent Document 1: Biotechnology and Bioengineering vol. 104, No. 4, p. 832-840). In addition, a technique has been developed in which hematopoietic stem cells (HSC) derived from cord blood are proliferated ex vivo to proliferate up to 49,000 times (Non-Patent Document 2: Plos One 12 (7): e0180832). However, these methods still do not provide sufficient numbers of neutrophils to treat GTX.
　ＥＳ細胞やｉＰＳ細胞などの多能性幹細胞を増殖させて、分化誘導を行って、様々な血液細胞好中球様細胞を生成する方法も開発されてきている（非特許文献３：J Clin Invest. (2009), 119(9), 2818-2829；非特許文献４：Blood vol. 113, No. 25, 6584-6592)。誘導多能性幹細胞（ｉＰＳ）は、自家移植を可能にする技術として特に着目されており、様々な血液の細胞成分を製造する方法について研究が行われている。ＧＴＸに供するための好中球を安定的に供給するために、誘導多能性幹細胞からＳＡＣ法を用いて造血前駆細胞へと誘導し、培地中のサイトカインを調整することで好中球特有の細胞マーカーを発現し、好中球の機能を発揮する好中球様細胞へと分化させる手法が開発されている（非特許文献５：Stem Cells (2016), vol.34, p.1513-1526; 非特許文献６：Stem Cells Translational Medicine (2019) vol. 8, 557-567）。また、ＥＴＶ２ｍｍＲＮＡを導入して骨髄内皮前駆細胞を誘導し、分化させる手法で好中球を取得する方法も開示されている。この手法では１０6個ｉＰＳ細胞から好中球への分化までに９～１４日かり、その間に一部の細胞が死滅するので、最終的に１．７×１０7細胞程度の好中球しか提供できていない（非特許文献７：Stem Cell Reports vol 13, 1099-1110）。 A method of proliferating pluripotent stem cells such as ES cells and iPS cells to induce differentiation to generate various blood cell neutrophil-like cells has also been developed (Non-Patent Document 3: J Clin Invest). (2009), 119 (9), 2818-2829; Non-Patent Document 4: Blood vol. 113, No. 25, 6584-6592). Induced pluripotent stem cells (iPS) have received particular attention as a technique that enables autologous transplantation, and studies have been conducted on methods for producing various blood cellular components. In order to stably supply neutrophils for GTX, induced pluripotent stem cells are induced into hematopoietic progenitor cells using the SAC method, and cytokines in the medium are adjusted to be specific to neutrophils. A method for expressing cell markers and differentiating them into neutrophil-like cells that exert neutrophil function has been developed (Non-Patent Document 5: Stem Cells (2016), vol.34, p.1513-1526). Non-Patent Document 6: Stem Cells Translational Medicine (2019) vol. 8, 557-567). Further, a method for obtaining neutrophils by introducing ETV2 mM mRNA to induce bone marrow endothelial progenitor cells and differentiating them is also disclosed. Rent 9-14 days 10 6 iPS cells to differentiate into neutrophils in this technique, since some cells are killed during which finally 1.7 × 10 7 cells about neutrophils only Not provided (Non-Patent Document 7: Stem Cell Reports vol 13, 1099-1110).
　幹細胞から分化させて好中球様細胞を取得するにあたり、十分な数まで好中球前駆細胞を増殖させ、また迅速に好中球様細胞に分化させる手法の開発が求められている。 In order to obtain neutrophil-like cells by differentiating from stem cells, it is required to develop a method for proliferating neutrophil progenitor cells to a sufficient number and rapidly differentiating them into neutrophil-like cells.
　本発明者らは、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現させた幹細胞由来の造血前駆細胞において、さらにＢＣＬ２ファミリーに属する少なくとも１の遺伝子を強制発現することで、増殖性が改善された好中球前駆細胞を取得できることを見出した。またこうして得られた好中球前駆細胞に対し、強制発現された遺伝子の発現を抑制又は停止することにより、速やかに好中球様細胞に分化することを見出した。さらにこうして得られた好中球様細胞が、好中球としての機能を発揮できることを確認した。そこで、本発明は下記に関する： The present inventors improved proliferativeness by forcibly expressing at least one gene belonging to the BCL2 family in stem cell-derived hematopoietic progenitor cells in which the c-Myc gene and the BMI1 gene were forcibly expressed. We have found that neutrophil progenitor cells can be obtained. It was also found that the neutrophil progenitor cells thus obtained rapidly differentiate into neutrophil-like cells by suppressing or stopping the expression of the forcibly expressed gene. Furthermore, it was confirmed that the neutrophil-like cells thus obtained can exert a function as neutrophils. Therefore, the present invention relates to the following:
［１]　幹細胞から分化誘導後に１２週を超えて増殖能を有し、１０13以上の細胞数への増殖可能であり、好中球様細胞への分化能を有する、幹細胞由来の好中球前駆細胞。
［２]　前記幹細胞が、誘導多能性幹細胞又は造血幹細胞である、項目１に記載の好中球前駆細胞。
［３]　ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子が強制発現されている、項目１に記載の好中球前駆細胞。
［４]　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子をコードするＤＮＡが発現可能に連結された発現ベクター由来ＤＮＡを含む、項目１～３のいずれか一項に記載の好中球前駆細胞。
［５]　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子をコードするＤＮＡが発現可能に連結された発現ベクター由来ＤＮＡを含む、好中球様細胞への分化能を有する、幹細胞由来の好中球前駆細胞。
［６]　前記幹細胞が、誘導多能性幹細胞又は造血幹細胞である、項目５に記載の好中球細胞。
［７]　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子が、ＢＣＬ－ＸＬ、ＢＣＬ２Ａ１、及びＭＣＬ１からなる群から選ばれる、項目３～６のいずれか一項に記載の好中球前駆細胞。
［８]　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子がＢＣＬ－ＸＬである、項目７に記載の好中球前駆細胞。
［９]　フィーダー細胞非存在下で増殖能を有する、項目８に記載の好中球前駆細胞。
［１０]　ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子をコードするＤＮＡが発現可能に連結された含む発現ベクターを含む、項目１～９のいずれか一項に記載の好中球前駆細胞。
［１１]　前記発現ベクターが、薬剤制御性の遺伝子発現ベクターである、項目４～１０のいずれか一項に記載の好中球前駆細胞。
［１２]　前記発現ベクターが、ゲノムに組み込まれている、項目１１に記載の好中球前駆細胞。
［１３]　ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子の遺伝子発現を抑制又は停止することにより、好中球へと分化する、項目１～１２のいずれか一項に記載の好中球前駆細胞。
［１４]　前記好中球様細胞が、遊走アッセイで測定した場合に、ヒト単離好中球と同等の遊走性を有する、項目１～１３のいずれか一項に記載の好中球前駆細胞。
［１５]　前記好中球様細胞が、ＬＦＡ－１インテグリン（ＣＤ１１ａ／ＣＤ１８）、インテグリンα－Ｍ（ＣＤ１１ｂ）、ＣＸＣＲ１、ＦＰＲ１、及びＬ－セレクチン（ＣＤ６２Ｌ)からなる群から選ばれる、少なくとも１の接着因子を発現する、項目１～１４のいずれか一項に記載の好中球前駆細胞。
［１６]　項目１～１５のいずれか一項に記載の好中球前駆細胞由来の好中球様細胞。
［１７]　ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子からなる群から選ばれる少なくとも１の遺伝子をコードするＤＮＡを発現可能に含む発現ベクターを含む、幹細胞由来の好中球様細胞。
［１８]　前記発現ベクターが、ゲノムに組み込まれている、項目１７に記載の好中球様細胞。
［１９]　遊走アッセイで測定した場合に、ヒト単離好中球と同等の遊走性を有する、項目１６～１８のいずれか一項に記載の好中球様細胞。
［２０]　ＬＦＡ－１インテグリン（ＣＤ１１ａ／ＣＤ１８）、インテグリンα－Ｍ（ＣＤ１１ｂ）、ＣＸＣＲ１、ＦＰＲ１、及びＬ－セレクチン（ＣＤ６２Ｌ)からなる群から選ばれる、少なくとも１の接着因子を発現する、項目１６～１９のいずれか一項に記載の好中球様細胞。
［２１]　幹細胞由来の造血前駆細胞、或いは生体由来の造血幹細胞及び/又は造血前駆細胞にｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子の強制発現下で培養する工程、
　さらに、ＢＣＬ２ファミリーに属する少なくとも１の遺伝子の遺伝子発現の強制発現下で培養する工程、
　を含む、好中球前駆細胞の製造方法。
［２２]　前記幹細胞が、誘導多能性幹細胞又は造血幹細胞である、項目２１に記載の製造方法。
［２３]　前記培養が、Ｇ－ＣＳＦ、ＳＣＦ、ＴＰＯ、及びＦＬＴ３－Ｌからなる群から選ばれる少なくとも１のサイトカイン添加培地で行われる、項目２１又は２２に記載の製造方法。
［２４]　ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子の強制発現、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子の強制発現が、遺伝子導入により行われる、項目２１～２３のいずれか一項に記載の製造方法。
［２５]　薬剤制御性ベクターを用いて遺伝子導入が行われ、薬剤の添加の有無により遺伝子発現を制御する、項目２４に記載の製造方法。
［２６]　前記好中球前駆細胞が、幹細胞からの分化誘導後、１２週を超えて増殖能を有し、１０13以上の細胞数への増殖可能である、項目２１～２５のいずれか一項に記載の製造方法。
［２７]　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子が、ＢＣＬ－ＸＬ、ＢＣＬ２Ａ１、及びＭＣＬ１からなる群から選ばれる、項目２１～２６のいずれか一項に記載の製造方法。
［２８]　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子がＢＣＬ－ＸＬである、項目２７に記載の製造方法。
［２９]　フィーダー細胞非存在下で培養する工程を含む、項目２８に記載の製造方法。
［３０]　項目２１～２９のいずれか一項に記載の製造方法により製造された好中球前駆細胞。
［３１]　項目１～１５、および３０いずれか一項に記載の好中球前駆細胞において、ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、並びに前記ＢＣＬ２ファミリーに属する少なくとも１の遺伝子の遺伝子発現を抑制又は停止する工程をさらに含む、好中球様細胞の製造方法。
［３２]　項目３１に記載の製造方法により製造された好中球様細胞。
［３３]　項目１６～２１、及び３１のいずれか一項に記載の好中球様細胞を含む、好中球減少、好中球機能不全、又は免疫不全状態により引き起こされる疾患の治療用組成物。
［３４]　前記疾患が、好中球減少症又は好中球減少性発熱である、項目３３に記載の治療用組成物。
［３５]　好中球減少、好中球機能不全、又は免疫不全状態により引き起こされる疾患を患う対象を治療する方法であって、
　項目１６～２１、及び３１のいずれか一項に記載の好中球様細胞を前記対象に投与する工程
　を含む、前記方法。
［３６]　項目１６～２１、及び３１のいずれか一項に記載の好中球様細胞を含む、研究用組成物。
「３７」　好中球減少、好中球機能不全、又は免疫不全状態により引き起こされる疾患の治療又は予防において使用するための、項目１６～２１、及び３１のいずれか一項に記載の好中球様細胞。
［３８］　好中球減少、好中球機能不全、又は免疫不全状態により引き起こされる疾患の治療又は予防用の医薬の製造のための、項目１６～２１、及び３１のいずれか一項に記載の好中球様細胞の使用。 [1] has the ability to grow beyond 12 weeks after induction of differentiation from stem cells, 10 13 or more is capable of growing to cell number, with the ability to differentiate into neutrophil-like cells, stem cell-derived neutrophil Progenitor cells.
[2] The neutrophil progenitor cell according to item 1, wherein the stem cell is an induced pluripotent stem cell or a hematopoietic stem cell.
[3] The neutrophil progenitor cell according to item 1, wherein the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family are forcibly expressed.
[4] The neutrophil progenitor cell according to any one of items 1 to 3, which comprises a DNA derived from an expression vector in which a DNA encoding at least one gene belonging to the BCL2 family is expressively linked.
[5] A stem cell-derived neutrophil progenitor cell having the ability to differentiate into a neutrophil-like cell, which comprises an expression vector-derived DNA in which a DNA encoding at least one gene belonging to the BCL2 family is expressively linked.
[6] The neutrophil cell according to item 5, wherein the stem cell is an induced pluripotent stem cell or a hematopoietic stem cell.
[7] The neutrophil progenitor cell according to any one of items 3 to 6, wherein at least one gene belonging to the BCL2 family is selected from the group consisting of BCL-XL, BCL2A1 and MCL1.
[8] The neutrophil progenitor cell according to item 7, wherein at least one gene belonging to the BCL2 family is BCL-XL.
[9] The neutrophil progenitor cell according to item 8, which has a proliferative ability in the absence of a feeder cell.
[10] The neutrophil progenitor cell according to any one of items 1 to 9, which comprises an expression vector containing an expressively linked DNA encoding the c-Myc gene and the BMI1 gene.
[11] The neutrophil progenitor cell according to any one of items 4 to 10, wherein the expression vector is a drug-regulatory gene expression vector.
[12] The neutrophil progenitor cell according to item 11, wherein the expression vector is integrated into the genome.
[13] The item according to any one of items 1 to 12, wherein the gene is differentiated into neutrophils by suppressing or stopping the gene expression of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family. Neutrophil progenitor cells.
[14] The neutrophil progenitor cell according to any one of items 1 to 13, wherein the neutrophil-like cell has migration properties equivalent to those of human isolated neutrophils when measured by a migration assay. ..
[15] At least one of the neutrophil-like cells selected from the group consisting of LFA-1 integrin (CD11a / CD18), integrin α-M (CD11b), CXCR1, FPR1, and L-selectin (CD62L). The neutrophil progenitor cell according to any one of items 1 to 14, which expresses an adhesion factor.
[16] The neutrophil-like cell derived from the neutrophil progenitor cell according to any one of items 1 to 15.
[17] Stem cell-derived neutrophils comprising an expression vector capable of expressing DNA encoding at least one gene selected from the group consisting of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family. Like cells.
[18] The neutrophil-like cell according to item 17, wherein the expression vector is integrated into the genome.
[19] The neutrophil-like cell according to any one of items 16 to 18, which has the same migration property as human isolated neutrophils when measured by a migration assay.
[20] Item 16 expressing at least one adhesion factor selected from the group consisting of LFA-1 integrin (CD11a / CD18), integrin α-M (CD11b), CXCR1, FPR1, and L-selectin (CD62L). The neutrophil-like cell according to any one of 19 to 19.
[21] A step of culturing a stem cell-derived hematopoietic progenitor cell or a living body-derived hematopoietic stem cell and / or a hematopoietic progenitor cell under forced expression of the c-Myc gene and the BMI1 gene.
Further, a step of culturing under forced expression of gene expression of at least one gene belonging to the BCL2 family,
A method for producing neutrophil progenitor cells, which comprises.
[22] The production method according to item 21, wherein the stem cell is an induced pluripotent stem cell or a hematopoietic stem cell.
[23] The production method according to item 21 or 22, wherein the culture is carried out in at least one cytokine-added medium selected from the group consisting of G-CSF, SCF, TPO, and FLT3-L.
[24] The production method according to any one of items 21 to 23, wherein the forced expression of the c-Myc gene and the BMI1 gene and the forced expression of at least one gene belonging to the BCL2 family are carried out by gene transfer.
[25] The production method according to item 24, wherein gene transfer is performed using a drug controllability vector, and gene expression is controlled depending on the presence or absence of drug addition.
[26] the neutrophil precursor cells, after induction of differentiation from stem cells, have the ability to grow beyond 12 weeks, it can be grown to 10 13 or more in the number of cells, one of the items 21-25 one The manufacturing method described in the section.
[27] The production method according to any one of items 21 to 26, wherein at least one gene belonging to the BCL2 family is selected from the group consisting of BCL-XL, BCL2A1 and MCL1.
[28] The production method according to item 27, wherein at least one gene belonging to the BCL2 family is BCL-XL.
[29] The production method according to item 28, which comprises the step of culturing in the absence of feeder cells.
[30] Neutrophil progenitor cells produced by the production method according to any one of items 21 to 29.
[31] In the neutrophil progenitor cell according to any one of items 1 to 15 and 30, the gene expression of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family is suppressed or stopped. A method for producing neutrophil-like cells, further comprising a step.
[32] Neutrophil-like cells produced by the production method according to item 31.
[33] A therapeutic composition for a disease caused by neutropenia, neutropenia, or immunodeficiency, comprising the neutrophil-like cells according to any one of items 16-21, 31. ..
[34] The therapeutic composition according to item 33, wherein the disease is neutropenia or neutropenic fever.
[35] A method of treating a subject suffering from a disease caused by neutropenia, neutrophil dysfunction, or immunodeficiency.
The method comprising the step of administering the neutrophil-like cell according to any one of items 16 to 21 and 31 to the subject.
[36] A research composition comprising the neutrophil-like cell according to any one of items 16 to 21 and 31.
"37" The neutrophil according to any one of items 16 to 21, and 31 for use in the treatment or prevention of diseases caused by neutropenia, neutrophil dysfunction, or immunodeficiency. Neutrophils.
[38] The item according to any one of items 16 to 21, and 31 for the manufacture of a medicament for the treatment or prevention of a disease caused by neutropenia, neutrophil dysfunction, or immunodeficiency. Use of neutrophil-like cells.
　本発明により、増殖性の高い好中球前駆細胞を提供する。好中球前駆細胞から治療や実験などに十分な数の好中球様細胞を提供することができ、かかる好中球様細胞は、生体の好中球と同等の機能・性質を有し、治療や実験に供することができる。 The present invention provides highly proliferative neutrophil progenitor cells. A sufficient number of neutrophil-like cells can be provided from neutrophil progenitor cells for treatment, experiments, etc., and such neutrophil-like cells have the same functions and properties as living neutrophils. Can be used for treatment and experiments.
図１Ａは、サイトカイン添加スケジュール及び遺伝子導入のタイミングについての実験設計を示す。ｃ－Ｍｙｃ及びＢＭＩ１遺伝子の導入後、ＮｅｕＣｓ－ＸＬへの分化を開始させるまで、ドキシサイクリンが添加されており、この期間は導入された遺伝子が強制発現される。分化を開始させる際にドキシサイクリンを含まない培地へと置換し、それにより導入遺伝子の発現が抑制又は停止される。図１Ｂは、代替となるサイトカイン添加スケジュールの例を示す。FIG. 1A shows an experimental design for cytokine addition schedule and gene transfer timing. After the introduction of the c-Myc and BMI1 genes, doxycycline is added until differentiation into NeuCs-XL is initiated, during which time the introduced genes are forcibly expressed. When initiating differentiation, the medium is replaced with a doxycycline-free medium, which suppresses or arrests the expression of the transgene. FIG. 1B shows an example of an alternative cytokine addition schedule. 図２Ａは、ｃ－Ｍｙｃ及びＢＭＩ１が遺伝子導入されたＮｅｕＰｓを、フィーダー細胞の存在下で１０週まで培養後、ＢＣＬ２ファミリー遺伝子として、ＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、及びＭＣＬ１を導入した場合の細胞数の変化を示す。図２Ｂは、ｃ－Ｍｙｃ及びＢＭＩ１が遺伝子導入されたＮｅｕＰｓをフィーダー細胞の存在下で１０週まで培養後、ＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、及びＭＣＬ１を導入し、フィーダー細胞の非存在下で培養した場合の細胞数の変化を示す。図２Ｃは、ｃ－Ｍｙｃ及びＢＭＩ１の遺伝子導入後、１０～１７日目にＢＣＬ２ファミリー遺伝子として、ＢＣＬ－ＸＬを発現させた場合の、さらに長期の増殖性を示す。図２Ｄは、ｃ－Ｍｙｃ及びＢＭＩ１の遺伝子導入後、１０～１７日目にＢＣＬ－ＸＬを遺伝子導入された臍帯血の造血幹前駆細胞由来のＮｅｕＰｓの増殖試験を示す。図２Ｅは、臍帯血の造血幹前駆細胞由来のＮｅｕＰｓに対し、ドキシサイクリン非存在下で培養し分化を誘導した際の細胞数変化を示す。図２Ｆは、臍帯血の造血幹前駆細胞由来のＮｅｕＰｓと、ドキシサイクリン不存在下で培養して得られたＮｅｕＣｓと、臍帯血ＣＤ３４陽性細胞について、顆粒球系表面抗原ＣＤ３３、及び赤芽球系表面抗原ＣＤ２３５Ａについてフローサイトメトリーにより分析した結果を示す。FIG. 2A shows the number of cells when BCL2A1, BCL-XL, and MCL1 were introduced as BCL2 family genes after culturing NeuPs into which c-Myc and BMI1 were introduced for up to 10 weeks in the presence of feeder cells. Show change. FIG. 2B shows the case where NeuPs into which c-Myc and BMI1 have been transgenic are cultured in the presence of feeder cells for up to 10 weeks, then BCL2A1, BCL-XL, and MCL1 are introduced and cultured in the absence of feeder cells. It shows the change in the number of cells in. FIG. 2C shows a longer-term proliferative property when BCL-XL is expressed as a BCL2 family gene 10 to 17 days after the gene transfer of c-Myc and BMI1. FIG. 2D shows a proliferation test of NeuPs derived from hematopoietic stem progenitor cells of cord blood into which BCL-XL was transgenic 10 to 17 days after transfection of c-Myc and BMI1. FIG. 2E shows changes in the number of cells of NeuPs derived from hematopoietic stem progenitor cells of cord blood when cultured in the absence of doxycycline to induce differentiation. FIG. 2F shows NeuPs derived from hematopoietic stem progenitor cells of cord blood, NeuCs obtained by culturing in the absence of doxycycline, cord blood CD34-positive cells, granulocytic surface antigen CD33, and erythrocytic surface. The results of analysis of the antigen CD235A by flow cytometry are shown. 図３Ａはドキシサイクリン未添加培地への置換前（Dox-on）と置換後２日目（Dox-off day2）、４日目（Dox-off day4）における好中球前駆細の導入遺伝子の細胞内タンパク質量の変化を示す。図３Ｂはドキシサイクリン未添加培地への置換前（Dox-on）と置換後（Dox-off day4）における、好中球前駆細の細胞表面マーカーの発現変化を示す。図３Ｃはドキシサイクリン未添加培地への置換前（Dox-on）と置換後（Dox-off day4）、置換後ＬＰＳ添加後（Dox-off day4＋LPS 300 ng/ml）における細胞及び核の形態変化を示す。FIG. 3A shows intracellular neutrophil precursor transgenes before replacement with doxycycline-free medium (Dox-on), 2 days after replacement (Dox-off day2), and 4th day (Dox-off day4). Shows changes in protein content. FIG. 3B shows changes in the expression of cell surface markers of neutrophil precursors before and after replacement with a doxycycline-free medium (Dox-on) and after replacement (Dox-off day 4). FIG. 3C shows changes in cell and nucleus morphology before replacement with doxycycline-free medium (Dox-on), after replacement (Dox-off day4), and after replacement with LPS (Dox-off day4 + LPS 300 ng / ml). .. 図４Ａは、ＮｅｕＰｓ－ＸＬからＮｅｕＣｓ－ＸＬへ分化した際の遺伝子発現プロファイルの変化を示しており、遺伝子オントロジー機能濃縮解析を行い、最も顕著な上位３０のＧＯカテゴリーを示す。図４ＢはＮｅｕＣｓ－ＸＬにおいて、ＬＰＳ刺激前後の遺伝子発現プロファイルの変化を示しており、遺伝子オントロジー機能濃縮解析を行い、最も顕著な上位３０のＧＯカテゴリーを示す。FIG. 4A shows the change in the gene expression profile when differentiating from NeuPs-XL to NeuCs-XL, and the gene ontology function enrichment analysis is performed to show the most prominent top 30 GO categories. FIG. 4B shows the changes in the gene expression profile before and after LPS stimulation in NeuCs-XL, and the gene ontology function enrichment analysis is performed to show the most prominent top 30 GO categories. 図４Ｃは、ＮｅｕＰｓ－ＸＬ、ＬＰＳ未刺激ＮｅｕＣｓ－ＸＬ、ＬＰＳ刺激ＮｅｕＣｓ－ＸＬの間の遺伝子発現プロファイルの変化について主成分分析を行った結果を示す。図４Ｄは、ＮｅｕＣｓ－ＸＬにおいて、ＬＰＳ刺激を付与前後におけるＮＦκＢ経路の遺伝子セット濃縮分析を示す。FIG. 4C shows the results of principal component analysis of changes in gene expression profiles between NeuPs-XL, LPS-unstimulated NeuCs-XL, and LPS-stimulated NeuCs-XL. FIG. 4D shows gene set enrichment analysis of the NFκB pathway before and after LPS stimulation in NeuCs-XL. 図５は、ＮｅｕＣｓ－ＸＬにおいて、ＬＰＳ刺激無し（（LPS(-)）又は刺激有（（LPS(＋)）における炎症性サイトカイン（ＩＬ－１Ａ、ＩＬ－１Ｂ、ＣＸＣＬ２、ＩＬ－８）の遺伝子発現変化を定量的ＰＣＲにより測定した結果を示す。FIG. 5 shows the genes for inflammatory cytokines (IL-1A, IL-1B, CXCL2, IL-8) in NeuCs-XL without LPS stimulation ((LPS (-)) or with stimulation ((LPS (+))). The result of measuring the expression change by quantitative PCR is shown. 図６は、ＮｅｕＣｓ－ＸＬにおいて、ＬＰＳ刺激前（（LPS(-)）、刺激後１５分、３０分、６０分における、細胞内のＮＦκＢ経路とＭＡＰＫ経路の各因子（ＮＦκＢ、ＩκＢα、ｐ３８、ＥＲＫ）のタンパク質量及びリン酸化されたタンパク質量（ｐ－）の変化をウエスタンブロットにより示す。FIG. 6 shows the intracellular NFκB and MAPK pathway factors (NFκB, IκBα, p38) before LPS stimulation ((LPS (-)) and 15 minutes, 30 minutes, and 60 minutes after stimulation in NeuCs-XL. Changes in the amount of ERK) protein and the amount of phosphorylated protein (p-) are shown by Western blot. 図７Ａは、フローサイトメトリーによりＮｅｕＰｓ－ＸＬからＮｅｕＣｓ－ＸＬへの分化前（Dox-on）、分化誘導４日後（Dox-off day 4)における、細胞表面マーカーであるＬＦＡ－１インテグリン（ＣＤ１１ａ／ＣＤ１８）、インテグリンα－Ｍ（ＣＤ１１ｂ）、ＣＸＣＲ１、ＦＰＲ１の変化を示す。図７Ｂは、フローサイトメトリーによりＮｅｕＰｓ－ＸＬからＮｅｕＣｓ－ＸＬへの分化前（Dox-on）、分化誘導４日後（Dox-off day 4)、分化誘導４日後かつＬＰＳ刺激４時間後（Dox-off day 4＋LPS4ｈｒ)における、細胞表面マーカーであるＬ－セレクチン（ＣＤ６２Ｌ)の存在量変化を示す。FIG. 7A shows LFA-1 integrin (CD11a /), which is a cell surface marker, before differentiation from NeuPs-XL to NeuCs-XL by flow cytometry (Dox-on) and 4 days after differentiation induction (Dox-off day 4). CD18), integrin α-M (CD11b), CXCR1 and FPR1 are shown. FIG. 7B shows before differentiation from NeuPs-XL to NeuCs-XL by flow cytometry (Dox-on), 4 days after differentiation induction (Dox-off day 4), 4 days after differentiation induction and 4 hours after LPS stimulation (Dox-). The change in the abundance of L-selectin (CD62L), which is a cell surface marker, in off day 4 + LPS4hr) is shown. 図８は、ＮｅｕＣｓ－ＸＬに対して行った遊走アッセイの結果を示す。下層チャンバーに生理食塩水（ＰＢＳ）、ウシ胎児血清（ＦＢＳ　０．１％）、ウシ胎児血清（ＦＢＳ　１％）、又はウシ胎児血清（ＦＢＳ　１０％）を添加した場合に、ＦＢＳの用量依存的に遊走活性が増加することを示す。FIG. 8 shows the results of a migration assay performed on NeuCs-XL. Dose-dependent FBS when saline (PBS), fetal bovine serum (FBS 0.1%), fetal bovine serum (FBS 1%), or fetal bovine serum (FBS 10%) is added to the lower chamber. It is shown that the migratory activity is increased. 図９は、ＮｅｕＰｓ－ＸＬに対し分化誘導前（NeuP on）と分化誘導後（NeuP off)において、真菌認識に関与するＴＬＲ２、ＴＬＲ４、及びＣＬＥＣ７Ａ（Ｄｅｃｔｒｉｎ－１）の発現変化を示す。FIG. 9 shows changes in the expression of TLR2, TLR4, and CLEC7A (Dextrin-1) involved in fungal recognition before differentiation induction (NeuP on) and after differentiation induction (NeuP off) with respect to NeuPs-XL. 図１０Ａ～Ｃは、ＮｅｕＰｓ－ＸＬとＮｅｕＣｓ－ＸＬが、細菌を貪食している画像を示す（Ａ：黄色ブドウ球菌（S. aureus)、Ｂ：大腸菌（E. coli)、Ｃ：カンジダ菌（C. albicans)）。10A-C show images of NeuPs-XL and NeuCs-XL phagocytosing bacteria (A: Staphylococcus aureus (S. aureus), B: Escherichia coli (E. coli), C: Candida (Candida albicans). C. albicans)). 図１０Ｄ～Ｆは、ＮｅｕＰｓ－ＸＬとＮｅｕＣｓ－ＸＬの貪食作用を定量したグラフを示す（Ｄ：黄色ブドウ球菌（S. aureus)、Ｅ：大腸菌（E. coli)、Ｆ：カンジダ菌（C. albicans)）。FIGS. 10D to 10F show graphs quantifying the phagocytic action of NeuPs-XL and NeuCs-XL (D: Staphylococcus aureus (S. aureus), E: Escherichia coli (E. coli), F: Candida (C. albicans)). 図１１Ａは、ＮｅｕＰｓ－ＸＬ、ＮｅｕＣｓ－ＸＬ、またはヒト末梢血好中球（Neutrophil）をＦＩＴＣ標識および熱不活化大腸菌懸濁液と混合し、大腸菌を貪食した細胞をフローサイトメトリーにより検出した結果を示す。図１１Ｂは、図１１Ａの貪食アッセイのフローサイトメトリーの結果を数値化して比較したグラフを示す。FIG. 11A shows the results of mixing NeuPs-XL, NeuCs-XL, or human peripheral blood neutrophils with FITC-labeled and heat-inactivated E. coli suspension, and detecting E. coli-phagocytotic cells by flow cytometry. Is shown. FIG. 11B shows a graph in which the flow cytometry results of the phagocytosis assay of FIG. 11A are quantified and compared. 図１２Ａは、ＮｅｕＰｓ－ＸＬ、ＮｅｕＣｓ－ＸＬ、またはヒト末梢血好中球（Neutrophil）における酸化的バーストアッセイの結果を示し、ＰＭＡの存在下又は非存在下におけるジヒドロローダミンの蛍光を示す。図１２Ｂは、図１２Ａのアッセイの結果を数値化して比較したグラフを示す。FIG. 12A shows the results of an oxidative burst assay in NeuPs-XL, NeuCs-XL, or human peripheral blood neutrophils, showing the fluorescence of dihydrorhodamine in the presence or absence of PMA. FIG. 12B shows a graph comparing the assay results of FIG. 12A quantified. 図１３Ａは、低用量（２．０×１０5個）のＮｅｕＣｓ投与後のインビボイメージングの実験設計を示す。この実験設計では、放射線（２．０Ｇｙ）照射されたＮＳＧマウスに対し、ＬＰＳ刺激後、Ｌｕｃ２発現ＮｅｕＣｓ－ＸＬが投与される。図１３Ｂは、ＬＰＳを腹腔内投与されたＮＳＧマウス（ＬＰＳ）において、白血球数が増加することを示すグラフである。図１３Ｃは、ＬＰＳ刺激無し（ＬＰＳ（－））と、ＬＰＳ刺激有（ＬＰＳi.p.)のＮＳＧマウスにおいて、２．０×１０5個ｌｕｃ２発現ＮｅｕＣｓ－ＸＬを腹腔内注射し、１時間後、２４時間後、４８時間後に撮像した、インビボイメージングの結果を示す。FIG. 13A shows an experimental design of in vivo imaging after low dose (2.0 × 10 5) administration of NeuCs. In this experimental design, Luc2-expressing NeuCs-XL is administered to NSG mice irradiated with radiation (2.0 Gy) after LPS stimulation. FIG. 13B is a graph showing an increase in white blood cell count in NSG mice (LPS) intraperitoneally administered with LPS. FIG. 13C shows an intraperitoneal injection of 2.0 × 10 5 luc2-expressing NeuCs-XL in NSG mice without LPS stimulation (LPS (−)) and with LPS stimulation (LPSi.p.) 1 hour later. The results of in vivo imaging taken after 24 hours and 48 hours are shown. 図１４Ａは、高用量（１．０×１０7個（５．０×１０8細胞/ｋｇに対応））のＮｅｕＣｓ投与後のインビボイメージングの実験設計を示す。図１４Ｂは、ＮｅｕＣｓ投与後１時間及び４８時間に撮像した、インビボイメージングの結果を示す。図１４Ｃは、ＮｅｕＣｓ投与後１時間及び４８時間後に採取されたマウス血液において、マウスＣＤ４５陽性細胞と、ヒトＣＤ４５陽性細胞とをそれぞれ標識してフローサイトメトリーにより分析した結果を示す。図１４Ｄは、ＮｅｕＣｓ投与後１時間及び４８時間後に採取された末梢血（ＰＢ）おけるヒトＣＤ４５+細胞の細胞数を示す。FIG. 14A shows an experimental design of in vivo imaging after administration of high doses (1.0 × 10 7 cells (corresponding to 5.0 × 10 8 cells / kg)) of NeuCs. FIG. 14B shows the results of in vivo imaging taken 1 hour and 48 hours after administration of NeuCs. FIG. 14C shows the results of flow cytometry analysis of mouse CD45-positive cells and human CD45-positive cells in mouse blood collected 1 hour and 48 hours after administration of NeuCs, respectively. FIG. 14D shows the number of human CD45 + cells in peripheral blood (PB) collected 1 hour and 48 hours after administration of NeuCs. 図１５Ａは、バイオフィルム慢性感染モデルにおけるＮｅｕＣｓ投与後のインビボイメージングの実験設計を示す。図１５Ｂは、バイオフィルム（細菌付着カテーテルセグメント）を皮下に移植した写真を示す。図１５Ｃは、バイオフィルム移植７日後、１×１０7個のｌｕｃ２発現ＮｅｕＣｓ－ＸＬをマウスに静脈内注射し、５分後、３０分後、６０分後、１２０分後に撮像したインビボイメージングの結果を示す。FIG. 15A shows an experimental design of in vivo imaging after NeuCs administration in a biofilm chronic infection model. FIG. 15B shows a photograph of a biofilm (bacterial adherent catheter segment) implanted subcutaneously. FIG. 15C shows the results of in vivo imaging in which 1 × 10 7 luc2-expressing NeuCs-XL were intravenously injected into mice 7 days after biofilm transplantation and imaged 5 minutes, 30 minutes, 60 minutes, and 120 minutes later. Is shown. 図１６Ａは、急性致死性腹膜炎モデルにおけるＮｅｕＣｓ－ＸＬ投与後の生存試験についての実験設計を示す。図１６Ｂは、急性致死性腹膜炎モデルにおけるＮｅｕＣｓ－ＸＬ投与後の生存試験の結果を示す。図１６Ｃは、急性致死性腹膜炎モデルにおけるＮｅｕＣｓ－ＸＬ投与後の腹腔内における細菌数を示すグラフである。FIG. 16A shows an experimental design for a survival test after administration of NeuCs-XL in an acute lethal peritonitis model. FIG. 16B shows the results of a survival test after administration of NeuCs-XL in a model of acute lethal peritonitis. FIG. 16C is a graph showing the number of bacteria in the abdominal cavity after administration of NeuCs-XL in an acute lethal peritonitis model. 図１７Ａは放射線誘発性血球減少症罹患マウスの急性致死性腹膜炎モデルにおける、ＮｅｕＣｓ－ＸＬ投与後の生存試験についての実験設計を示す。図１７Ｂは、放射線照射後の白血球数の推移を示す。図１７Ｃは、放射線照射後の好中球数の推移を示す。図１７Ｄは、放射線誘発性血球減少症罹患マウスの急性致死性腹膜炎モデルにおける、ＮｅｕＣｓ－ＸＬ投与後の生存試験結果を示す。FIG. 17A shows an experimental design for a survival test after administration of NeuCs-XL in a model of acute lethal peritonitis in mice with radiation-induced cytopenia. FIG. 17B shows the transition of the white blood cell count after irradiation. FIG. 17C shows the transition of the number of neutrophils after irradiation. FIG. 17D shows the results of a survival test after administration of NeuCs-XL in a model of acute lethal peritonitis in mice suffering from radiation-induced cytopenia.
　本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解される。従って、他に定義されない限り、本明細書中で使用されるすべての専門用語及び科学技術用語は、本発明の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。 It is understood that the terms used in the present specification are used in the meanings commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
（定義）
［好中球前駆細胞（Neutrophil-primed progenitors：ＮｅｕＰｓ）］
　本発明において好中球前駆細胞（ＮｅｕＰｓ）は、自己増殖性と好中球又は好中球様細胞への分化性を有する細胞である。本発明では、好中球前駆細胞は、幹細胞、特に誘導多能性幹細胞や胚性幹細胞等から造血前駆細胞を経て製造されるか、生体由来の造血幹細胞又は造血前駆細胞から製造される。より具体的に、血管内皮増殖因子（ＶＥＧＦ）の存在下で誘導多能性幹細胞等の幹細胞を培養することで分化を誘導し、造血前駆細胞（ＨＰＣ）を取得する。次いで、ＨＰＣに対してｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現することにより、好中球前駆細胞（ＮｅｕＰｓ）を得ることができる。別法では、生体由来の造血幹細胞及び／又は造血前駆細胞（造血幹前駆細胞）に、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現することにより、好中球前駆細胞（ＮｅｕＰｓ）を得ることができる。ＮｅｕＰｓは１２週まで増殖可能であり、１２週までの増殖により最大で１０12個まで増殖可能である。一方、ＮｅｕＰｓに対し、さらにＢＣＬ２－ファミリー遺伝子を強制発現することで、１２週を超え、好ましくは１３週を超え、より好ましくは１５週を超え、さらに好ましくは１７週を超え増殖可能な好中球前駆細胞を得た。その結果、ＢＣＬ－ファミリー遺伝子を強制発現された好中球前駆細胞は、１０13個以上、１０15個以上、好ましくは１０18個以上、１０20個以上、１０23個以上、さらに好ましくは１０25個まで細胞数へと増殖が可能になる。これらの好中球前駆細胞は、強制発現している遺伝子の発現を停止又は抑制することにより、すみやかに好中球様細胞（Neutrophil-like cells：ＮｅｕＣｓ）に分化する。遺伝子発現されたＢＣＬ２－ファミリー遺伝子の種類に応じて、例えばＢＣＬ－ＸＬを強制発現されて得た好中球前駆細胞を特にＮｅｕＰｓ－ＸＬとし、さらに分化されて得た好中球様細胞を特にＮｅｕＣｓ－ＸＬとする。 (Definition)
[Neutrophil-primed progenitors (NeuPs)]
In the present invention, neutrophil progenitor cells (NeuPs) are cells having self-proliferation and differentiation into neutrophils or neutrophil-like cells. In the present invention, neutrophil progenitor cells are produced from stem cells, particularly induced pluripotent stem cells, embryonic stem cells, etc. via hematopoietic progenitor cells, or from living body-derived hematopoietic stem cells or hematopoietic progenitor cells. More specifically, differentiation is induced by culturing stem cells such as induced pluripotent stem cells in the presence of vascular endothelial growth factor (VEGF) to obtain hematopoietic progenitor cells (HPC). Next, neutrophil progenitor cells (NeuPs) can be obtained by forcibly expressing the c-Myc gene and the BMI1 gene in HPC. Alternatively, neutrophil progenitor cells (NeuPs) can be obtained by forcibly expressing the c-Myc gene and the BMI1 gene in living body-derived hematopoietic stem cells and / or hematopoietic progenitor cells (hematopoietic progenitor cells). .. NeuPs can grow up to 12 weeks, and up to 10 12 can grow by growing up to 12 weeks. On the other hand, by forcibly expressing the BCL2-family gene in NeuPs, it is possible to proliferate over 12 weeks, preferably over 13 weeks, more preferably over 15 weeks, and even more preferably over 17 weeks. Sphere progenitor cells were obtained. As a result, the number of neutrophil progenitor cells for which the BCL-family gene was forcibly expressed was 10 13 or more, 10 15 or more, preferably 10 18 or more, 10 20 or more, 10 23 or more, and more preferably 10. It is possible to grow up to 25 cells. These neutrophil progenitor cells rapidly differentiate into neutrophil-like cells (NeuCs) by stopping or suppressing the expression of the forcibly expressed gene. Depending on the type of BCL2-family gene expressed, for example, neutrophil progenitor cells obtained by forcibly expressing BCL-XL are designated as NeuPs-XL, and neutrophil-like cells obtained by further differentiation are particularly designated as NeuPs-XL. Let it be NeuCs-XL.
［好中球様細胞（Neutrophil-like cells：ＮｅｕＣｓ）］
　本発明において好中球様細胞（ＮｅｕＣｓ）は、造血前駆細胞又は好中球前駆細胞から分化し、好中球の機能及び性質の一部又は全部を発揮する細胞をいう。造血前駆細胞から分化誘導させる場合、造血前駆細胞をＧ－ＣＳＦ存在下で培養することにより、９～１４日目で好中球様細胞へと分化する。一方で、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現された好中球前駆細胞では、これらの遺伝子の発現を停止又は抑制することで、４日以内に好中球様細胞に分化する。ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現後、さらにＢＣＬ２ファミリー遺伝子を強制発現された好中球前駆細胞でも、これらの遺伝子の発現を停止又は抑制することで、４日以内に好中球様細胞に分化する。好中球の機能としては、感染症を抑制する作用を有する。より具体的に、好中球は、感染症部位に集積し、細菌・真菌に対して殺菌能を有する。より細かく好中球の機能を分類すると、好中球様細胞は、接着能、遊走能、貪食能、及び殺菌能からなる群から選ばれる少なくとも１の機能を発揮する。好中球様細胞は、生体内から単離された好中球と同様の細胞マーカーを発現する。このような細胞マーカーとしては、ＣＤ１６ｂ、ＣＤ６６ｂなどが挙げられる。 [Neutrophil-like cells (NeuCs)]
In the present invention, neutrophil-like cells (NeuCs) refer to cells that differentiate from hematopoietic progenitor cells or neutrophil progenitor cells and exert some or all of the functions and properties of neutrophils. When inducing differentiation from hematopoietic progenitor cells, the hematopoietic progenitor cells are cultured in the presence of G-CSF to differentiate into neutrophil-like cells on days 9 to 14. On the other hand, neutrophil progenitor cells in which the c-Myc gene and the BMI1 gene are forcibly expressed are differentiated into neutrophil-like cells within 4 days by stopping or suppressing the expression of these genes. Neutrophil-like cells within 4 days by stopping or suppressing the expression of these genes even in neutrophil progenitor cells in which the c-Myc gene and BMI1 gene are forcibly expressed and then the BCL2 family gene is forcibly expressed. Differentiate into. The function of neutrophils is to suppress infectious diseases. More specifically, neutrophils accumulate at the site of infection and have a bactericidal ability against bacteria and fungi. When the functions of neutrophils are further classified, neutrophil-like cells exert at least one function selected from the group consisting of adhesive ability, migration ability, phagocytosis ability, and bactericidal ability. Neutrophil-like cells express cell markers similar to neutrophils isolated in vivo. Examples of such a cell marker include CD16b, CD66b and the like.
［幹細胞］
　本発明において幹細胞は、自己増殖性と血液細胞、特に好中球への分化能を有する細胞を指す。より具体的に、誘導多能性幹細胞（induced pluripotent cells: iPSCs)、胚性幹細胞（ESCｓ）、胚性生殖幹細胞（EGCｓ）、体性幹細胞が挙げられる。体性幹細胞としては、造血幹細胞、脂肪幹細胞、間葉系幹細胞、歯髄幹細胞などが挙げられる。 [Stem cells]
In the present invention, stem cells refer to cells that are self-proliferating and have the ability to differentiate into blood cells, particularly neutrophils. More specifically, induced pluripotent cells (iPSCs), embryonic stem cells (ESCs), embryonic germ stem cells (EGCs), somatic stem cells can be mentioned. Examples of somatic stem cells include hematopoietic stem cells, adipose stem cells, mesenchymal stem cells, and dental pulp stem cells.
［誘導多能性幹細胞（induced pluripotent cells: iPSCs)」
　本発明において誘導多能性幹細胞(iPSCs)は、体細胞を初期化することにより製造された幹細胞をいう。ここで用いた体細胞の種類は特に限定されず、任意の体細胞を用いることができる。一例として、骨髄由来ＣＤ３４＋細胞、臍帯血由来細胞、及び皮膚線維芽細胞などの体細胞を用いて誘導多能性幹細胞を確立することができる。本発明の方法により製造された好中球様細胞を、好中球減少、好中球機能不全、又は免疫不全状態に関する疾患を患う患者に投与する治療を行う場合には、該疾患を患う患者自身から分離した体細胞を用いることが好ましい。体細胞から誘導多能性幹細胞を製造する方法は、本技術分野において既知の方法に基づいて製造される。一例として、少なくとも１種類以上の初期化遺伝子、好ましくは幾つかの初期化遺伝子を組み合わせて体細胞に導入し発現させる。初期化遺伝子とは、Oct遺伝子、Klf遺伝子、Sox遺伝子、Myc遺伝子、NANOG遺伝子、LIN28遺伝子、hTERT遺伝子、SV40largeT遺伝子などが挙げられるが、これらに限られることを意図するものではない。Oct遺伝子、Klf遺伝子、Sox遺伝子及びMyc遺伝子にはそれぞれ、複数のファミリー遺伝子が含まれており、ファミリー遺伝子の中から適宜選択することができる。一例として、下記の遺伝子の組み合わせを使用することができる：
（ｉ）Oct遺伝子、Klf遺伝子、Sox遺伝子、Myc遺伝子
（ii）Oct遺伝子、Sox遺伝子、NANOG遺伝子、LIN28遺伝子
（iii）Oct遺伝子、Klf遺伝子、Sox遺伝子、Myc遺伝子、hTERT遺伝子、SV40largeT遺伝
子
（iv）Oct遺伝子、Klf遺伝子、Sox遺伝子
特に好ましくは、初期化遺伝子として、Oct3/4、KLF4、SOX2、ｃ－Mycが使用されうる。 [Induced pluripotent cells (iPSCs)]
Induced pluripotent stem cells (iPSCs) in the present invention refer to stem cells produced by reprogramming somatic cells. The type of somatic cell used here is not particularly limited, and any somatic cell can be used. As an example, somatic cells such as bone marrow-derived CD34 + cells, cord blood-derived cells, and cutaneous fibroblasts can be used to establish induced pluripotent stem cells. When the neutrophil-like cells produced by the method of the present invention are administered to a patient suffering from a disease related to neutropenia, neutrophil dysfunction, or immunodeficiency, the patient suffering from the disease is treated. It is preferable to use somatic cells isolated from themselves. Methods for producing induced pluripotent stem cells from somatic cells are produced based on methods known in the art. As an example, at least one or more reprogramming genes, preferably several reprogramming genes, are combined and introduced into somatic cells for expression. The reprogramming gene includes, but is not intended to be limited to, the Oct gene, the Klf gene, the Sox gene, the Myc gene, the NANOG gene, the LIN28 gene, the hTERT gene, the SV40largeT gene, and the like. Each of the Oct gene, Klf gene, Sox gene, and Myc gene contains a plurality of family genes, and can be appropriately selected from the family genes. As an example, the following gene combinations can be used:
(I) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40largeT gene (iv) ) Oct gene, Klf gene, Sox gene Particularly preferably, Oct3 / 4, KLF4, SOX2, c-Myc can be used as the reprogramming gene.
［造血幹細胞］
　造血幹細胞とは、自己増殖性と全ての血液細胞への分化する細胞をいう。造血幹細胞は、造血前駆細胞、特にリンパ球系前駆細胞と骨髄球系前駆細胞に分化する。リンパ球系前駆細胞は、Ｔリンパ球、Ｂリンパ球に分化する。骨髄球系前駆細胞は、顆粒球、単球、マクロファージ、赤血球、血小板などへと分化する。造血幹細胞は、生体内の骨髄に存在し、また臍帯血中にも存在する。造血幹細胞は、一例として骨髄又は臍帯血からはＣＤ３４陽性細胞として単離することができる。骨髄又は臍帯血からＣＤ３４陽性細胞として単離された細胞には、造血幹細胞と造血前駆細胞の両者が含まれるため、造血幹前駆細胞とも呼ぶことができる。 [Hematopoietic stem cells]
Hematopoietic stem cells are cells that are self-proliferating and differentiate into all blood cells. Hematopoietic stem cells differentiate into hematopoietic progenitor cells, especially lymphocytic progenitor cells and myeloid progenitor cells. Lymphocyte progenitor cells differentiate into T lymphocytes and B lymphocytes. Myeloid progenitor cells differentiate into granulocytes, monocytes, macrophages, erythrocytes, platelets and the like. Hematopoietic stem cells are present in the bone marrow in vivo and also in cord blood. Hematopoietic stem cells can be isolated as CD34-positive cells from bone marrow or cord blood, for example. Cells isolated as CD34-positive cells from bone marrow or umbilical cord blood contain both hematopoietic stem cells and hematopoietic progenitor cells, and thus can also be referred to as hematopoietic stem progenitor cells.
　以下に本発明の好ましい実施形態を説明する。以下に提供される実施形態は、本発明のよりよい理解のために提供されるものであり、本発明の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本発明の範囲内で適宜改変を行うことができることは明らかである。また、以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。 A preferred embodiment of the present invention will be described below. It is understood that the embodiments provided below are provided for a better understanding of the invention and that the scope of the invention should not be limited to the following description. Therefore, it is clear that a person skilled in the art can appropriately make modifications within the scope of the present invention in consideration of the description in the present specification. It is also understood that the following embodiments may be used alone or in combination.
　本発明は、幹細胞由来の好中球前駆細胞、或いは生体の造血幹細胞及び／又は造血前駆細胞由来の好中球様細胞に関する。好中球前駆細胞は、自己増殖性を有し、好中球様細胞へ分化することができる細胞をいう。より具体的に、本発明の好中球前駆細胞は、造血前駆細胞に対し分化誘導後に１２週を超えて増殖能を有し、１０13以上の細胞数への増殖可能である。分化誘導後とは、幹細胞由来の造血前駆細胞に、又は生体由来の造血幹細胞及び／又は造血前駆細胞にｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現した後のことをいう。このような増殖性は、単に幹細胞を好中球前駆細胞へと分化させることによって達成できるものではなく、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を導入し、さらにＢＣＬ２ファミリーに属する少なくとも１を強制発現された結果達成される。 The present invention relates to stem cell-derived neutrophil progenitor cells, or living hematopoietic stem cells and / or neutrophil-like cells derived from hematopoietic progenitor cells. Neutrophil progenitor cells are cells that are self-proliferating and capable of differentiating into neutrophil-like cells. More specifically, neutrophil progenitor cells of the present invention have the ability to grow beyond 12 weeks after induction of differentiation to hematopoietic progenitor cells, it can be grown to 10 13 or more in the number of cells. After induction of differentiation means after forcibly expressing the c-Myc gene and the BMI1 gene in hematopoietic progenitor cells derived from stem cells, or in hematopoietic stem cells and / or hematopoietic progenitor cells derived from living organisms. Such proliferative potential was not achieved by simply differentiating stem cells into neutrophil progenitor cells, but by introducing the c-Myc gene and the BMI1 gene, and forcibly expressing at least one belonging to the BCL2 family. The result is achieved.
　本発明において強制発現とは、任意の方法で細胞において特定の遺伝子の発現を増加させることをいう。一例として、発現ベクターの細胞への導入や、薬剤導入により特定遺伝子の発現を増加させることができる。遺伝子の発現の増加と停止又は抑制する観点から、可逆的又は不可逆的な発現制御性の遺伝子発現ベクターを用いることが好ましい。発現制御性の遺伝子発現ベクターとして、薬剤制御性、光制御性、又は温度感受性の遺伝子発現ベクターを用いることができる。これらの遺伝子発現ベクターは、薬剤導入、光照射、温度変化など特定の条件でのみ遺伝子発現を誘導することができる。一例として、ドキシサイクリン制御性のTet on/off発現ベクターを使用することができる。Tet on/off発現ベクターを用いることで、ドキシサイクリンの存在下では発現ベクターに組み込まれた遺伝子の発現を促進する一方、ドキシサイクリンの非存在下では発現ベクター組み込まれた遺伝子の発現を抑制することができる。さらに別の態様では、特定条件で導入された遺伝子発現ベクターが除去又は破壊されるように設計された遺伝子発現ベクターを用いることで、遺伝子発現を制御することができる。 In the present invention, forced expression means increasing the expression of a specific gene in a cell by an arbitrary method. As an example, the expression of a specific gene can be increased by introducing an expression vector into cells or introducing a drug. From the viewpoint of increasing, stopping or suppressing gene expression, it is preferable to use a reversible or irreversible expression-regulating gene expression vector. As the expression-regulated gene expression vector, a drug-controlled, photoregulated, or temperature-sensitive gene expression vector can be used. These gene expression vectors can induce gene expression only under specific conditions such as drug introduction, light irradiation, and temperature change. As an example, a doxycycline-controlled Tet on / off expression vector can be used. By using the Tet on / off expression vector, the expression of the gene integrated into the expression vector can be promoted in the presence of doxycycline, while the expression of the gene integrated into the expression vector can be suppressed in the absence of doxycycline. .. In yet another embodiment, gene expression can be controlled by using a gene expression vector designed to remove or disrupt the gene expression vector introduced under specific conditions.
　発現ベクターの種類は特に限定されず、ウイルスベクターでもプラスミドベクターでもよい。導入した遺伝子が細胞の染色体に組み込まれるようなウイルスベクターを用いることもできる。本発明で使用できるウイルスベクターとしては、レトロウィルスベクター（レンチウィルスベクターを含む）、アデノウイルスベクター、アデノ随伴ウイルスベクターなどを挙げることができる。本発明で導入される遺伝子は、１つの発現ベクターに搭載されていてもよいし、２又は３の発現ベクターに分けて搭載されていてもよい。 The type of expression vector is not particularly limited, and may be a viral vector or a plasmid vector. A viral vector can also be used in which the introduced gene is integrated into the chromosome of the cell. Examples of the virus vector that can be used in the present invention include a retrovirus vector (including a lentivirus vector), an adenovirus vector, and an adeno-associated virus vector. The gene introduced in the present invention may be loaded on one expression vector, or may be loaded separately on two or three expression vectors.
　本発明に係る幹細胞由来の好中球前駆細胞、又は当該好中球前駆細胞から分化された好中球様細胞は、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子を遺伝子発現ベクターで強制発現させた場合には、導入された遺伝子発現ベクターに起因するＤＮＡを含む。したがって、本発明にかかる好中球前駆細胞又は好中球様細胞は、ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子からなる群から選択される少なくとも１、少なくとも２、又は全ての遺伝子をコードするＤＮＡが発現可能に連結された発現ベクター由来のＤＮＡを含む。このようなＤＮＡはゲノムに組み込まれていてもよいし、プラスミドとして存在していてもよい。 The stem cell-derived neutrophil progenitor cell according to the present invention, or the neutrophil-like cell differentiated from the neutrophil progenitor cell, has a c-Myc gene, a BMI1 gene, and at least one gene belonging to the BCL2 family. When forcibly expressed with an expression vector, it contains DNA derived from the introduced gene expression vector. Therefore, the neutrophil progenitor cell or neutrophil-like cell according to the present invention is selected from the group consisting of at least one gene belonging to the c-Myc gene, the BMI1 gene, and the BCL2 family, or at least 1, at least 2, or. Includes DNA from an expression vector in which DNA encoding all genes is expressively linked. Such DNA may be integrated into the genome or may exist as a plasmid.
　ＢＣＬ２ファミリー遺伝子は、アポトーシスの抑制又は誘導を担う遺伝子ファミリーであり、ＢＣＬ２、ＢＡＸ、ＢＡＫ、ＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、ＭＣＬ１などが知られている。ＢＣＬ２ファミリー遺伝子に属する少なくとも１の遺伝子を、単にＢＣＬ２ファミリー遺伝子ということもできる。ＢＣＬ２は、濾胞性リンパ腫の染色体転座に関与するタンパク質として同定された。アポトーシス促進作用を有するＢＣＬ２ファミリーは、ミトコンドリア外膜に局在し、膜の透過性を高めることでアポトーシスカスケードのシグナルとなる。一方で、アポトーシス抑制作用を有するＢＣＬ２ファミリーは、アポトーシス促進作用を有するＢＣＬ２ファミリーの作用を阻害すると考えられている。アポトーシス抑制性のＢＣＬ２ファミリーとして、ＢＣＬ２Ａ１（NM_004049）、ＢＣＬ－ＸＬ（NM_001317919）、ＭＣＬ１（NM_021960）が挙げられ、これらの遺伝子が、好中球前駆細胞において発現することで、アポトーシス抑制作用を発揮すると考えられている。 The BCL2 family gene is a gene family responsible for suppressing or inducing apoptosis, and BCL2, BAX, BAK, BCL2A1, BCL-XL, MCL1 and the like are known. At least one gene belonging to the BCL2 family gene can also be simply referred to as a BCL2 family gene. BCL2 has been identified as a protein involved in the chromosomal translocation of follicular lymphoma. The BCL2 family, which has an apoptosis-promoting effect, is localized in the outer mitochondrial membrane and becomes a signal of the apoptosis cascade by increasing the permeability of the membrane. On the other hand, the BCL2 family having an apoptosis-suppressing action is considered to inhibit the action of the BCL2 family having an apoptosis-promoting action. Examples of the anti-apoptotic BCL2 family include BCL2A1 (NM_004049), BCL-XL (NM_001317919), and MCL1 (NM_021960), and when these genes are expressed in neutrophil progenitor cells, they exert an anti-apoptotic effect. It is considered.
　ｃ－Ｍｙｃ遺伝子（NM_002467）は、細胞の分化及び増殖に関与する転写制御因子であり、多能性維持に関与している。がん細胞において恒常的に発現していることも知られている。ｃ－Ｍｙｃは、Ｍｙｃファミリーの一種であり、他にｌ－Ｍｙｃやｎ－Ｍｙｃといった遺伝子がＭｙｃファミリーとして知られている。 The c-Myc gene (NM_002467) is a transcriptional regulator involved in cell differentiation and proliferation, and is involved in the maintenance of pluripotency. It is also known to be constitutively expressed in cancer cells. c-Myc is a kind of Myc family, and other genes such as l-Myc and n-Myc are known as Myc family.
　ＢＭＩ１遺伝子（NM_005180）は、ポリコーム複合体１を形成するタンパク質の一つであり、ポリコームグループリングフィンガータンパク質（PCGF4）又はリングフィンガー0タンパク質５１（RNF51）とも呼ばれている。細胞周期阻害遺伝子であるｐ１６及びｐ１９を制御することが知られており、がん遺伝子として報告されている。 The BMI1 gene (NM_005180) is one of the proteins that form the polycomb complex 1, and is also called the polycomb group ring finger protein (PCGF4) or ring finger 0 protein 51 (RNF51). It is known to regulate cell cycle inhibitory genes p16 and p19, and has been reported as an oncogene.
　本発明のさらに別の態様では、本発明は好中球前駆細胞の製造方法にも関する。具体的に、下記の工程：
　幹細胞由来の造血前駆細胞、或いは生体由来の造血幹細胞及び／又は造血前駆細胞をｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子の強制発現下で培養する工程、
　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子の強制発現下で培養する工程
　を含む。 In yet another aspect of the invention, the invention also relates to a method for producing neutrophil progenitor cells. Specifically, the following steps:
A step of culturing a hematopoietic progenitor cell derived from a stem cell or a hematopoietic stem cell and / or a hematopoietic progenitor cell derived from a living body under forced expression of the c-Myc gene and the BMI1 gene.
Includes the step of culturing under forced expression of at least one gene belonging to the BCL2 family.
　幹細胞由来の造血前駆細胞は、幹細胞、特にｉＰＳ細胞などをＶＥＧＦ存在下で培養し造血前駆細胞を得る工程により得ることができる。したがって、前記好中球前駆細胞の製造方法は、幹細胞をＶＥＧＦ存在下で培養し造血前駆細胞を得る工程を含んでいてもよい。より具体的に、幹細胞をＶＥＧＦ添加培地中で１０日～２０日、好ましくは１２日～約２週間培養し、ＣＤ３４＋／ＣＤ４３＋の細胞を造血前駆細胞として分離することができる。将来的に好中球様細胞に分化させる観点から、骨髄球造血前駆細胞であることが好ましい。ＶＥＧＦ添加培地は、ＶＥＧＦが添加された既知の血液細胞培養培地である。 Stem cell-derived hematopoietic progenitor cells can be obtained by culturing stem cells, especially iPS cells, in the presence of VEGF to obtain hematopoietic progenitor cells. Therefore, the method for producing neutrophil progenitor cells may include a step of culturing stem cells in the presence of VEGF to obtain hematopoietic progenitor cells. More specifically, stem cells can be cultured in VEGF-added medium for 10 to 20 days, preferably 12 days to about 2 weeks, and CD34 + / CD43 + cells can be separated as hematopoietic progenitor cells. From the viewpoint of differentiating into neutrophil-like cells in the future, myelocyte hematopoietic progenitor cells are preferable. The VEGF-added medium is a known blood cell culture medium to which VEGF has been added.
　生体由来の造血幹細胞又は造血前駆細胞は、主に臍帯血又は骨髄に由来する造血幹細胞及び/又は造血前駆細胞を指す。これらの細胞は、臍帯血又は骨髄から、ＣＤ３４陽性細胞として分離することで得ることができる。造血幹細胞と造血前駆細胞は、区別されてもよいし、区別されずに「造血幹前駆細胞」と呼ばれてもよい。造血幹細胞と造血前駆細胞に対し、ｃ－Ｍｙｃ遺伝子及びＢＭＩ遺伝子を強制発現させることにより、いずれも好中球前駆細胞（ＮｅｕＰｓ）に分化する。 Living body-derived hematopoietic stem cells or hematopoietic progenitor cells mainly refer to umbilical cord blood or bone marrow-derived hematopoietic stem cells and / or hematopoietic progenitor cells. These cells can be obtained by isolation from cord blood or bone marrow as CD34-positive cells. Hematopoietic stem cells and hematopoietic progenitor cells may be distinguished or may be referred to as "hematopoietic stem progenitor cells" without distinction. By forcibly expressing the c-Myc gene and the BMI gene in hematopoietic stem cells and hematopoietic progenitor cells, both are differentiated into neutrophil progenitor cells (NeuPs).
　血液細胞培養培地は、本技術分野において周知の任意の培養培地を使用することができる。血液細胞培養培地には、血液細胞分化を促進するサイトカインが添加されうる。このようなサイトカインとして、ＳＣＦ、ＴＰＯ、ＦＬＴ３－Ｌ、及びＧＣＳＦからなる群から選ばれる少なくとも１のサイトカインを適切なスケジュールで使用することができる。サイトカインの添加スケジュールは、好中球前駆細胞の高い増殖能を維持できるよう当業者であれば適宜選択することができる。サイトカイン添加スケジュールについての例を図１Ａ、１Ｂに示す。幹細胞から好中球前駆細胞へと分化させる間、細胞を維持、増殖、分化を促進するためフィーダー細胞と共培養される。使用されるフィーダー細胞は、本技術分野に周知の任意のフィーダー細胞を使用しうる。 As the blood cell culture medium, any culture medium well known in the art can be used. Cytokines that promote blood cell differentiation can be added to the blood cell culture medium. As such cytokines, at least one cytokine selected from the group consisting of SCF, TPO, FLT3-L, and GCSF can be used on an appropriate schedule. Cytokine addition schedules can be appropriately selected by those skilled in the art so as to maintain the high proliferative capacity of neutrophil progenitor cells. Examples of cytokine addition schedules are shown in FIGS. 1A and 1B. During differentiation from stem cells to neutrophil progenitor cells, the cells are co-cultured with feeder cells to maintain, proliferate and promote differentiation. As the feeder cell used, any feeder cell known in the art can be used.
　造血前駆細胞及び/又は造血幹細胞にｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現させる工程は、ｃ－Ｍｙｃ及びＢＭＩ１遺伝子発現用ベクターを導入することにより行われる。具体的にはＴｅｔ－ｏｎ／ｏｆｆシステム制御下にｃ－Ｍｙｃ及びＢＭＩ１遺伝子を作動可能に配置した遺伝子発現用ベクターを備えたレンチウイルスに感染させ、ドキシサイクリン存在下で培養することで、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現することができる。ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現下で造血前駆細胞及び/又は造血幹細胞を培養することにより、造血前駆細胞及び/又は造血幹細胞は好中球前駆細胞へと分化し、こうして得られた好中球前駆細胞は、遺伝子導入後１１週まで培養することができるが、１２週以降の増殖能は有さず、１０13個を超える増殖能を有さない。 The step of forcibly expressing the c-Myc gene and the BMI1 gene in the hematopoietic progenitor cells and / or the hematopoietic stem cells is performed by introducing a vector for expressing the c-Myc and BMI1 genes. Specifically, c-Myc is infected with a lentivirus equipped with a gene expression vector in which the c-Myc and BMI1 genes are operably arranged under the control of the Tet-on / off system, and cultured in the presence of doxycycline. The gene and the BMI1 gene can be forcibly expressed. By culturing hematopoietic progenitor cells and / or hematopoietic stem cells under forced expression of the c-Myc gene and BMI1 gene, the hematopoietic progenitor cells and / or hematopoietic stem cells differentiated into neutrophil progenitor cells. neutrophil progenitor cells can be cultured up to 11 weeks after the gene introduction, proliferative capacity after 12 weeks no, no proliferative capacity in excess of 10 13.
　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子（ＢＣＬ２ファミリー遺伝子）の遺伝子発現を強制発現する工程は、好中球前駆細胞が確立する前又は確立した後に導入することができる。ＢＣＬ２ファミリー遺伝子の遺伝子発現を強制発現する工程を行う際に、前工程で行われたｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子は、継続して強制発現されていてもよいし、発現が停止されていてもよい。操作の単純化の観点から、ＢＣＬ２ファミリー遺伝子の遺伝子発現を強制発現する工程において、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子が継続して発現していることが好ましい。具体的に、ＢＣＬ２ファミリー遺伝子の遺伝子発現を強制発現する工程は、ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子導入後に、ＢＣＬ２ファミリー遺伝子の遺伝子発現用ベクターを導入することにより行われる。より具体的にはＴｅｔ－ｏｎ／ｏｆｆシステム制御下にＢＣＬ２ファミリー遺伝子を作動可能に配置した遺伝子発現用ベクターを備えたレンチウイルスに感染させ、ドキシサイクリン存在下で培養することで、ＢＣＬ２ファミリー遺伝子を強制発現することができる。ＢＣＬ２ファミリー遺伝子の強制発現は、一例としてｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子を強制発現後１０日目以降に行われ、好中球前駆細胞の増殖能が失われる前までに行われうる。ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子が強制発現されて分化した好中球前駆細胞は、遺伝子導入後１２週まで培養することができるが、さらにＢＣＬ２ファミリー遺伝子を強制発現することで、１２週以降も増殖能を有し、１０13以上の細胞数、１０15以上、好ましくは１０18以上、１０20以上、１０23以上、さらに好ましくは１０25まで細胞数への増殖可能になる。 The step of forcibly expressing the gene expression of at least one gene belonging to the BCL2 family (BCL2 family gene) can be introduced before or after the establishment of neutrophil progenitor cells. When the step of forcibly expressing the gene expression of the BCL2 family gene is performed, the c-Myc gene and the BMI1 gene carried out in the previous step may be continuously forcibly expressed or may be stopped. Good. From the viewpoint of simplifying the operation, it is preferable that the c-Myc gene and the BMI1 gene are continuously expressed in the step of forcibly expressing the gene expression of the BCL2 family gene. Specifically, the step of forcibly expressing the gene expression of the BCL2 family gene is performed by introducing the gene expression vector of the BCL2 family gene after the introduction of the c-Myc gene and the BMI1 gene. More specifically, the BCL2 family gene is forced by infecting it with a lentivirus equipped with a gene expression vector in which the BCL2 family gene is operably arranged under the control of the Tet-on / off system and culturing it in the presence of doxycycline. Can be expressed. Forcible expression of the BCL2 family gene can be performed, for example, 10 days after the forced expression of the c-Myc gene and the BMI1 gene, and before the proliferative capacity of the neutrophil progenitor cells is lost. The neutrophil progenitor cells differentiated by forced expression of the c-Myc gene and BMI1 gene can be cultured up to 12 weeks after gene transfer, but by further forced expression of the BCL2 family gene, they can proliferate after 12 weeks. It has the ability to grow to a cell number of 10 13 or more, preferably 10 15 or more, preferably 10 18 or more, 10 20 or more, 10 23 or more, and more preferably 10 25.
　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子として、ＢＣＬ－ＸＬを遺伝子導入した場合、得られた好中球前駆細胞は、フィーダー細胞非依存的に増殖が可能となる。フィーダー細胞非依存的増殖が可能な好中球前駆細胞は、品質管理の観点で好ましい。また、好中球前駆細胞から分化させた好中球様細胞を、治療用途で投与する場合に、フィーダー細胞の混入を避ける観点でより好ましい。したがって、フィーダー細胞非依存的増殖を可能にする観点から、本発明において、ＢＣＬ２ファミリー遺伝子として、ＢＣＬ－ＸＬを用いることが好ましい。 When BCL-XL is introduced as at least one gene belonging to the BCL2 family, the obtained neutrophil progenitor cells can proliferate independently of feeder cells. Neutrophil progenitor cells capable of feeder cell-independent proliferation are preferable from the viewpoint of quality control. Further, when neutrophil-like cells differentiated from neutrophil progenitor cells are administered for therapeutic purposes, it is more preferable from the viewpoint of avoiding contamination with feeder cells. Therefore, from the viewpoint of enabling feeder cell-independent proliferation, it is preferable to use BCL-XL as the BCL2 family gene in the present invention.
　本発明のさらなる態様は、本発明は好中球様細胞の製造方法にも関する。ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１、好ましくは少なくとも２、さらに好ましくは全ての遺伝子が強制発現されて製造され好中球前駆細胞において、強制発現された遺伝子の発現を停止又は抑制することにより、好中球様細胞へと分化させることができる。したがって、本発明の好中球前駆細胞の製造方法において、さらにｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１、好ましくは少なくとも２、さらに好ましくは全ての遺伝子の遺伝子の強制発現を抑制する工程を含めることで、好中球様細胞を製造することができる。 A further aspect of the present invention also relates to a method for producing neutrophil-like cells. At least 1, preferably at least 2, and more preferably all genes belonging to the c-Myc gene, BMI1 gene, and BCL2 family are forcibly expressed and produced, and the expression of the forcibly expressed gene is stopped in neutrophil progenitor cells. Alternatively, by suppressing it, it can be differentiated into neutrophil-like cells. Therefore, in the method for producing neutrophil progenitor cells of the present invention, the forced expression of genes of at least 1, preferably at least 2, and more preferably all genes belonging to the c-Myc gene, BMI1 gene, and BCL2 family is further suppressed. Neutrophil-like cells can be produced by including the step of performing.
　強制発現の抑制は、本技術分野に周知の任意の方法で行われうる。例えば、発現ベクターが細胞内から除去又は破壊されてもよいし、発現を抑制する処理を行うこともできる。一例としてTet on/off発現ベクターを使用している場合には、ドキシサイクリン非存在下で細胞を培養することで、強制発現された遺伝子の発現を停止又は抑制することができる。 Suppression of forced expression can be performed by any method well known in the art. For example, the expression vector may be removed or destroyed from the cell, or a treatment for suppressing expression may be performed. When a Tet on / off expression vector is used as an example, the expression of the forcibly expressed gene can be stopped or suppressed by culturing the cells in the absence of doxycycline.
　本発明の好中球様細胞の製造方法は、具体的に下記の工程：
　幹細胞由来の造血前駆細胞、或いは生体由来の造血幹細胞及び/又は造血前駆細胞をｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子の強制発現下で培養する工程、
　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子の強制発現下で培養する工程、
　ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子の強制発現を抑制して培養する工程
　を含む。強制発現を抑制して培養する工程以外の工程は、好中球前駆細胞の製造方法と同じ工程であってよく、同じ特徴を有する。したがって、好中球様細胞の製造方法において、ＢＣＬ２ファミリー遺伝子の遺伝子発現を強制発現する工程を行う際に、前工程で行われたｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子は、継続して強制発現されていてもよいし、発現が停止されていてもよい。 The method for producing neutrophil-like cells of the present invention specifically describes the following steps:
A step of culturing a hematopoietic progenitor cell derived from a stem cell or a hematopoietic stem cell and / or a hematopoietic progenitor cell derived from a living body under forced expression of the c-Myc gene and the BMI1 gene.
The step of culturing under forced expression of at least one gene belonging to the BCL2 family,
It includes a step of suppressing the forced expression of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family and culturing. The steps other than the step of suppressing forced expression and culturing may be the same steps as the method for producing neutrophil progenitor cells, and have the same characteristics. Therefore, in the method for producing neutrophil-like cells, when the step of forcibly expressing the gene expression of the BCL2 family gene is performed, the c-Myc gene and the BMI1 gene performed in the previous step are continuously forcibly expressed. The expression may be stopped.
　本発明により得られた好中球様細胞は、好中球の機能や性質の一部又は全部を発揮する。好中球の主な機能は、感染症の抑制であるが、感染症の抑制には、感染症部位に集積し、細菌に対して殺菌することによる。より詳細に分類すると、好中球の感染症の抑制に関わる機能は、接着能、遊走能、貪食能、及び殺菌能に分類することができる。 The neutrophil-like cells obtained by the present invention exert some or all of the functions and properties of neutrophils. The main function of neutrophils is to control infectious diseases, but the control of infectious diseases is by accumulating at the site of infectious disease and killing bacteria. More specifically, the functions involved in the suppression of neutrophil infections can be classified into adhesive ability, migratory ability, phagocytic ability, and bactericidal ability.
　したがって、本発明の好中球様細胞は、その接着能で特定することができる。接着能の評価として、ＬＦＡ－１インテグリン（ＣＤ１１ａ／ＣＤ１８）、インテグリンα－Ｍ（ＣＤ１１ｂ）、ＣＸＣＲ１、ＦＰＲ１、及びＬ－セレクチン（ＣＤ６２Ｌ)からなる群から選ばれる、少なくとも１の接着因子の発現又は存在量により、評価することができる。接着因子の発現又は存在量は、本技術分野に既知の任意の方法によって行うことができる。一例として、定量的ＰＣＲや、抗体に基づいて、イムノブロット、ＦＡＣＳなどで存在量を決定することもできる。 Therefore, the neutrophil-like cells of the present invention can be identified by their adhesive ability. As an evaluation of adhesiveness, expression of at least one adhesion factor selected from the group consisting of LFA-1 integrin (CD11a / CD18), integrin α-M (CD11b), CXCR1, FPR1, and L-selectin (CD62L) or It can be evaluated by the abundance. The expression or abundance of adhesion factors can be achieved by any method known in the art. As an example, the abundance can be determined by quantitative PCR, immunoblot, FACS, etc. based on the antibody.
　さらに別の実施形態では、本発明の好中球様細胞は、その遊走能により特定することができる。遊走能は、細孔膜で遮られたチャンバーを有するマイグレーションプレートを用いた遊走アッセイを行うことで決定することができる。遊走アッセイとしては、一例としてCytoSelect(商標）（Cell Biolabs, Inc.）のポアサイズ３μｍを用いた遊走アッセイにおいて、血液から単離された好中球、好ましくはヒト単離好中球と同程度の遊走アッセイを有する細胞を好中球様細胞としてすることができる。 In yet another embodiment, the neutrophil-like cells of the present invention can be identified by their migratory ability. The migration ability can be determined by performing a migration assay using a migration plate having a chamber blocked by a pore membrane. As an example of the migration assay, in the migration assay using a pore size of 3 μm of CytoSelect ™ (Cell Biolabs, Inc.), neutrophils isolated from blood, preferably human isolated neutrophils, are comparable in size. Cells having a migration assay can be neutrophil-like cells.
　さらに別の実施形態では、本発明の好中球様細胞は、その貪食能により特定することができる。貪食能は食作用アッセイにより決定することができる。一例として、細菌と、好中球様細胞とを懸濁し、好中球様細胞が貪食した細胞数を決定することにより行われる。具体的に、細菌をグラム染色などで染色して計数することもできるし、予め蛍光標識された細菌を使用し、蛍光に基づいて計数することもできる。単離された好中球、好ましくはヒト単離好中球と同程度の貪食能を有する細胞を好中球様細胞として特定することができる。 In yet another embodiment, the neutrophil-like cells of the present invention can be identified by their phagocytic ability. Phagocytosis can be determined by a phagocytosis assay. As an example, it is carried out by suspending bacteria and neutrophil-like cells and determining the number of cells phagocytosed by the neutrophil-like cells. Specifically, bacteria can be counted by staining with Gram stain or the like, or bacteria pre-fluorescently labeled can be used and counted based on fluorescence. Isolated neutrophils, preferably cells having the same phagocytic capacity as human isolated neutrophils, can be identified as neutrophil-like cells.
　さらに別の実施形態では、本発明の好中球様細胞は、その殺菌能により特定することができる。好中球様細胞の殺菌能は、酸化的バーストアッセイにより決定することができる。単離された好中球、好ましくはヒト単離好中球と同程度の殺菌能を有する細胞を好中球様細胞として特定することができる。 In yet another embodiment, the neutrophil-like cells of the present invention can be identified by their bactericidal activity. The bactericidal ability of neutrophil-like cells can be determined by an oxidative burst assay. Isolated neutrophils, preferably cells having similar bactericidal activity as human isolated neutrophils, can be identified as neutrophil-like cells.
　本発明の別の態様では、本発明は好中球様細胞を含む組成物に関する。一の実施形態では、本発明の組成物は、好中球を輸血により補充する顆粒球輸血療法（Granulocyte Transfusion Therapy：ＧＴＸ）に用いられ、好中球減少・機能不全に関わる疾患の治療又は予防用組成物である。好中球減少・機能不全に関わる疾患としては好中球減少症、好中球減少性発熱、免疫不全などが挙げられる。本発明の組成物は、抗癌剤投与および／または放射線治療によるがん治療を受けている患者に予防的に投与されてもよい。本発明の組成物は、細胞医薬ということもでき、患者本人の体細胞又は造血幹細胞から作成された好中球様細胞を用いてもよいし、免疫反応が最小限になるよう選択された他家細胞を用いてもよい。本発明の好中球様細胞は、ＧＴＸ治療に有効とされる十分な細胞数で提供することができるという利点を有する。また別の態様では、好中球様細胞を含む研究用組成物に関してもよい。本発明に係る好中球様細胞を、好中球に対する研究、好中球を用いた薬剤スクリーニングなどにおいて使用するにあたり、一定の品質を有する好中球として大量の細胞数で提供可能であるという利点を有する。 In another aspect of the invention, the invention relates to a composition comprising neutrophil-like cells. In one embodiment, the compositions of the invention are used in Granulocyte Transfusion Therapy (GTX), which replaces neutrophils by blood transfusion, to treat or prevent diseases associated with neutropenia / dysfunction. Composition for use. Diseases related to neutropenia / dysfunction include neutropenia, neutropenic fever, and immunodeficiency. The compositions of the present invention may be prophylactically administered to a patient undergoing cancer treatment with anti-cancer drug administration and / or radiation therapy. The composition of the present invention may be a cell medicine, and neutrophil-like cells prepared from the patient's own somatic cells or hematopoietic stem cells may be used, or the immune response is selected to be minimized. Somatic cells may be used. The neutrophil-like cells of the present invention have the advantage that they can be provided in a sufficient number of cells that are effective for GTX treatment. In yet another aspect, a research composition containing neutrophil-like cells may be used. When the neutrophil-like cells according to the present invention are used in research on neutrophils, drug screening using neutrophils, etc., it is possible to provide neutrophils having a certain quality in a large number of cells. Has advantages.
　本発明の別の態様では、本発明は好中球様細胞を投与することを含む治療又は予防方法に関していてもよい。好中球様細胞は、好中球減少・機能不全を引き起こしているか、又は引き起こすリスクのある対象に投与される。このような対象としては、一例として、抗癌剤投与および／または放射線治療によるがん治療を受けている患者、骨髄移植の移植前処理を受けることで骨髄にダメージを受けている患者、がんの存在による好中球減少・機能不全を伴う免疫不全患者、遺伝性疾患による先天性免疫不全患者が挙げられる。 In another aspect of the invention, the invention may relate to a therapeutic or prophylactic method comprising administering neutrophil-like cells. Neutrophil-like cells are administered to subjects who have or are at risk of causing neutropenia / dysfunction. Such subjects include, for example, patients undergoing cancer treatment with anticancer drug administration and / or radiotherapy, patients with bone marrow damage due to pre-transplantation of bone marrow transplantation, and the presence of cancer. Examples include immunodeficiency patients with neutropenia and dysfunction due to hereditary diseases, and congenital immunodeficiency patients due to hereditary diseases.
材料と方法
（１）ヒトの一次サンプル
　一次試料はインフォームドコンセント後に採取した。なお、ヒト細胞を用いた試験はすべて東京大学の治験審査委員会において審査され、承認された(承認された治験実施計画書No.2771及び2314)。 Materials and methods (1) Human primary sample The primary sample was collected after informed consent. All studies using human cells were reviewed and approved by the clinical trial review committee of the University of Tokyo (approved clinical trial protocol Nos. 2771 and 2314).
（２）マウス
　BALB/cマウス、NOD.Cg‐PrkdcscidIl2rgtm1Wjl/Szj(NSG)マウスは、それぞれ日本SLC,Inc.およびチャールス・リバーラボラトリーズ・ジャパン(株)から購入した。使用したすべてのマウスは８～１２週齢であり、すべての動物実験は東京大学の動物実験のガイドラインを遵守した。 (2) Mice BALB / c mice and NOD.Cg-Prkdc scid Il2rg tm1Wjl / Szj (NSG) mice were purchased from Japan SLC, Inc. and Charles River Laboratories Japan Co., Ltd., respectively. All mice used were 8-12 weeks old and all animal studies adhered to the University of Tokyo animal testing guidelines.
（３）細胞株
　下記２つのｉＰＳＣｓクローンを使用した：
　　骨髄ＣＤ３４（＋）細胞由来ｉＰＳＣ；及び
　　臍帯血由来ｉＰＳＣ（６１０Ｂ１）
　骨髄ＣＤ３４（＋）細胞由来ｉＰＳＣは、Stem Cell Reports 10, (2018）に従い製造された。臍帯血由来ｉＰＳＣ（６１０Ｂ１）は、ｉＰＳ細胞研究応用センター（京都大学）から提供された。
　フィーダー細胞として、マウスC3H10T1/2細胞を用いた。 (3) Cell line The following two iPSCs clones were used:
Bone marrow CD34 (+) cell-derived iPSC; and cord blood-derived iPSC (610B1)
Bone marrow CD34 (+) cell-derived iPSCs were produced according to Stem Cell Reports 10, (2018). Cord blood-derived iPSC (610B1) was provided by the Center for iPS Cell Research and Application (Kyoto University).
Mouse C3H10T1 / 2 cells were used as feeder cells.
（４）プラスミド
　ＦＬＡＧ標識ヒトＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、ＭＣＬ１の配列を含むプラスミドをInvitrogenから購入した。ｃ－Ｍｙｃの配列を含むプラスミドを日本癌研究資源バンク（JCRB)から購入した。ＦＬＡＧ標識ＢＭＩ１の配列を含むプラスミドを、Blood 117, 3617-3628 (2011)に従い調製した。ｌｕｃ２を含むプラスミドをPromegaから購入した。構成性または誘導性タンパク質発現レンチウイルスを産生するために、以下のレンチウイルスベクタープラスミドをそれぞれ用いた:CSII-EF-MCS-IRES2-Venus(RIKEN BRC)またはCSIV-TRE-RfA-UbC-KT(RIKEN BRC)。ヒトＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、ＭＣＬ１、ｃ－Ｍｙｃ、及びＢＭＩ１それぞれを作動可能にレンチウイルスベクタープラスミドに組み込んだ。構築されたすべてのプラスミドをサンガー配列決定により検証した。 (4) plasmid A plasmid containing the FLAG-labeled human BCL2A1, BCL-XL, and MCL1 sequences was purchased from Invitrogen. A plasmid containing the c-Myc sequence was purchased from the Japan Cancer Research Resource Bank (JCRB). A plasmid containing the sequence of FLAG-labeled BMI1 was prepared according to Blood 117, 3617-3628 (2011). A plasmid containing luc2 was purchased from Promega. The following lentiviral vector plasmids were used to produce constitutive or inducible protein-expressing lentiviruses, respectively: CSII-EF-MCS-IRES2-Venus (RIKEN BRC) or CSIV-TRE-RfA-UbC-KT ( RIKEN BRC). Human BCL2A1, BCL-XL, MCL1, c-Myc, and BMI1 were each operably integrated into a lentiviral vector plasmid. All plasmids constructed were validated by Sanger sequencing.
（５）レンチウイルス
　ＨＥＫ２９３Ｔ細胞に、導入遺伝子を組み込んだレンチウイルスベクタープラスミド、ＭＤ２Ｇパッケージングプラスミド(Invitrogen)およびＰＡＸ２エンベローププラスミド(Invitrogen)で一過性にトランスフェクトし、レンチウイルス上清を得た。４８時間後、ウイルス上清を回収し、感染に利用した。ベクターを形質導入された細胞を選別し、ｉｎ　ｖｉｔｒｏ培養に供した。ドキシサイクリン誘導性レンチウイルスベクターを用いた場合、導入遺伝子発現を誘導するために１μｇ／ｍｌのドキシサイクリン（Ｄｏｘ）を添加した。 (5) Lentivirus HEK293T cells were transiently transfected with a lentiviral vector plasmid, MD2G packaging plasmid (Invitrogen) and PAX2 envelope plasmid (Invitrogen) in which the transgene was incorporated to obtain a lentivirus supernatant. After 48 hours, the virus supernatant was collected and used for infection. Cells transduced with the vector were selected and subjected to in vitro culture. When a doxycycline-inducible lentiviral vector was used, 1 μg / ml doxycycline (Dox) was added to induce transgene expression.
（６）統計解析
　Ｐｒｉｓｍ８ソフトウェアを用いて、図に示すように一元配置分散分析検定、対応のない両側t検定、ログランク検定を実施した。 (6) Statistical analysis Using Prism8 software, one-way ANOVA test, unpaired two-sided t-test, and log rank test were performed as shown in the figure.
実施例１：好中球前駆細胞（ＮｅｕＰｓ）の樹立
（１）誘導多能性幹細胞（ｉＰＳＣ）から造血前駆細胞（ＨＰＣ）への分化誘導
　１％インスリン-トランスフェリン-セレン-エタノールアミン(ITSX;Life Technologies)、１％ペニシリン/ストレプトマイシン/グルタミン(PSG;Life Technologies)、０．４５ｍｍｏｌ／Ｌモノチオグリセロール(Sigma-Aldrich)、５０ｍｇ／ｍＬアスコルビン酸(Sigma-Aldrich)、及び１５％高度濾過済みＦＢＳ（Thermo Fisher Scientific)を含有し、２０ｎｇ／ｍＬヒト組換え血管内皮増殖因子(VEGF;Peprotech)を添加されたイスコブ改変ダルベッコ培地(Sigma-Aldrich)において、マイトマイシンＣで処理したＣ３Ｈ１０Ｔ１／２細胞上にｉＰＳＣのクラスターを移し、造血分化のために共培養した。細胞培養は３７℃で５％ＣＯ2を行い、培地を４、７、１０、１２、１４日目に交換した。１４～１５日間の培養後、ピペットチップで崩したｉＰＳＣ嚢を細胞ストレイナーでろ過し、ＣＤ３４＋／ＣＤ４３＋ＨＰＣを蛍光活性化細胞選別（ＦＡＣＳ）により単離し、ｉＰＳＣｓ由来ＨＰＣｓを得た。ＦＡＣＳは、FACSAria IIおよびFACSAria III細胞ソーター(BD Biosciences)を用いて行った。データをFlowJo(TreeStar,Ashland,OR,USA)で分析した。ｉＰＳＣｓ由来ＨＰＣｓを単離するために、ＡＰＣ結合抗ヒトＣＤ３４(4H11;eBioscience)抗体及びＰＥ結合抗ヒトＣＤ４３(DTF1;Beckman Coulter)抗体を利用した。 Example 1: Establishment of neutrophil progenitor cells (NeuPs) (1) Induction of differentiation from pluripotent stem cells (iPSC) to hematopoietic progenitor cells (HPC) 1% insulin-transferase-selenium-ethanolamine (ITSX; Life) Technologies), 1% penicillin / streptomycin / glutamine (PSG; Life Technologies), 0.45 mmol / L monothioglycerol (Sigma-Aldrich), 50 mg / mL ascorbic acid (Sigma-Aldrich), and 15% highly filtered FBS ( IPSCs on C3H10T1 / 2 cells treated with mitomycin C in Iscob-modified Darbecco medium (Sigma-Aldrich) containing Thermo Fisher Scientific) and supplemented with 20 ng / mL human recombinant vascular endothelial growth factor (VEGF; Peprotech). Clusters were transferred and co-cultured for hematopoietic differentiation. The cell culture was subjected to 5% CO 2 at 37 ° C., and the medium was changed on the 4, 7, 10, 12, and 14 days. After culturing for 14 to 15 days, the iPSC sac broken with a pipette tip was filtered through a cell strainer, and CD34 + / CD43 + HPC was isolated by fluorescence activated cell selection (FACS) to obtain iPSCs-derived HPCs. FACS was performed using FACSAria II and FACSAria III cell sorters (BD Biosciences). The data were analyzed by FlowJo (TreeStar, Ashland, OR, USA). APC-binding anti-human CD34 (4H11; eBioscience) antibody and PE-binding anti-human CD43 (DTF1; Beckman Coulter) antibody were used to isolate iPSCs-derived HPCs.
（２）造血前駆細胞（ＨＰＣ）から好中球前駆細胞への分化誘導
　ｉＰＳＣｓ由来ＨＰＣｓを、０～２日目と同じ培養条件で１２時間前培養し、ｃ－ＭｙｃおよびＢＭＩ１のレンチウイルスを導入した。各実験において、ｃ－ＭｙｃおよびＢＭＩ１の最初の導入後、単一細胞選別または限界希釈を３日間実施した。１％ＩＴＳＸ（Life Technologies)、１％ＰＳＧ（Life Technologies)、及び１５％高度濾過ＦＢＳ(Thermo Fisher Scientific）を含むＩＭＤＭ(Sigma-Aldrich)を用い、３７℃、５％ＣＯ2雰囲気下で、１μｇ／ｍｌドキシサイクリン存在下でマイトマイシンＣ処理ＯＰ９フィーダー細胞上で共培養した。組換えヒトＳＣＦ(Peprotech)、ＴＰＯ(Peprotech)、ＦＬＴ３－Ｌ(Peprotech)、Ｇ－ＣＳＦ(Peprotech)を培地に添加した。サイトカイン添加スケジュールとして、０～２日において５０ｎｇ／ｍｌ　ＳＣＦ、５０ｎｇ／ｍｌ　ＴＰＯ、及び５０ｎｇ／ｍｌ　ＦＬＴ３－Ｌを用い、３～５日において５０ｎｇ／ｍｌ　ＳＣＦ、５０ｎｇ／ｍｌ　ＦＬＴ３－Ｌ、１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを用い、６日以降は１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを用いた。ＢＣＬ－ＸＬのレンチウイルス導入を、１０日目から１７日目まで行い、好中球前駆細胞（ＮｅｕＰｓ）を得た。サイトカイン添加スケジュールを図１Ａに示す。 (2) Induction of differentiation of hematopoietic progenitor cells (HPC) into neutrophil progenitor cells iPSCs-derived HPCs were pre-cultured for 12 hours under the same culture conditions as on days 0 to 2, and c-Myc and BMI1 lentivirus were introduced. did. In each experiment, single cell selection or limiting dilution was performed for 3 days after the initial introduction of c-Myc and BMI1. Using IMDM (Sigma-Aldrich) containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), and 15% highly filtered FBS (Thermo Fisher Scientific), 1 μg in a 37 ° C., 5% CO 2 atmosphere. Co-cultured on mitomycin C-treated OP9 feeder cells in the presence of / ml doxycycline. Recombinant human SCF (Peprotech), TPO (Peprotech), FLT3-L (Peprotech), and G-CSF (Peprotech) were added to the medium. As a cytokine addition schedule, 50 ng / ml SCF, 50 ng / ml TPO, and 50 ng / ml FLT3-L were used in 0 to 2 days, and 50 ng / ml SCF, 50 ng / ml FLT3-L, 100 ng / ml in 3 to 5 days. G-CSF was used, and 100 ng / ml G-CSF was used after 6 days. Lentivirus introduction of BCL-XL was carried out from the 10th day to the 17th day to obtain neutrophil progenitor cells (NeuPs). The cytokine addition schedule is shown in FIG. 1A.
（２－２）サイトカインスケジュールの調整
　サイトカインスケジュールについては、５０ｎｇ／ｍｌ　ＳＣＦ、５０ｎｇ／ｍｌ　ＴＰＯ、５０ｎｇ／ｍｌ　ＦＬＴ３－Ｌ、及び１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを図１Ｂに示された期間に使用し、サイトカインスケジュールを最適化した。 (2-2) Adjustment of Cytokine Schedule For the cytokine schedule, 50 ng / ml SCF, 50 ng / ml TPO, 50 ng / ml FLT3-L, and 100 ng / ml G-CSF were used during the period shown in FIG. 1B. Optimized cytokine schedule.
（２－３）拡張可能な好中球前駆細胞の樹立の限界希釈分析
　ｃ－ＭｙｃおよびＢＭＩ１のレンチウイルスの導入の３日後に、ＫｕＯ（＋）ＨＰＣを９６ウェルプレート上で１、５、または２５細胞／ウェルで８回の繰り返し実験で、播種した。最初のレンチウイルス誘発後２８日目に、各々のウェルにおける拡張可能な好中球前駆細胞をチェックし、ＥＬＤＡソフトウエアを用いて拡張可能な好中球前駆細胞の絶対頻度を計算した。 (2-3) Limit Dilution Analysis of Extendable Neutrophil Progenitor Cell Establishment Three days after introduction of c-Myc and BMI1 lentivirus, KuO (+) HPC was placed 1, 5, or on a 96-well plate. Seeded in 8 repeated experiments at 25 cells / well. Twenty-eight days after the first lentivirus induction, dilatable neutrophil progenitor cells in each well were checked and the absolute frequency of dilatable neutrophil progenitor cells was calculated using ELDA software.
（３）好中球前駆細胞（ＮｅｕＰｓ）の長期増殖アッセイ
　１００００個のＮｅｕＰｓを、１％ＩＴＳＸ(Life Technologies)、１％ＰＳＧ(Life Technologies)、および１５％高度濾過ＦＢＳ(Thermo Fisher Scientific)を含み、１００ｎｇ／ｍｌのＧ－ＣＳＦを添加されたＩＭＤＭ(Sigma-Aldrich技術)中で、マイトマイシンＣ処理ＯＰ９フィーダー細胞上に、３７℃、５％ＣＯ2で１μｇ／ｍｌのＤｏｘの存在下で培養した。２日目と４日目に培地を交換し、ＮｅｕＰｓをトリパンブルー染色を用いて計数した。推定細胞数を、培養の１週間前の推定細胞数にそれらの増殖比率を乗じたものとして計算した。長期増殖は、その増殖比率が１未満になると停止した。初回導入後５週までに５０万個以上に達したＮｅｕＰｓの数を確認し、初回導入後５週における開始細胞数として５０万を設定した。初回導入後、７０日後（１０週）に、ＮｅｕＰｓに、ＢＣＬ２ファミリー遺伝子としてＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、及びＭＣＬ１を導入した。導入には、各ＢＣＬファミリー遺伝子のＤＮＡを作動可能に連結されたＣＳＩＩ‐ＥＦ‐ＭＣＳ‐ＩＲＥＳ２‐Ｖｅｎｕｓレンチウイルスベクターを用いた。３０００個のＶｅｎｕｓ（＋）細胞をＦＡＣＳで播種し、１％ＩＴＳＸ(Life Technologies)、１％ＰＳＧ(Life Technologies)、１５％高度濾過ＦＢＳ(Thermo Fisher Scientific)を含み、１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを添加されたＩＭＤＭ(Sigma-Aldrich)中で、マイトマイシンＣ処理ＯＰ９フィーダー細胞上にて、３７℃、５％ＣＯ2雰囲気下、１μｇ／ｍｌドキシサイクリン存在下で培養し、１週ごとに計数した。各計数は２回反復実験し、２回の実験の平均として細胞数を決定した。結果を図２に示す。図２Ａは、ＢＣＬ２ファミリー遺伝子として、ＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、及びＭＣＬ１を導入した場合の細胞数の変化を示す。次いで、７０日後にＢＣＬ２Ａ１、ＢＣＬ－ＸＬ、及びＭＣＬ１遺伝子を導入後、フィーダー細胞非存在下で培養した場合の細胞数の変化を図２Ｂに示す。図２Ｃは、ＢＣＬ２ファミリー遺伝子として、ＢＣＬ－ＸＬを発現させた場合の、さらに長期の増殖性を示す。１６週間の培養で１０23細胞個に達し、ヒト移植に必要な好中球数の１０12倍に達した。なお、以下の実験において、ＢＣＬ２ファミリーとしてＢＣＬ－ＸＬを発現させて得た好中球前駆細胞をＮｅｕＰｓ－ＸＬとし、これを分化させて得た好中球様細胞をＮｅｕＣｓ－ＸＬとした。 (3) Long-term proliferation assay of neutrophil progenitor cells (NeuPs) 10000 NeuPs containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), and 15% highly filtered FBS (Thermo Fisher Scientific). , 100 ng / ml G-CSF added, cultured on mitomycin C-treated OP9 feeder cells in IMDM (Sigma-Aldrich technology) at 37 ° C., 5% CO 2 in the presence of 1 μg / ml Dox. .. Medium was exchanged on days 2 and 4, and NeuPs were counted using trypan blue staining. The estimated number of cells was calculated as the estimated number of cells one week before culturing multiplied by their growth ratio. Long-term growth stopped when its growth ratio was less than 1. The number of NeuPs that reached 500,000 or more by 5 weeks after the initial introduction was confirmed, and 500,000 was set as the starting cell number 5 weeks after the initial introduction. Seventy days (10 weeks) after the initial introduction, BCL2A1, BCL-XL, and MCL1 were introduced into NeuPs as BCL2 family genes. For introduction, a CSII-EF-MCS-IRES2-Venus lentiviral vector operably linked with the DNA of each BCL family gene was used. 3000 Venus (+) cells were seeded by FACS, containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), 15% highly filtered FBS (Thermo Fisher Scientific), 100 ng / ml G-CSF. In the added IMDM (Sigma-Aldrich), the cells were cultured on mitomycin C-treated OP9 feeder cells at 37 ° C. in a 5% CO 2 atmosphere in the presence of 1 μg / ml doxycycline, and counted weekly. Each count was repeated twice and the cell count was determined as the average of the two experiments. The results are shown in FIG. FIG. 2A shows changes in the number of cells when BCL2A1, BCL-XL, and MCL1 are introduced as BCL2 family genes. Then, after introducing the BCL2A1, BCL-XL, and MCL1 genes 70 days later, the change in the number of cells when cultured in the absence of feeder cells is shown in FIG. 2B. FIG. 2C shows a longer-term proliferative property when BCL-XL is expressed as a BCL2 family gene. After 16 weeks of culture, the number of cells reached 10 23, which was 10 12 times the number of neutrophils required for human transplantation. In the following experiments, Neutrophil progenitor cells obtained by expressing BCL-XL as the BCL2 family were designated as NeuPs-XL, and neutrophil-like cells obtained by differentiating them were designated as NeuCs-XL.
（４）ＮｅｕＰｓからのＮｅｕＣｓへの分化誘導
　１０－１７日目にＢＣＬ－ＸＬのレンチウイルスを導入し、１４日間以上培養後、ドキシサイクリン未添加培地へと置換して、ｃ－Ｍｙｃ、ＢＭＩ１、及びＢＣＬ－ＸＬのサイレンシングを行った。抗Ｍｙｃ‐Ｔａｇ抗体（９Ｂ１１；Cell Signaling）、抗Ｂｃｌ‐ｘＬ抗体（５４Ｈ６；Cell Signaling）、抗Ｂｍｉ１抗体（Ｄ２０Ｂ７；Cell Signaling）、抗β‐アクチン抗体を一次抗体として、二次抗体としてHRP結合抗ウサギ(Santa Cruz Biotechnology,sc-516102)又は抗マウス抗体(Santa Cruz Biotechnology,sc-2357)を用い、イムノブロッティングを行った。Immobilon(Millipore)及びLAS-4000画像解析装置(FUJIFILM、東京、日本)を用いて撮像した（図３）。ドキシサイクリン未添加培地へ置換後４日目には、ｃ－Ｍｙｃ、ＢＭＩ１、及びＢＣＬ－ＸＬがサイレンシングされた。 (4) Induction of differentiation of NeuPs into NeuCs On days 10-17, the lentivirus of BCL-XL was introduced, cultured for 14 days or more, and then replaced with a doxycycline-free medium to c-Myc, BMI1, and c-Myc. BCL-XL was silenced. Anti-Myc-Tag antibody (9B11; Cell Signaling), anti-Bcl-xL antibody (54H6; Cell Signaling), anti-Bmi1 antibody (D20B7; Cell Signaling), anti-β-actin antibody as primary antibody, HRP binding as secondary antibody Immunobrotting was performed using anti-rabbit (Santa Cruz Biotechnology, sc-516102) or anti-mouse antibody (Santa Cruz Biotechnology, sc-2357). Images were taken using Immobilon (Millipore) and LAS-4000 image analyzer (FUJIFILM, Tokyo, Japan) (Fig. 3). On the 4th day after replacement with the doxycycline-free medium, c-Myc, BMI1, and BCL-XL were silenced.
（５）臍帯血の造血幹細胞又は造血前駆細胞からのＮｅｕＰｓ及びＮｅｕＰｃの樹立
　臍帯血からフローサイトメトリーにより、ＣＤ３４をマーカーとして単離された臍帯血由来の造血幹細胞又は造血前駆細胞（造血幹前駆細胞（ＵＣＢ－ＨＳＰＣ））を、０～２日目と同じ培養条件で１２時間前培養し、ｃ－ＭｙｃおよびＢＭＩ１のレンチウイルスを導入した。各実験において、ｃ－ＭｙｃおよびＢＭＩ１の最初の導入後、単一細胞選別または限界希釈を３日間実施した。１％ＩＴＳＸ（Life Technologies)、１％ＰＳＧ（Life Technologies)、及び１５％高度濾過ＦＢＳ(Thermo Fisher Scientific）を含むＩＭＤＭ(Sigma-Aldrich)を用い、３７℃、５％ＣＯ2雰囲気下で、１μｇ／ｍｌドキシサイクリン存在下でマイトマイシンＣ処理ＯＰ９フィーダー細胞上で共培養した。組換えヒトＳＣＦ(Peprotech)、ＴＰＯ(Peprotech)、ＦＬＴ３－Ｌ(Peprotech)、Ｇ－ＣＳＦ(Peprotech)を培地に添加した。サイトカイン添加スケジュールとして、０～２日において５０ｎｇ／ｍｌ　ＳＣＦ、５０ｎｇ／ｍｌ　ＴＰＯ、及び５０ｎｇ／ｍｌ　ＦＬＴ３－Ｌを用い、３～５日において５０ｎｇ／ｍｌ　ＳＣＦ、５０ｎｇ／ｍｌ　ＦＬＴ３－Ｌ、１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを用い、６日以降は１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを用いた。ＢＣＬ－ＸＬのレンチウイルス導入を、１０日目から１７日目まで行い、臍帯血由来の造血幹前駆細胞由来の好中球前駆細胞（ＵＣＢ－ＨＳＰＣ－ＮｅｕＰｓ）を得た。サイトカイン添加スケジュールを図１Ａに示す。 (5) Establishment of NeuPs and NeuPc from cord blood hematopoietic stem cells or cord blood progenitor cells Cord blood-derived hematopoietic stem cells or hematopoietic progenitor cells isolated from cord blood by flow cytometry using CD34 as a marker. (UCB-HSPC)) was pre-cultured for 12 hours under the same culture conditions as on the 0th to 2nd days, and the lentivirus of c-Myc and BMI1 was introduced. In each experiment, single cell selection or limiting dilution was performed for 3 days after the initial introduction of c-Myc and BMI1. Using IMDM (Sigma-Aldrich) containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), and 15% highly filtered FBS (Thermo Fisher Scientific), 1 μg in a 37 ° C., 5% CO 2 atmosphere. Co-cultured on mitomycin C-treated OP9 feeder cells in the presence of / ml doxycycline. Recombinant human SCF (Peprotech), TPO (Peprotech), FLT3-L (Peprotech), and G-CSF (Peprotech) were added to the medium. As a cytokine addition schedule, 50 ng / ml SCF, 50 ng / ml TPO, and 50 ng / ml FLT3-L were used in 0 to 2 days, and 50 ng / ml SCF, 50 ng / ml FLT3-L, 100 ng / ml in 3 to 5 days. G-CSF was used, and 100 ng / ml G-CSF was used after 6 days. Lentivirus introduction of BCL-XL was carried out from the 10th day to the 17th day to obtain neutrophil progenitor cells (UCB-HSPC-NeuPs) derived from hematopoietic stem progenitor cells derived from cord blood. The cytokine addition schedule is shown in FIG. 1A.
（６）臍帯血由来の造血幹細胞由来好中球前駆細胞（ＮｅｕＰｓ）の長期増殖アッセイ
　１００００個のＮｅｕＰｓを、１％ＩＴＳＸ(Life Technologies)、１％ＰＳＧ(Life Technologies)、および１５％高度濾過ＦＢＳ(Thermo Fisher Scientific)を含み、１００ｎｇ／ｍｌのＧ－ＣＳＦを添加されたＩＭＤＭ(Sigma-Aldrich Technologies)中で、マイトマイシンＣ処理ＯＰ９フィーダー細胞上に、３７℃、５％ＣＯ2で１μｇ／ｍｌのＤｏｘの存在下で培養した。２日目と４日目に培地を交換し、ＮｅｕＰｓをトリパンブルー染色を用いて計数した。初回導入後、１０～１７日に、ＮｅｕＰｓに、ＢＣＬ－ＸＬを遺伝子導入した。導入には、ＢＣＬ－ＸＬのＤＮＡを作動可能に連結されたＣＳＩＩ‐ＥＦ‐ＭＣＳ‐ＩＲＥＳ２‐Ｖｅｎｕｓレンチウイルスベクターを用いた。３０００個のＶｅｎｕｓ（＋）細胞をＦＡＣＳで播種し、１％ＩＴＳＸ(Life Technologies)、１％ＰＳＧ(Life Technologies)、１５％高度濾過ＦＢＳ(Thermo Fisher Scientific)を含み、１００ｎｇ／ｍｌ　Ｇ－ＣＳＦを添加されたＩＭＤＭ(Sigma-Aldrich)中で、マイトマイシンＣ処理ＯＰ９フィーダー細胞上にて、３７℃、５％ＣＯ2雰囲気下、１μｇ／ｍｌドキシサイクリン存在下で培養し、１週ごとに計数した（図２Ｄ）。ＢＣＬ－ＸＬのレンチウイルスを導入し、１４日間以上培養後、ドキシサイクリン未添加半固形培地（Methocult4434 classic）へと置換して、ｃ－Ｍｙｃ、ＢＭＩ１、及びＢＣＬ－ＸＬのサイレンシング及び分化誘導を行い、臍帯血由来の造血幹前駆細胞由来の好中球様細胞（ＵＣＢ－ＨＳＰＣ－ＮｅｕＣｓ）を得た。分化誘導の間の細胞数の変化を図２Ｅに示す。顆粒球系の細胞マーカーであるＣＤ３３及び赤芽球系の細胞マーカーであるＣＤ２３５Ａについて、フローサイトメトリーにより発現を調べた（図２Ｆ）。 (6) Long-term proliferation assay of cord blood-derived hematopoietic stem cell-derived neutrophil progenitor cells (NeuPs) 10000 NeuPs, 1% ITSX (Life Technologies), 1% PSG (Life Technologies), and 15% highly filtered FBS 1 μg / ml at 37 ° C., 5% CO 2 on mitomycin C-treated OP9 feeder cells in IMDM (Sigma-Aldrich Technologies) containing (Thermo Fisher Scientific) and supplemented with 100 ng / ml G-CSF. It was cultured in the presence of Dox. Medium was exchanged on days 2 and 4, and NeuPs were counted using trypan blue staining. BCL-XL was gene-introduced into NeuPs 10 to 17 days after the initial introduction. For introduction, a CSII-EF-MCS-IRES2-Venus lentiviral vector operably linked with BCL-XL DNA was used. 3000 Venus (+) cells were seeded by FACS, containing 1% ITSX (Life Technologies), 1% PSG (Life Technologies), 15% highly filtered FBS (Thermo Fisher Scientific), 100 ng / ml G-CSF. In the added IMDM (Sigma-Aldrich), the cells were cultured on mitomycin C-treated OP9 feeder cells at 37 ° C. in a 5% CO 2 atmosphere in the presence of 1 μg / ml doxycycline, and counted weekly (Fig.). 2D). BCL-XL lentivirus was introduced, and after culturing for 14 days or more, it was replaced with a semi-solid medium (Methocult4434 classic) without docycyclin, and c-Myc, BMI1, and BCL-XL were silenced and induced to differentiate. , UCB-HSPC-NeuCs derived from hematopoietic stem progenitor cells derived from cord blood were obtained. The change in cell number during the induction of differentiation is shown in FIG. 2E. The expression of CD33, which is a granulocyte-based cell marker, and CD235A, which is an erythroblast-based cell marker, was examined by flow cytometry (Fig. 2F).
実施例２：ＮｅｕＣｓの性質の特定
（１）フローサイトメトリー
　ｃ－Ｍｙｃ、ＢＭＩ１、及びＢＣＬ－ＸＬのサイレンシング前のＮｅｕＰｓ－ＸＬと、サイレンシングの４日後のＮｅｕＣｓ－ＸＬについて、細胞表面マーカーを分析するために、ＡＰＣ結合抗ヒトＣＤ１６ｂ抗体(REA584;MiltenyiBiotec)、ＡＰＣ結合抗ヒトＣＤ６６ｂ抗体（G10F5;Biolegend)を用い、フローサイトメトリーを行った（図３Ｂ）。ＮｅｕＰｓ－ＸＬ（Ｄｏｘ－ｏｎ）では、ＣＤ１６ｂ及びＣＤ６６ｂの発現がみられなかった一方、ＮｅｕＣｓ（Ｄｏｘ－ｏｆｆ）ではＣＤ１６ｂ及びＣＤ６６ｂの発現が増加し、好中球様細胞（ＮｅｕＣｓ－ＸＬ）に分化していることが示された。 Example 2: Identification of the properties of NeuCs (1) Flow cytometry Cell surface markers were used for NeuPs-XL before silencing of c-Myc, BMI1, and BCL-XL, and NeuCs-XL 4 days after silencing. For analysis, flow cytometry was performed using APC-binding anti-human CD16b antibody (REA584; Miltenyi Biotec) and APC-binding anti-human CD66b antibody (G10F5; Biolegend) (Fig. 3B). In NeuPs-XL (Dox-on), the expression of CD16b and CD66b was not observed, while in NeuCs (Dox-off), the expression of CD16b and CD66b was increased, and the expression was divided into neutrophil-like cells (NeuCs-XL). It was shown that it has become.
（２）ＮｅｕＰｓ、ＮｅｕＣｓの形態、並びにＬＰＳ反応後の形態変化
　ｃ－Ｍｙｃ、ＢＭＩ１、及びＢＣＬ－ＸＬのサイレンシング前のＮｅｕＰｓ－ＸＬと、サイレンシングの４日後のＮｅｕＣｓ－ＸＬ、並びにサイレンシング４日後に３００ｎｇ/ｍｌのＬＰＳ（FUJIFILM）を添加後、Wright Giemsa染色を行って撮影した（スケールバー＝１０μｍ）（図３Ｃ）。サイレンシング４日後のＮｅｕＣｓ－ＸＬは分節核を有し、リポ多糖（ＬＰＳ）に応答して毒性顆粒と空胞化を形成した。 (2) Morphology of NeuPs, NeuCs, and morphological changes after LPS reaction NeuPs-XL before silencing of c-Myc, BMI1, and BCL-XL, NeuCs-XL 4 days after silencing, and silencing 4 After a day, 300 ng / ml LPS (FUJIFILM) was added, followed by Wright Giemsa staining (scale bar = 10 μm) (Fig. 3C). After 4 days of silencing, NeuCs-XL had segmental nuclei and formed toxic granules and vacuoles in response to lipopolysaccharide (LPS).
（３）ＮｅｕＰｓからＮｅｕＣｓへ分化誘導後、ＮｅｕＣｓのＬＰＳ反応後の遺伝子発現プロファイルの変化
（ａ）ＲＮＡ配列決定
　遺伝子発現プロファイリングのために、Illumina HiSeqプラットフォームを使用して、ＮｅｕＰｓ－ＸＬ、２４時間３００ｎｇ／ｍｌのＬＰＳ刺激したＮｅｕＣｓ－ＸＬ及び刺激なしのＮｅｕＣｓ－ＸＬについて、ＲＮＡ配列決定を行った。Bcl2fastq(v2.17.1.14)を用いて、ベース呼び出しおよび予備品質分析用にオリジナル画像データを処理した。Illumina内蔵ソフトウェアは、最初の２５塩基の品質に基づいて、配列決定断片（すなわち、ｒｅａｄｓ）の各々を保存するか廃棄するかを決定した。このステップから得られた生データ(Pass Filter Data)を、塩基配列および対応する配列決定品質情報を含むＦＡＳＴＱフォーマットで保存した。データフィルタリングはCutadaptソフトウェア(version1.9.1)により行い、参照ゲノムへのクリーンデータのアラインメントは、初期パラメータでHisat2(v2.0.1)を用い行った。遺伝子発現計算を行い、読み取り数に基づいてＦＰＫＭ（１００万読み取りあたりのフラグメント/キロベース)を算出した。ＧＯＳｅｑソフトウェアを用いて遺伝子オントロジー機能濃縮解析を行い、最も顕著な上位３０のＧＯカテゴリーを図に示した（図４Ａ、Ｂ）。ＧＳＥＡソフトウェアを用いて遺伝子集合濃縮解析を行った。 (3) Changes in gene expression profile after induction of differentiation from NeuPs to NeuCs and after LPS reaction of NeuCs (a) RNA sequencing Using the Illumina HiSeq platform, NeuPs-XL, 24 hours 300 ng RNA sequencing was performed on / ml LPS-stimulated NeuCs-XL and unstimulated NeuCs-XL. Bcl2fastq (v2.17.1.14) was used to process the original image data for base recall and preliminary quality analysis. The Illumina built-in software decided whether to store or discard each of the sequencing fragments (ie, reads) based on the quality of the first 25 bases. The raw data (Pass Filter Data) obtained from this step was stored in FASTQ format containing the nucleotide sequence and the corresponding sequencing quality information. Data filtering was performed by Cutadap software (version 1.9.1), and clean data was aligned to the reference genome using Hisat2 (v2.0.1) as the initial parameter. Gene expression was calculated and FPKM (fragment / kilobase per million reads) was calculated based on the number of reads. Gene ontology function enrichment analysis was performed using GOSeq software, and the most prominent top 30 GO categories are shown in the figure (FIGS. 4A, B). Reassortment enrichment analysis was performed using GSEA software.
　ＢＣＬ－ＸＬを導入されたＮｅｕＰｓ－ＸＬ（ＮｅｕＰｓ－ＸＬ (Dox on））と、サイレンシング４日後のＮｅｕＣｓ－ＸＬ（Dox off day 4)、並びにサイレンシング４日後にさらにＬＰＳを添加されたＮｅｕＣｓ－ＸＬ（Dos-off day4) LPS（+））について、遺伝子発現プロファイリングをグローバルに実施した。すべての遺伝子発現量について各細胞集団で最も分散が大きくなる第一主成分と二番目に分散が大きくなる第二主成分に変換し二次元上にプロットする主成分分析の結果、各群は別個のサブ集団として位置し（図４Ｃ）、遺伝子オントロジー解析の結果、ＮｅｕＣｓ－ＸＬは、複数の生物学的処理、細胞成分、及び分子機能に関与する遺伝子、並びに好中球の成熟に関与する遺伝子の発現に有意な変化が認められた（図４Ａ～Ｃ）。特に、ＮｅｕＣｓ－ＸＬにおけるＬＰＳ刺激は、炎症性および自然免疫応答、ケモカイン活性および走化性、細胞接着、サイトカイン活性など、重要な好中球機能に関連する生物学的プロセスの有意な変化を誘導した（図４Ｂ）。 NeuPs-XL (NeuPs-XL (Dox on)) introduced with BCL-XL, NeuCs-XL (Dox off day 4) 4 days after silencing, and NeuCs-added LPS 4 days after silencing. Gene expression profiling was performed globally for XL (Dos-off day4) LPS (+). As a result of principal component analysis, which converts all gene expression levels into the first principal component with the largest dispersion in each cell population and the second principal component with the second largest dispersion and plots them two-dimensionally, each group is separate. As a result of Gene Ontology analysis, NeuCs-XL is a gene involved in multiple biological processes, cell components, and molecular functions, as well as genes involved in neutrophil maturation. Significant changes were observed in the expression of (FIGS. 4A to 4C). In particular, LPS stimulation in NeuCs-XL induces significant changes in biological processes associated with important neutrophil function, such as inflammatory and innate immune responses, chemokine activity and chemotaxis, cell adhesion, and cytokine activity. (Fig. 4B).
　次に、ＮｅｕＣｓ－ＸＬにおける分子シグナリング経路をＲＮＡシークエンシングによる遺伝子発現量に基づいて、GSEAソフトウェア（https://www.gsea-msigdb.org/gsea/index.jsp）を用いたgene set enrichment analysisによって検討した。遺伝子セット濃縮分析は、好中球におけるＬＰＳ刺激の重要な下流成分であるＮＦκＢ経路のＬＰＳ誘導活性化を意味した（図４Ｄ）。
（ｂ）定量的ＰＣＲ
　RNAeasy試薬(QIAGEN)で全ＲＮＡを抽出後、ReverTra Ace qPCR RT Master Mix(TOYOBO)で逆転写を行った。ＰＣＲに用いたプライマー配列を表１に列挙した。定量的リアルタイムＰＣＲを、製品説明書(TOYOBO)に従ってTHUNDERBIRD SYBR qPCR MixでLightCycler480システム(Roche社)で実施した。結果をＧＡＰＤＨで正規化した（図５）。
Figure JPOXMLDOC01-appb-T000001
 　ＲＮＡ配列決定データと一致し、定量的ＰＣＲはＮｅｕＣｓ－ＸＬにおける炎症性サイトカインのＬＰＳ誘導アップレギュレーションを示した(図５)。 Next, a gene set enrichment analysis using GSEA software (https://www.gsea-msigdb.org/gsea/index.jsp) based on the gene expression level of the molecular signaling pathway in NeuCs-XL by RNA sequencing. Considered by. Geneset enrichment analysis meant LPS-induced activation of the NFκB pathway, an important downstream component of LPS stimulation in neutrophils (Fig. 4D).
(B) Quantitative PCR
After extracting total RNA with RNAeasy reagent (QIAGEN), reverse transcription was performed with ReverTra Ace qPCR RT Master Mix (TOYOBO). The primer sequences used for PCR are listed in Table 1. Quantitative real-time PCR was performed on the LightCycler 480 system (Roche) with THUNDERBIRD SYBR qPCR Mix according to the product description (TOYOBO). The results were normalized by GAPDH (Fig. 5).
Figure JPOXMLDOC01-appb-T000001
 Consistent with RNA sequencing data, quantitative PCR showed LPS-induced upregulation of inflammatory cytokines in NeuCs-XL (FIG. 5).
（ｃ）ウエスタンブロットによる解析
　ＮｅｕＣｓ－ＸＬに対し、ＬＰＳ刺激前、及び刺激後１５分、３０分、及び６０分後におけるＮＦκＢ及びＭＡＰＫ経路に関与するタンパク質のリン酸化を解析するため、ウエスタンブロットを行った。抗P‐NFκB抗体(93H1; Cell signaling)、抗NFκB抗体(D14E12; Cell signaling)、抗IκBα抗体(44D4; Cell signaling)、抗P38抗体(3D7; Cell signaling)、抗P38抗体、抗P‐Erk1/2抗体(197G2; Cell signaling)、抗Erk1/2抗体(137F5; Cell signaling)および抗β‐アクチン抗体を一次抗体として用い、二次抗体としてHRP結合抗ウサギ(Santa Cruz Biotechnology,sc-516102)又は抗マウス抗体(Santa Cruz Biotechnology,sc-2357)を用い、ウエスタンブロットを行った。Immobilon(Millipore)及びLAS-4000画像解析装置(FUJIFILM、東京、日本)を用いて撮影した（図６)）。ウェスタンブロット法により、ＬＰＳ刺激は、ＬＰＳ刺激に関連する別の下流経路群であるＮＦκＢおよびＭＡＰＫ経路を１５分以内に速やかに活性化することが示された（図６）。 (C) Analysis by Western Blotting Western blots were used to analyze the phosphorylation of proteins involved in the NFκB and MAPK pathways before LPS stimulation and 15 minutes, 30 minutes, and 60 minutes after stimulation with NeuCs-XL. went. Anti-P-NFκB antibody (93H1; Cell signaling), anti-NFκB antibody (D14E12; Cell signaling), anti-IκBα antibody (44D4; Cell signaling), anti-P38 antibody (3D7; Cell signaling), anti-P38 antibody, anti-P-Erk1 / 2 antibody (197G2; Cell signaling), anti-Erk1 / 2 antibody (137F5; Cell signaling) and anti-β-actin antibody were used as the primary antibody, and HRP-binding anti-rabbit (Santa Cruz Biotechnology, sc-516102) was used as the secondary antibody. Alternatively, Western blot was performed using an anti-mouse antibody (Santa Cruz Biotechnology, sc-2357). The images were taken using an Immobilon (Millipore) and a LAS-4000 image analyzer (FUJIFILM, Tokyo, Japan) (Fig. 6). Western blotting showed that LPS stimulation rapidly activates another downstream pathway group associated with LPS stimulation, the NFκB and MAPK pathways, within 15 minutes (Fig. 6).
　以上の実験結果をまとめると、遺伝子発現プロファイリングおよびシグナル伝達経路分析により、ＮｅｕＣｓ－ＸＬが様々な機能と迅速な分子応答とを有し、その両方がプライマリな好中球と共通することが示された。 Summarizing the above experimental results, gene expression profiling and signal transduction pathway analysis show that NeuCs-XL has various functions and rapid molecular response, both of which are common to primary neutrophils. It was.
実施例３：ＮｅｕＣｓに対するインビトロ機能アッセイ
（１)接着分子についてのフローサイトメトリー
　ＮｅｕＰｓ－ＸＬ（Dox-on）からＮｅｕＣｓ－ＸＬ（Dox-off day 4）に分化した際の接着性の変化を調べるため、ＮｅｕＰｓ－ＸＬ及びＮｅｕＣｓ－ＸＬに対し、接着分子であるＬＦＡ－１インテグリン（ＣＤ１１ａ／ＣＤ１８）およびインテグリンα－Ｍ（ＣＤ１１ｂ）、ならびにケモカインおよびケモアトラクタント受容体であるＣＸＣＲ１およびＦＰＲ１のタンパク質についてフローサイトメトリーを行った。ＡＰＣ結合抗ＣＤ１１ａ／１８抗体(m24;Biolegend)、ＡＰＣ結合抗ＣＤ１１ｂ抗体(ICRF44;Biolegend)及びＡＰＣ結合抗ＦＰＲ１抗体(W15086B;Biolegended)、ＦＩＴＣ結合抗ヒトＣＸＣＲ１抗体(8F1;Biolegend)を用いた。ＮｅｕＰｓ－ＸＬでは細胞表面に存在していなかったＣＤ１１ａ／ＣＤ１８、ＣＤ１１ｂ、ＣＸＣＲ１およびＦＰＲ１のタンパク質がそれぞれ、ＮｅｕＣｓ－ＸＬに分化すると発現増加した（図７Ａ)。好中球の初期接着及びローリングに関与するＬ－セレクチン（ＣＤ６２Ｌ）についての変化を調べるため、ＮｅｕＰｓ－ＸＬに対し培地にＬＰＳ（FUJIFILM）１００ｎｇ／ｍｌを添加し、５％ＣＯ2、３７℃雰囲気下で４時間培養し、ＬＰＳ刺激ＮｅｕＣｓ－ＸＬ（Dox-off day4 + LPS 4h)を得た。ＮｅｕＰｓ－ＸＬ（Dox-on）、ＬＰＳ刺激ＮｅｕＣｓ－ＸＬ（Dox-off day4 + LPS 4h)、未刺激ＮｅｕＣｓ－ＸＬ(Dox-off day 4)について、ｐａｃｉｆｉｃ　ｂｌｕｅ結合抗ヒトＣＤ６２Ｌ（DREG-56;Biolegended)抗体を用いてフローサイトメトリーを行った。ＮｅｕＰｓ－ＸＬから、ＮｅｕＣｓ－ＸＬ（Dox-off day4）へと分化すると、Ｌ－セレクチン（ＣＤ６２Ｌ)を発現するようになるが、ＬＰＳ刺激ＮｅｕＣｓ－ＸＬ（Dox-off day4 + LPS 4h)では、ＬＰＳ刺激に応答してＬ－セレクチンが速やかに脱落することが示された(図７Ｂ)。 Example 3: Integrin Function Assay for NeuCs (1) Flow Cytometry for Adhesion Molecules To investigate changes in adhesion during differentiation from NeuPs-XL (Dox-on) to NeuCs-XL (Dox-off day 4) Flow for the adhesion molecules LFA-1 integrin (CD11a / CD18) and integrin α-M (CD11b), and the proteins of the chemokine and chemotractant receptors CXCR1 and FPR1 against NeuPs-XL and NeuCs-XL. Cytometry was performed. APC-binding anti-CD11a / 18 antibody (m24; Biolegend), APC-binding anti-CD11b antibody (ICRF44; Biolegend), APC-binding anti-FPR1 antibody (W15086B; Biolegended), and FITC-binding anti-human CXCR1 antibody (8F1; Biolegend) were used. In NeuPs-XL, the expression of CD11a / CD18, CD11b, CXCR1 and FPR1 proteins, which were not present on the cell surface, increased when they differentiated into NeuCs-XL (Fig. 7A). To investigate changes in L-selectin (CD62L) involved in initial adhesion and rolling of neutrophils, 100 ng / ml of LPS (FUJIFILM) was added to the medium for NeuPs-XL, 5% CO 2 , 37 ° C atmosphere. The cells were cultured under 4 hours to obtain LPS-stimulated NeuCs-XL (Dox-off day4 + LPS 4h). For NeuPs-XL (Dox-on), LPS-stimulated NeuCs-XL (Dox-off day4 + LPS 4h), and unstimulated NeuCs-XL (Dox-off day 4), cyclic blue-bound anti-human CD62L (DREG-56; Biolegended) ) Flow cytometry was performed using the antibody. Differentiation from NeuPs-XL to NeuCs-XL (Dox-off day4) results in the expression of L-selectin (CD62L), but LPS-stimulated NeuCs-XL (Dox-off day4 + LPS 4h) causes LPS. It was shown that L-selectins rapidly shed in response to stimuli (Fig. 7B).
（２）遊走アッセイ
　CytoSelect（商標）96ウェル細胞遊走試験（３μｍ）チャンバー(Cell Biolabs)の下部トレイに、ウシ胎児血清０．１％、１％、または１０％を含むＰＢＳのみを配置した。１．０×１０5細胞のＮｅｕＣｓ－ＸＬに１００μｌの無血清培地を加え、上部膜チャンバーに配置し、そして２時間インキュベートした。遊走した細胞をＣｙＱｕａｎｔ　ＧＲ色素（Ｃｅｌl Biolabs)で製造プロトコルに従って染色し、４８０ｎｍ／５２０ｎｍにて蛍光プレートリーダーARVO‐X(PerkinElmer)で蛍光を測定した。ＮｅｕＣｓ－ＸＬは、ウシ胎児血清含有ＰＢＳに対して用量依存的に遊走した（図８）。 (2) Migration Assay Only PBS containing 0.1%, 1%, or 10% fetal bovine serum was placed in the lower tray of the CytoSelect ™ 96-well Cell Migration Test (3 μm) chamber (Cell Biolabs). 100 μl of serum-free medium was added to NeuCs-XL of 1.0 × 10 5 cells, placed in an upper membrane chamber and incubated for 2 hours. The migrating cells were stained with CyQuant GR dye (Cell Biolabs) according to the production protocol, and the fluorescence was measured with a fluorescence plate reader ARVO-X (PerkinElmer) at 480 nm / 520 nm. NeuCs-XL migrated dose-dependently with respect to fetal bovine serum-containing PBS (Fig. 8).
（３－１）貪食能に関与する遺伝子発現
　真菌認識に関与するＤｅｃｔｒｉｎ－１、ＴＬＲ２、及びＴＬＲ４について、ＲＮＡ配列決定に基づいて、遺伝子発現量を決定した。具体的に、ＮｅｕＰｓ－ＸＬ（Dox-on）とＮｅｕＰｓ－ＸＬ(Dox-off day 4)について、Bcl2fastq(v2.17.1.14)を用いて、ベース呼び出しおよび予備品質分析用にオリジナル画像データを処理した。Illumina内蔵ソフトウェアは、最初の２５塩基の品質に基づいて、配列決定断片（すなわち、ｒｅａｄｓ）の各々を保存するか廃棄するかを決定した。このステップから得られた生データ(Pass Filter Data)を、塩基配列および対応する配列決定品質情報を含むＦＡＳＴＱフォーマットで保存した。データフィルタリングはCutadaptソフトウェア(version1.9.1)により行い、参照ゲノムへのクリーンデータのアラインメントは、初期パラメータでHisat2(v2.0.1)を用い行った。遺伝子発現計算を行い、読み取り数に基づいてＦＰＫＭ（１００万読み取りあたりのフラグメント/キロベース)を算出した。これによりＤｅｃｔｒｉｎ－１、ＴＬＲ２、及びＴＬＲ４の遺伝子発現量を測定した（図９）。好中球前駆細胞から好中球様細胞へと分化することで、真菌の認識に関与する認識分子の遺伝子発現が増大した。 (3-1) Gene expression involved in phagocytosis The gene expression levels of Vectorin-1, TLR2, and TLR4 involved in fungal recognition were determined based on RNA sequencing. Specifically, for NeuPs-XL (Dox-on) and NeuPs-XL (Dox-off day 4), Bcl2fastq (v2.17.1.14) is used to process the original image data for base recall and preliminary quality analysis. did. The Illumina built-in software decided whether to store or discard each of the sequencing fragments (ie, reads) based on the quality of the first 25 bases. The raw data (Pass Filter Data) obtained from this step was stored in FASTQ format containing the nucleotide sequence and the corresponding sequencing quality information. Data filtering was performed by Cutadap software (version 1.9.1), and clean data was aligned to the reference genome using Hisat2 (v2.0.1) as the initial parameter. Gene expression was calculated and FPKM (fragment / kilobase per million reads) was calculated based on the number of reads. As a result, the gene expression levels of Dextrin-1, TLR2, and TLR4 were measured (Fig. 9). Differentiation from neutrophil progenitor cells to neutrophil-like cells increased gene expression of recognition molecules involved in fungal recognition.
（３－２）グラム染色による食作用アッセイ
　細菌を用いた食作用アッセイを１２ウェルプレートで行った。各ウェルには、２０％ＦＢＳを添加された１ｍＬのＩＭＤＭを加え、１．０×１０6個のＮｅｕＰｓ－ＸＬまたはＮｅｕＣｓ－ＸＬと、指数期の細菌(１．０×１０7ＣＦＵの黄色ブドウ球菌 ((S.aureus：Xen-29、PerkinElmer)、２．０×１０7ＣＦＵの大腸菌(E.coli：X-1ブルー、NIPPON GENE))、又は１×１０7ＣＦＵのカンジダ菌（Candida albicans (ATCC 24433)を添加した。大腸菌及び黄色ブドウ球菌の場合３７℃で２時間インキュベートした後、カンジダ菌については３０分３７℃でインキュベートした後、試料をＰＢＳで洗浄し、１００×ｇで５分間２回遠心分離し、顕微鏡スライド上にサイトスピンし、グラム染色（FUJIFILM）を行い撮影した（図１０Ａ～Ｃ）。食作用陽性細胞の割合を評価するために２００個の細胞を計数した。ＮｅｕＣｓ－ＸＬは黄色ブドウ球菌、大腸菌、及びカンジダ菌の食作用を示した(図１０Ｄ～Ｆ）。 (3-2) Phagocytosis assay by Gram stain Phagocytosis assay using bacteria was performed on a 12-well plate. In each well, 1 mL of IMDM supplemented with 20% FBS was added, 1.0 × 10 6 NeuPs-XL or NeuCs-XL, and exponential bacteria (1.0 × 10 7 CFU Staphylococcus aureus). Staphylococcus aureus ((S.aureus: Xen-29, PerkinElmer), 2.0 x 10 7 CFU E. coli (E. coli: X-1 blue, NIPPON GENE)) or 1 x 10 7 CFU Candida albicans (ATCC 24433) was added. For Escherichia coli and Staphylococcus aureus, it was incubated at 37 ° C for 2 hours, for Candida, it was incubated for 30 minutes at 37 ° C, and then the sample was washed with PBS and 100 × g for 5 minutes. Centrifuge twice, cytospin on microscopic slides, gram-stained (FUJIFILM) and photographed (FIGS. 10A-C). 200 cells were counted to assess the proportion of food-positive cells. NeuCs. -XL showed the feeding action of Staphylococcus aureus, Escherichia coli, and Candida albicans (FIGS. 10D to F).
（４）フローサイトメトリーによる食作用アッセイ
　ＮｅｕＰｓ－ＸＬ、ＮｅｕＣｓ－ＸＬ、またはヒト末梢血好中球をＦＩＴＣ標識および熱不活化大腸菌懸濁液（Cayman）と混合し、製造プロトコルに従い１：１０の最終希釈とした。１００ｎｇ／ｍｌのＧ－ＣＳＦを添加して１時間のインキュベート後、表面結合Ｅ．ｃｏｌｉをクエンチするためにトリパンブルー溶液を添加し、ＦＩＴＣ取り込みをフローサイトメトリーにより分析した。フローサイトメトリーにより、ＮｅｕＣｓ－ＸＬによるＦＩＴＣ標識Ｅ．ｃｏｌｉの食作用が末梢血（ＰＢ）の初代ヒト好中球と同程度であることが確認された（図１１Ａ、Ｂ）。 (4) Phagocytosis assay by flow cytometry NeuPs-XL, NeuCs-XL, or human peripheral blood neutrophils are mixed with FITC-labeled and heat-inactivated Escherichia coli suspension (Cayman) and 1:10 according to the production protocol. It was the final dilution. After adding 100 ng / ml of G-CSF and incubating for 1 hour, surface-bonded E.I. A trypan blue solution was added to quench the coli and FITC uptake was analyzed by flow cytometry. FITC labeling with NeuCs-XL by flow cytometry. It was confirmed that the phagocytosis of colli was comparable to that of primary human neutrophils in peripheral blood (PB) (FIGS. 11A, B).
（５）酸化的バーストアッセイ
　殺菌機能に関連する酸化的破裂を評価するために、ヒト末梢血（ＰＢ）中の赤血球を溶解し、白血球を分析した。１．０×１０5個のＮｅｕＰｓ、ＮｅｕＰｓ－ＸＬ、ＮｅｕＣｓ、ＮｅｕＣｓ－ＸＬ又はヒト白血球を０．５％ウシ血清アルブミン(FUJIFILM)、1単位/mlカタラーゼ(Santa)及び１００μＭジヒドロローダミン１２３（ＤＨＲ１２３）塩酸塩(FUJIFILM)を含むＨＢＳＳ(FUJIFILM)に懸濁した。３．２μＭのＰＭＡ(Santa Curz)またはＰＢＳを細胞懸濁液に添加し、５％ＣＯ2、３７℃で２０分間インキュベートした後、ＤＨＲ１２３の蛍光をフローサイトメトリーにより分析した。ＮｅｕＣｓ－ＸＬもＰＭＡ刺激酸化的バーストを示した。これは殺菌機能と関連している（図１２Ａ、Ｂ）。これらの結果は、ＮｅｕＣｓ－ＸＬが初代ヒト好中球と同様の抗細菌活性を発揮できることを強く示唆する。 (5) Oxidative Burst Assay To evaluate oxidative rupture associated with bactericidal function, erythrocytes in human peripheral blood (PB) were lysed and leukocytes were analyzed. 1.0 × 10 5 NeuPs, NeuPs-XL, NeuCs, NeuCs-XL or human leukocytes 0.5% bovine serum albumin (FUJIFILM), 1 unit / ml catalase (Santa) and 100 μM dihydrorhodamine 123 (DHR123) Suspended in HBSS (FUJIFILM) containing hydrochloride (FUJIFILM). After adding 3.2 μM PMA (Santa Curz) or PBS to the cell suspension and incubating at 5% CO 2 , 37 ° C. for 20 minutes, the fluorescence of DHR123 was analyzed by flow cytometry. NeuCs-XL also showed a PMA-stimulated oxidative burst. This is associated with bactericidal function (FIGS. 12A, B). These results strongly suggest that NeuCs-XL can exert antibacterial activity similar to that of primary human neutrophils.
実施例４：ＮｅｕＣｓに対するインビボ機能アッセイ
（１）ｌｕｃ２発現ＮｅｕＣｓ－ＸＬのインビボイメージング
（ａ）ｌｕｃ２発現ＮｅｕＰｓ－ＸＬ及びｌｕｃ２発現ＮｅｕＣｓ－ＸＬの樹立
　ＣＳＩＩ－ＥＦ－ＭＣＳ－ＩＲＥＳ２－Ｖｅｎｕｓを用いて、ＮｅｕＰｓ－ＸＬにｌｕｃ２レンチウイルスを導入した。Ｖｅｎｕｓを発現するＮｅｕＰｓ－ＸＬの単一セルをＦＡＣＳによって単離し、増幅させ、そしてＮｅｕＣｓ－ＸＬに分化させた。インビトロにおいて、Ｄ‐ルシフェリン（VivoGIo（商標）Luciferin;Promega）曝露によりＮｅｕＣｓ－ＸＬにおけるｌｕｃ２発現を確認した。 Example 4: In vivo Function Assay for NeuCs (1) In vivo Imaging of Luc2-Expressed NeuCs-XL (a) Establishment of Luc2-Expressed NeuPs-XL and Luc2-Expressed NeuCs-XL Using CSII-EF-MCS-IRES2-Venus, NeuPs -The luc2 lentivirus was introduced into XL. A single cell of NeuPs-XL expressing Venus was isolated by FACS, amplified and differentiated into NeuCs-XL. In vitro, luc2 expression in NeuCs-XL was confirmed by exposure to D-luciferin (VivoGIo ™ Luciferin; Promega).
（ｂ）ＬＰＳ誘発炎症モデル
　２．０Ｇｙのガンマ線を照射した免疫不全性ＮＳＧ（ＮＳＧ）マウスの左下腹部に2.５ｍｇ／ｋｇのＬＰＳを腹腔内注射することにより、ＬＰＳ誘発炎症のマウスモデルを確立した。 (B) LPS-induced inflammation model A mouse model of LPS-induced inflammation was established by intraperitoneally injecting 2.5 mg / kg of LPS into the left lower abdomen of immunodeficient NSG (NSG) mice irradiated with 2.0 Gy gamma rays. did.
（ｃ）インビボイメージング（低用量）
　ＬＰＳ注射３０分後、２．０×１０5個ｌｕｃ２発現ＮｅｕＣｓ－ＸＬを腹腔内注射した（図１３Ａ）。ＬＰＳ注射の２４時間後に末梢血中の白血球数を測定し、ＬＰＳ誘導炎症を確認した（図１３Ｂ）。ｌｕｃ２発現ＮｅｕＣｓ－ＸＬを移植したマウスを、Ｄ‐ルシフェリン（VivoGIo（商標）Luciferin；Promega）の腹腔内注射後、製品説明書に従ってｉｎ　ｖｉｖｏイメージングシステム（ＰｅｒｋｉｎＥｌｍｅｒ）で生体内イメージングを行い、発光をリビング画像ソフトウェア(PerkinElmer)を用いて定量化した。マウスにＬＰＳを腹腔内注射した後、ｌｕｃ２発現ＮｅｕＣｓ－ＸＬを注射したＬＰＳ誘発炎症モデルは、１時間以内に炎症部位にＮｅｕＣｓ－ＸＬを蓄積し、ＬＰＳ注射なしのマウスにおけるＮｅｕＣｓ－ＸＬの拡散分布と著しい対照を示した。注射されたＮｅｕＣｓ－ＸＬの炎症部位での持続的な生存も明らかであった（図１３Ｃ）。 (C) In vivo imaging (low dose)
Thirty minutes after LPS injection, 2.0 × 10 5 luc2-expressing NeuCs-XL were injected intraperitoneally (Fig. 13A). The white blood cell count in the peripheral blood was measured 24 hours after the LPS injection to confirm LPS-induced inflammation (Fig. 13B). After intraperitoneal injection of D-luciferin (VivoGIo ™ Luciferin; Promega) in mice transplanted with luc2-expressing NeuCs-XL, in vivo imaging was performed with an in vivo imaging system (PerkinElmer) according to the product description, and luminescence was emitted. It was quantified using image software (PerkinElmer). An LPS-induced inflammation model in which mice were injected intraperitoneally with LPS and then injected with luc2-expressing NeuCs-XL accumulated NeuCs-XL at the site of inflammation within 1 hour, and the diffusion distribution of NeuCs-XL in mice without LPS injection. Showed a remarkable contrast. Persistent survival of the injected NeuCs-XL at the site of inflammation was also apparent (Fig. 13C).
（ｄ）インビボイメージング（高用量）
　２．０Ｇｙのガンマ線を照射したＮＳＧマウスにｌｕｃ２発現ＮｅｕＣｓ－ＸＬを静脈内注射した（図５ａ）。注入されたＮｅｕＣｓ－ＸＬの数は１．０×１０7個（５．０×１０8個／ｋｇ）であり、ヒトにおける効果的な顆粒球輸注に用いられた力価とほぼ同等の数とした。大部分の静脈内注射ＮｅｕＣｓ－ＸＬは肺に捕捉されたが、一部は末梢血（ＰＢ）中を循環した（図１４Ａ）
（ｄ－２）生体内での寿命解析
　静脈内注射された１．０×１０7個のｌｕｃ２発現ＮｅｕＣｓ－ＸＬは２日後のｉｎ　ｖｉｖｏイメージングのいずれにおいても、もはや検出できなかった（図１４Ｂ）。注射１時間後および４８時間後に末梢血を採取し、赤血球を溶解し、ＰＥ結合抗ヒトＣＤ４５およびＡＰＣ結合抗マウスＣＤ４５で末梢血（ＰＢ）を染色した。１００，０００以上の事象をフローサイトメトリーにより分析した。ｌｕｃ２発現ＮｅｕＣｓ－ＸＬは、４８時間後のフローサイトメトリーでももはや検出できなかった（図１４Ｃ、Ｄ）。これらの所見は、ＮｅｕＣｓ－ＸＬが生体内での寿命が短いことを示唆しており、これは臨床適用のための安全性の問題に関して有利である可能性を示している。 (D) In vivo imaging (high dose)
Luc2-expressing NeuCs-XL was intravenously injected into NSG mice irradiated with 2.0 Gy gamma rays (Fig. 5a). The number of NeuCs-XL injected was 1.0 × 10 7 (5.0 × 10 8 / kg), which was almost the same as the titer used for effective granulocyte infusion in humans. did. Most intravenously injected NeuCs-XL were captured in the lungs, but some circulated in the peripheral blood (PB) (FIG. 14A).
(D-2) Lifespan analysis in vivo Intravenously injected 1.0 × 10 7 luc2-expressing NeuCs-XL could no longer be detected by any of the in vivo imaging after 2 days (Fig. 14B). .. Peripheral blood was collected 1 hour and 48 hours after injection, erythrocytes were lysed, and peripheral blood (PB) was stained with PE-bound anti-human CD45 and APC-bound anti-mouse CD45. Over 100,000 events were analyzed by flow cytometry. Luc2-expressing NeuCs-XL could no longer be detected by flow cytometry after 48 hours (FIGS. 14C, D). These findings suggest that NeuCs-XL has a short lifespan in vivo, suggesting that it may be advantageous in terms of safety issues for clinical application.
（２）慢性バイオフィルム感染モデルにおけるin vivo遊走試験
（ａ）慢性バイオフィルム感染モデルの製造
　慢性皮下バイオフィルム感染モデルマウスをInfect. Immun. 71, 882-890 (2003)に従って確立した。要約すると、１４ゲージ静脈カテーテル（テルモ）を１ｃｍのセグメントに切断した。増殖指数期の１０4ＣＦＵ／ｍＬの黄色ブドウ球菌S.aureus(ATCC51153)を入れた試験管中で、前記セグメントを３７℃で３時間インキュベートすることにより、細菌バイオフィルムをカテーテルセグメント上に定着させた。定着させたカテーテルをさらに３７℃の新鮮な培地で２日間、培地を１２時間ごとに交換しつつインキュベートした。インキュベート後、カテーテルセグメントをＮＳＧマウス皮下に移植し（図１５Ｂ）、慢性皮下バイオフィルム感染モデルとした。 (2) In vivo migration test in a chronic biofilm infection model (a) Production of a chronic biofilm infection model A chronic subcutaneous biofilm infection model mouse was established according to Infect. Immun. 71, 882-890 (2003). In summary, a 14 gauge venous catheter (Terumo) was cut into 1 cm segments. Bacterial biofilms were colonized onto catheter segments by incubating the segments at 37 ° C. for 3 hours in tubes containing 10 4 CFU / mL Staphylococcus aureus S. aureus (ATCC 51153) during the growth index phase. It was. The colonized catheter was further incubated in fresh medium at 37 ° C. for 2 days, changing medium every 12 hours. After incubation, catheter segments were implanted subcutaneously in NSG mice (FIG. 15B) to create a chronic subcutaneous biofilm infection model.
（ｂ）インビボイメージング
　カテーテルセグメントをＮＳＧマウス皮下に移植７日後、1×１０7個のｌｕｃ２発現ＮｅｕＣｓ－ＸＬをマウスに静脈内注射し（図１５Ａ）、インビボイメージングシステム（PerkinElmer）で解析した（図１５Ｃ）。Ｌｕｃ２発現ＮｅｕＣｓ－ＸＬを免疫不全ＮＳＧマウスに静脈内注射したところ、ＮｅｕＣｓ－ＸＬが静脈内注射後１５分以内に感染部位に速やかに移動することを示した（図１５Ｃ）。 (B) In vivo Imaging 7 days after subcutaneously transplanting the catheter segment into NSG mice, 1 × 10 7 luc2-expressing NeuCs-XL were intravenously injected into the mice (Fig. 15A) and analyzed by an in vivo imaging system (PerkinElmer) (Fig. 15A). 15C). When Luc2-expressing NeuCs-XL was intravenously injected into immunodeficient NSG mice, it was shown that NeuCs-XL rapidly migrated to the infected site within 15 minutes after the intravenous injection (Fig. 15C).
（３）急性致死性腹膜炎モデルにおけるインビボモデル
（ａ）正常マウスにおける急性致死性腹膜炎モデル
　増殖指数期の2×１08CFU(最小致死量)の黄色ブドウ球菌(Xen-29、PerkinElmer)をBALB/cマウスに腹腔内注射することにより、細菌性腹膜炎のマウスモデルを確立した。その後、ＮｅｕＣｓ－ＸＬを腹腔内注射し、最初の２４時間は８時間ごと、その後の１３日間は２４時間ごとに生存を確認した（図１６Ａ）。細菌数の評価のため、５ｍｌのＰＢＳで腹腔内を洗浄して腹膜細菌を採取した。連続希釈試料をカナマイシン含有プレート上に平板培養し、カナマイシン耐性細菌コロニーを２４時間培養後に計数した。ＮｅｕＣｓ－ＸＬを腹腔内注射すると、用量依存的に生存率が改善し、ＮｅｕＣｓ－ＸＬを十分に大量に投与すると完全生存が得られた（図１６Ｂ）。さらに、ＮｅｕＣｓ－ＸＬを腹腔内注射すると、注射１６時間後に腹腔内のS.aureusのＣＦＵが低下することを確認した（図１６Ｃ）。
（ｂ）放射線誘発性血球減少症下マウスにおける急性致死性腹膜炎モデル
　ＢＡＬＢ／ｃマウスに準致死性（６．５Ｇｙ）で放射線を照射し、白血球の分画数を追跡した（図１７Ｂ）。５日後、増殖指数期に黄色ブドウ球菌(Xen-29、PerkinElmer)５×１０7ＣＦＵ（最小致死量に相当)を腹腔内注射することにより細菌性腹膜炎が成立した。その後、１×１０7ＮｅｕＣｓ－ＸＬを腹腔内注射し、１時間ごとに生存を確認した（図１７Ａ）。ＮｅｕＣｓ－ＸＬの腹腔内投与日より、生存率も改善した(図１７Ｄ)。これらの結果は、投与されたＮｅｕＣｓ－ＸＬが迅速に作用し、致死性感染を予防するという説得力のある証拠を提供した。 (3) an in vivo model of acute lethality peritonitis model (a) Staphylococcus aureus 2 × 10 8 CFU of acute lethal peritonitis model growth exponential phase in normal mice (minimum lethal dose) (Xen-29, PerkinElmer) BALB / A mouse model of bacterial peritonitis was established by intraperitoneal injection into c mice. Then, NeuCs-XL was injected intraperitoneally, and survival was confirmed every 8 hours for the first 24 hours and every 24 hours for the following 13 days (Fig. 16A). To evaluate the number of bacteria, peritoneal bacteria were collected by washing the abdominal cavity with 5 ml of PBS. Serially diluted samples were plate-cultured on a plate containing kanamycin, and kanamycin-resistant bacterial colonies were counted after culturing for 24 hours. Intraperitoneal injection of NeuCs-XL improved the survival rate in a dose-dependent manner, and administration of a sufficiently large amount of NeuCs-XL resulted in complete survival (Fig. 16B). Furthermore, it was confirmed that intraperitoneal injection of NeuCs-XL reduced the CFU of S. aureus in the abdominal cavity 16 hours after the injection (Fig. 16C).
(B) Acute lethal peritonitis model in radiation-induced cytopenia mice BALB / c mice were irradiated with quasi-lethal (6.5 Gy) and the number of leukocyte fractions was followed (Fig. 17B). Five days later, bacterial peritonitis was established by intraperitoneal injection of Staphylococcus aureus (Xen-29, PerkinElmer) 5 × 10 7 CFU (corresponding to the minimum lethal dose) during the growth index phase. Then, 1 × 10 7 NeuCs-XL was injected intraperitoneally, and survival was confirmed every hour (Fig. 17A). The survival rate was also improved from the day of intraperitoneal administration of NeuCs-XL (Fig. 17D). These results provided compelling evidence that the administered NeuCs-XL acted rapidly and prevented lethal infections.

Claims (15)

  1. 　誘導多能性幹細胞又は造血幹細胞などの幹細胞由来の造血前駆細胞を分化誘導後に１２週を超えて増殖能を有し、１０13以上の細胞数への増殖可能であり、好中球様細胞への分化能を有する、幹細胞由来の好中球前駆細胞。 Stem cell-derived hematopoietic progenitor cells such as induced pluripotent stem cells or hematopoietic stem cells have the ability to proliferate for more than 12 weeks after induction of differentiation, and can proliferate to a cell number of 10 13 or more, becoming neutrophil-like cells. Stem cell-derived neutrophil progenitor cells capable of differentiating.
  2. 　ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子、例えばＢＣＬ－ＸＬ、ＢＣＬ２Ａ１、及びＭＣＬ１が強制発現された、請求項１に記載の好中球前駆細胞。 The neutrophil progenitor cell according to claim 1, wherein the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family, such as BCL-XL, BCL2A1, and MCL1, are forcibly expressed.
  3. 　ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子、例えばＢＣＬ－ＸＬ、ＢＣＬ２Ａ１、及びＭＣＬ１からなる群から選ばれる少なくとも１をコードするＤＮＡが発現可能に連結された遺伝子発現ベクター、例えば薬剤制御性の遺伝子発現ベクター由来ＤＮＡを含む、好中球様細胞への分化能を有する、誘導多能性幹細胞又は造血幹細胞などの幹細胞由来の好中球前駆細胞。 A gene expression vector in which at least one gene belonging to the c-Myc gene, BMI1 gene, and BCL2 family, for example, a DNA encoding at least one selected from the group consisting of BCL-XL, BCL2A1, and MCL1 is expressively linked. For example, neutrophil precursor cells derived from stem cells such as induced pluripotent stem cells or hematopoietic stem cells, which have the ability to differentiate into neutrophil-like cells, including DNA derived from a drug-regulated gene expression vector.
  4. 　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子がＢＣＬ－ＸＬであり、フィーダー細胞非存在下で増殖能を有する、請求項２又は３に記載の好中球前駆細胞。 The neutrophil progenitor cell according to claim 2 or 3, wherein at least one gene belonging to the BCL2 family is BCL-XL and has a proliferative ability in the absence of a feeder cell.
  5. 　請求項１～４のいずれか一項に記載の好中球前駆細胞由来の好中球様細胞。 The neutrophil-like cell derived from the neutrophil progenitor cell according to any one of claims 1 to 4.
  6. 　以下の：
    　ＬＦＡ－１インテグリン（ＣＤ１１ａ／ＣＤ１８）、インテグリンα－Ｍ（ＣＤ１１ｂ）、ＣＸＣＲ１、ＦＰＲ１、及びＬ－セレクチン（ＣＤ６２Ｌ)からなる群から選ばれる、少なくとも１の接着因子を発現する、請求項５に記載の好中球様細胞。     below:
    5. The fifth aspect of claim 5, which expresses at least one adhesion factor selected from the group consisting of LFA-1 integrin (CD11a / CD18), integrin α-M (CD11b), CXCR1, FPR1 and L-selectin (CD62L). Neutrophil-like cells.
  7. 　遊走アッセイで測定した場合に、ヒト単離好中球と同等の遊走性を有し、及び／又は
    　生体内での遊走性、殺菌能力、生存延長効果を示す、請求項５又は６に記載の好中球様細胞。     The fifth or sixth claim, which has the same migratory properties as human isolated neutrophils when measured by a migration assay, and / or exhibits in vivo migration, bactericidal ability, and survival-prolonging effect. Neutrophil-like cells.
  8. 　幹細胞由来の造血前駆細胞、或いは生体由来の造血幹細胞及び/又は造血前駆細胞にｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子の強制発現下で培養する工程、
    　さらに、ＢＣＬ２ファミリーに属する少なくとも１の遺伝子、例えばＢＣＬ－ＸＬ、ＢＣＬ２Ａ１、及びＭＣＬ１の遺伝子発現の強制発現下で培養する工程、
    　を含む、好中球前駆細胞の製造方法。     A step of culturing a hematopoietic progenitor cell derived from a stem cell or a hematopoietic stem cell derived from a living body and / or a hematopoietic progenitor cell under forced expression of the c-Myc gene and the BMI1 gene.
    Further, a step of culturing under forced expression of gene expression of at least one gene belonging to the BCL2 family, for example, BCL-XL, BCL2A1 and MCL1.
    A method for producing neutrophil progenitor cells, which comprises.
  9. 　前記培養が、Ｇ－ＣＳＦ、ＳＣＦ、ＴＰＯ、及びＦＬＴ３－Ｌからなる群から選ばれる少なくとも１のサイトカイン添加培地で行われる、請求項８に記載の製造方法。 The production method according to claim 8, wherein the culture is carried out in at least one cytokine-added medium selected from the group consisting of G-CSF, SCF, TPO, and FLT3-L.
  10. 　ｃ－Ｍｙｃ遺伝子及びＢＭＩ１遺伝子の強制発現、及びＢＣＬ２ファミリーに属する少なくとも１の遺伝子の強制発現が、薬剤制御性ベクターを用いた遺伝子導入により行われ、薬剤の添加の有無により遺伝子発現を制御する、請求項９に記載の製造方法。 Forced expression of the c-Myc gene and BMI1 gene, and forced expression of at least one gene belonging to the BCL2 family are performed by gene transfer using a drug control vector, and gene expression is controlled by the presence or absence of drug addition. The manufacturing method according to claim 9.
  11. 　ＢＣＬ２ファミリーに属する少なくとも１の遺伝子がＢＣＬ－ＸＬであり、ＢＣＬ－ＸＬ強制発現下においては、フィーダー細胞非存在下で培養する工程を含む、請求項８～１０のいずれか一項に記載の製造方法。 The production according to any one of claims 8 to 10, wherein at least one gene belonging to the BCL2 family is BCL-XL, and under the forced expression of BCL-XL, the step of culturing in the absence of feeder cells is included. Method.
  12. 　請求項８～１１のいずれか一項に記載の製造方法により製造された好中球前駆細胞。 Neutrophil progenitor cells produced by the production method according to any one of claims 8 to 11.
  13. 　請求項１～４、及び１２のいずれか一項に記載の好中球前駆細胞において、ｃ－Ｍｙｃ遺伝子、ＢＭＩ１遺伝子、並びにＢＣＬ２ファミリーに属する少なくとも１の遺伝子の遺伝子発現を抑制又は停止する工程をさらに含む、好中球様細胞の製造方法。 The step of suppressing or stopping the gene expression of the c-Myc gene, the BMI1 gene, and at least one gene belonging to the BCL2 family in the neutrophil progenitor cell according to any one of claims 1 to 4 and 12. A method for producing neutrophil-like cells, which further comprises.
  14. 　請求項５～７のいずれか一項に記載の好中球様細胞、又は請求項１３に記載の製造方法により製造された好中球様細胞を含む、好中球減少により引き起こされる疾患、例えば好中球減少症又は好中球減少性発熱の治療又は予防用組成物、或いは研究用組成物。 A disease caused by neutropenia, including the neutrophil-like cell according to any one of claims 5 to 7, or the neutrophil-like cell produced by the production method according to claim 13. A composition for the treatment or prevention of neutropenia or neutropenic fever, or a composition for research.
  15. 　好中球減少症又は好中球減少性発熱などの好中球減少を引き起こしているか、引き起こすリスクのある対象を治療又は予防する方法であって、
    　請求項５～７のいずれか一項に記載の好中球様細胞を前記対象に投与する工程
    　を含む、前記方法。     A method of treating or preventing a subject who is or is at risk of causing neutropenia, such as neutropenia or neutropenic fever.
    The method comprising the step of administering the neutrophil-like cell according to any one of claims 5 to 7 to the subject.
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