WO2019127664A1 - 一种多能干细胞及其分化的t细胞和应用 - Google Patents

一种多能干细胞及其分化的t细胞和应用 Download PDF

Info

Publication number
WO2019127664A1
WO2019127664A1 PCT/CN2018/072254 CN2018072254W WO2019127664A1 WO 2019127664 A1 WO2019127664 A1 WO 2019127664A1 CN 2018072254 W CN2018072254 W CN 2018072254W WO 2019127664 A1 WO2019127664 A1 WO 2019127664A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
medium
cell
stem cell
recombinant mouse
Prior art date
Application number
PCT/CN2018/072254
Other languages
English (en)
French (fr)
Inventor
王金勇
郭荣群
张梦云
刘丽娟
刘晓飞
吕翠
杜鹃
Original Assignee
中国科学院广州生物医药与健康研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院广州生物医药与健康研究院 filed Critical 中国科学院广州生物医药与健康研究院
Priority to US16/312,794 priority Critical patent/US11299709B2/en
Priority to JP2020535615A priority patent/JP7098187B2/ja
Publication of WO2019127664A1 publication Critical patent/WO2019127664A1/zh

Links

Images

Classifications

    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the technical field of medical bioengineering and relates to a pluripotent stem cell and a differentiated T cell and application thereof.
  • Pluripotent stem cells are a class of cells with unlimited proliferative potential, differentiated into different lineages, and are easily genetically modified. They are hotspots in stem cell research. Inducing the differentiation of autologous pluripotent stem cells into different tissues can not only avoid ethical controversy, but also reduce the risk of immune rejection, which is a hotspot in the field of regenerative medicine. As an emerging immune cell therapy, CAR-T has a strong specificity and high cancer cell removal efficiency, and has received extensive attention.
  • the immune cells of CAR-T therapy are mainly derived from the patient's own T cells, but some patients (such as infants, advanced tumor immunodeficiency patients and severe chemotherapy patients) are unable to provide effective doses of T cells, and are expensive, which greatly limits the The application of therapy.
  • the above problems can be solved by obtaining functional T cells by pluripotent stem cells.
  • HSPCs hematopoietic stem and progenitor cells with multi-lineage hematopoietic reconstitution by expressing transcription factors ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1 in human pluripotent stem-derived hematopoietic endothelium.
  • HSPCs after transplantation, can produce multiple hematopoietic lineage cells (including T cells) (R. Sugimura et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature, 545, 432-438 (2017)), but the above The study requires up to seven transcription factors for stem cell induction, complex operation, poor stability, and low efficiency.
  • hematopoietic multipotent progenitors with partial lineage hematopoietic reconstitution can be obtained by expressing transcription factors FOSB, GFI1, RUNX1 and SPI1 in endothelial cells.
  • FOSB transcription factors
  • GFI1 transcription factors required to express hematopoietic stem cells.
  • the present invention provides a pluripotent stem cell and a differentiated T cell thereof, and the obtained pluripotent stem cell-derived T cell not only functions normally, but also has no tumorigenic risk.
  • the invention provides a vector comprising Runx1 and Hoxa9 co-expressed in tandem.
  • the cDNA sequences of Runx1 and Hoxa9 are expressed in tandem in the same vector for infecting host cells, and host cells stably expressing Runx1 and Hoxa9 can be obtained, which are simple and efficient, and the obtained host cells have differentiation into T cells.
  • Ability the expression of Runx1 and Hoxa9
  • the invention provides a nucleic acid expressing a vector of the first aspect.
  • the present invention provides a host cell, comprising the vector of the first aspect
  • the host cell is a pluripotent stem cell.
  • the present invention provides a method for directional differentiation of T cells by pluripotent stem cells, comprising the steps of:
  • step (1) Directly differentiating the pluripotent stem cells of step (1) into hematopoietic stem cell precursor cells;
  • step (3) co-culturing the hematopoietic stem cell precursor cells of step (2) with mouse bone marrow stromal cells to obtain T-lineage progenitor cells;
  • T-lineage progenitor cells are differentiated into T cells.
  • the obtained hematopoietic stem cell precursor cells are co-cultured with the OP9-DL1 cell line to obtain T-lineage progenitor cells, and the normalized T cells are obtained after differentiation. No tumorigenic risk.
  • the expression vector of Runx1 and Hoxa9 tandem in step (1) is integrated into the Rosa26 site of pluripotent stem cells.
  • the pluripotent stem cell of step (1) is a genetically edited induced pluripotent stem cell and/or an embryonic pluripotent stem cell line.
  • the method of integration according to step (1) comprises any one of homologous recombination, CRISPR/Cas9, TALEN, transfection or viral infection or a combination of at least two, preferably homologous recombination.
  • the resistance screening described in step (1) employs hygromycin B.
  • the method for directing differentiation according to step (2) comprises sequentially culturing pluripotent stem cells using D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium.
  • the hematopoietic stem cell precursor cell comprises sequentially culturing pluripotent stem cells using D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium.
  • the DO medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), and the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng. /mL, preferably 5 ng/mL.
  • BMP4 bone morphogenetic protein 4
  • the D2.5 medium is a basic differentiation medium containing 3-8 ng/mL activin A (Activin A) and 3-8 ng/mL basic fibroblast growth factor (bFGF), the activin
  • the concentration of A may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of the basic fibroblast growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL. It is preferably 5 ng/mL.
  • the D3 medium is containing 3-8 ng/mL activin A (Activin A), 3-8 ng/mL bone morphogenetic protein 4 (BMP4) and 3-8 ng/mL vascular endothelial growth factor (VEGF).
  • Activin A activin A
  • BMP4 bone morphogenetic protein 4
  • VEGF vascular endothelial growth factor
  • the concentration of the activin A may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng.
  • the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL.
  • the D4 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4) and 3-8 ng/mL vascular endothelial growth factor (VEGF), the bone morphogenetic protein 4
  • BMP4 bone morphogenetic protein 4
  • VEGF vascular endothelial growth factor
  • the concentration may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL. .
  • the D5 medium contains 3-8 ng/mL bone morphogenetic protein 4 (BMP4), 3-8 ng/mL vascular endothelial growth factor (VEGF), and 10-30 ng/mL recombinant mouse interleukin 3 (mIL3).
  • BMP4 bone morphogenetic protein 4
  • VEGF vascular endothelial growth factor
  • mIL3 mouse interleukin 3
  • mIL6 mouse interleukin 6
  • mSCF mouse stem cell factor
  • hTPO human thrombopoietin
  • 10-30 ng/mL human a basal differentiation medium of Fms-associated tyrosine kinase 3 ligand (hFlt3L) which may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of the recombinant mouse interleukin 3 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL.
  • the concentration of the recombinant mouse interleukin 6 can be 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, and the concentration of the recombinant mouse stem cell factor can be, for example, 10 ng.
  • the concentration of the recombinant human thrombopoietin may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, hFlt3L It may be, for example, the concentration of 10ng / mL, 20ng / mL or 30ng / mL, preferably 20ng / mL.
  • the D6 medium contains 3-8 ng/mL bone morphogenetic protein 4 (BMP4), 3-8 ng/mL vascular endothelial growth factor (VEGF), and 10-30 ng/mL recombinant mouse interleukin 3 (mIL3).
  • BMP4 bone morphogenetic protein 4
  • VEGF vascular endothelial growth factor
  • mIL3 mouse interleukin 3
  • the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • the concentration of the recombinant mouse interleukin 3 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL.
  • the concentration of the recombinant mouse interleukin 6 can be 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, and the concentration of the recombinant mouse stem cell factor can be, for example, 10 ng.
  • the concentration of the recombinant human thrombopoietin may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, hFlt3L It may be, for example, the degree of 10ng / mL, 20ng / mL or 30ng / mL, preferably 20ng / mL, the concentration of doxycycline may be for example 1 ⁇ g / mL or 2 ⁇ g / mL, preferably 1 ⁇ g / mL.
  • the D7 medium comprises 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF).
  • mIL3 mouse interleukin 3
  • mIL6 mouse interleukin 6
  • mSCF mouse stem cell factor
  • the concentration of the recombinant mouse interleukin 6 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL.
  • the concentration of the recombinant mouse stem cell factor may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL
  • the concentration of the recombinant human thrombopoietin may be, for example, 10 ng/mL, 20 ng/
  • the concentration of the hFlt3L may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL
  • the concentration of the doxycycline may be, for example, 1 ⁇ g/mL, or 30 ng/mL, preferably 20 ng/mL.
  • mL or 2 ⁇ g/mL preferably 1 ⁇ g/mL.
  • the basal differentiation medium comprises 10-20% fetal calf serum, 180-220 ⁇ g/mL iron-saturated transferrin, 4.5 ⁇ 10 ⁇ 4 M thioglycerol, 1-3 mM GlutaMAX TM -I additive, IMDM medium of 0.4-0.6 mM ascorbic acid
  • the concentration of the fetal bovine serum may be, for example, 10%, 15% or 20%, preferably 15%
  • the concentration of the iron-saturated transferrin may be, for example, Is 180 ⁇ g / mL, 200 ⁇ g / mL or 220 ⁇ g / mL, preferably 200 ⁇ g / mL
  • the concentration of the thioglycerol may be, for example, 4 ⁇ 10 -4 M, 4.5 ⁇ 10 -4 M or 5 ⁇ 10 -4 M,
  • it is 4.5 ⁇ 10 ⁇ 4 M
  • the concentration of the GlutaMAX TM ⁇ I additive may be, for
  • the inventors designed and optimized the hematopoietic differentiation system by changing the additive substance in the culture medium, and induced the hematopoietic differentiation of the pluripotent stem cells into hematopoietic stem cell precursor cells, which further penetrate the mouse bone marrow matrix.
  • the cells were co-cultured to obtain T-lineage progenitor cells.
  • the stromal cells of step (3) are OP9-DL1 cells.
  • the co-cultivation process in step (3) is carried out using doxycycline.
  • the T cells in step (4) are mainly CD3 + T cells.
  • the T cell is a TCR ⁇ cell and/or a TCR ⁇ / ⁇ cell.
  • the present invention provides a method for directional differentiation of T cells by pluripotent stem cells, comprising the following steps:
  • pluripotent stem cells according to step (1) are sequentially cultured in D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium, in the first 11 days of directed differentiation into hematopoietic stem cell precursor cells;
  • step (3) co-culturing the hematopoietic stem cell precursor cells in step (2) with OP9-DL1 cells, and inducing with doxycycline to obtain T-lineage progenitor cells;
  • T-lineage progenitor cells are differentiated into T cells, which are TCR ⁇ cells and/or TCR ⁇ / ⁇ cells.
  • the present invention provides a T-lineage progenitor cell and/or a T cell prepared by the method of the first aspect.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the vector of the first aspect, the host cell of the third aspect, the T-lineage progenitor cell or the T cell of the fifth aspect Any one or a combination of at least two.
  • the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides a pharmaceutical composition according to the fourth aspect for use in the preparation of a medicament for enhancing an immune response, preferably for the preparation of a medicament for enhancing an immune response of a T cell.
  • the pharmaceutical composition can be used to enhance the immune response, particularly to enhance the T cell immune response.
  • the present invention provides a pharmaceutical composition according to the fourth aspect for use in the preparation of a medicament for preventing and/or treating immunodeficiency, preferably for the preparation of a medicament for preventing and/or treating T cell immunodeficiency.
  • the pharmaceutical composition can be used for the prevention and/or treatment of immunodeficiency, particularly prevention and/or treatment of T cell immunodeficiency.
  • the present invention provides a pharmaceutical composition according to the fourth aspect for use in the preparation of a T cell immunotherapy for treating a tumor.
  • the pharmaceutical composition can be used for T cell immunotherapy.
  • the present invention has the following beneficial effects:
  • the present invention introduces an exogenous Runx1 and Hoxa9 co-expression vector into a pluripotent stem cell, and successfully constructs a pluripotent stem cell which inducibly expresses exogenous Runx1 and Hoxa9, and the pluripotent stem cell has the ability to differentiate into a T cell. And can be used to prepare drugs that enhance immune effects, prevent and/or treat immunodeficiency, and treat tumors;
  • the present invention adopts a directed differentiation system and a co-culture method to differentiate the pluripotent stem cells into T-lineage progenitor cells, and the T-lineage progenitor cells can be induced to differentiate into T cells, and can be used for preparing an enhanced immune effect, A drug that prevents and/or treats immunodeficiencies and treats tumors;
  • the pluripotent stem cell-derived T cells obtained by the method of the present invention have normal functions and no tumorigenic risk, and can be used for preparing a drug for enhancing an immune effect, preventing and/or treating immunodeficiency, and treating a tumor.
  • Figure 1 (A) is a schematic diagram of an inducible expression system for site-directed knock-in of the pluripotent stem cell Rosa26 site, which uses the p2a sequence to tandem the Runx1 and Hoxa9 cDNA sequences, and uses doxycycline to induce gene expression
  • Figure 1 (B) is a light field map of iRunx1-p2a-Hoxa9 pluripotent stem cells obtained by hygromycin resistance screening
  • Figure 1 (C) shows the relative expression levels of Runx1 and Hoxa9 after 24 hours of treatment with doxycycline;
  • Fig. 2(A) is a schematic diagram showing the embryoid body-monolayer culture system for inducing iRunx1-p2a-Hoxa9 pluripotent stem cells to differentiate into hematopoietic precursors, hematopoietic stem cell precursor cells and blood cells
  • Fig. 2(B) is an induced iRunx1-
  • the p2a-Hoxa9 pluripotent stem cells were differentiated to the cell morphology map on the 11th day
  • Fig. 2(C) was the flow cytometry analysis of the composition and proportion of hematopoietic-related cells on the 11th day of directional differentiation;
  • Figure 3 (A) is a flow sorting strategy for hematopoietic stem cell precursor cells
  • Figure 3 (B) is a sort of hematopoietic stem cell precursor cell population (CD31 + CD41 low CD45 - c-Kit + CD201 high ) and OP9-DL1 Schematic diagram of co-culture of cell lines
  • Figure 3 (C) shows the number of cobblestone-like regions observed under microscope after co-culture of hematopoietic stem cell precursor cell population with OP9-DL1 cell line for 10 days
  • Figure 3 (D) shows hematopoietic stem cell precursor. Light field map of the cobblestone-like formation area observed under the microscope after co-culture of the cell population with the OP9-DL1 cell line for 10 days.
  • Figure 4 (A) shows the transplantation of T-lineage progenitor cells into CD45.1 + NOD/SCID immunodeficient mice after co-culture of hematopoietic stem cell precursor cells with OP9-DL1 cell line, and Figure 4 (B) is transplanted for 4 weeks.
  • hemagglutination-derived blood cells were identified by flow cytometry, hematopoietic chimerism was detected in the iRunx1-p2a-Hoxa9 group, and peripheral blood, bone marrow, and spleen of the sacrificial recipient mice were observed in FIG. 4(C) after 5 weeks.
  • Fig. 4 (D) is the PCR and sequencing identification of the genome of blood cells derived from pluripotent stem cells.
  • Fig. 5(A) shows the DN cell population (DN1/DN2/DN3/DN4) analysis of pluripotent stem cells in the thymus of the victim recipient mice after 4 weeks
  • Fig. 5(B) shows the victim mice after 4 weeks.
  • Figure 5 (C) is a mixed lymphocyte reaction experiment in sacrificed mice 4 weeks later.
  • MLR MLR
  • PSC-T is Runx1-p2a-Hoxa9-induced pluripotent stem cell line differentiation.
  • T-lineage progenitor cells were transplanted into NOD-SCID recipient mice for 6 weeks, and magnetic beads-enriched CD3 + T cells were spleen.
  • Figure 6 is a representative cytokine secreted by ELISA in vitro stimulated T cells, wherein IL10-interleukin 10, IFN- ⁇ - ⁇ interferon, IL-2-interleukin 2, TNF- ⁇ -tumor necrosis factor ⁇ .
  • the inducible expression sequence was specifically inserted into the Rosa26 locus of pluripotent stem cells.
  • the knock-in sequence contains the Runx1-p2a-Hoxa9 tandem sequence and is used for anti- sexually screened hygromycin B resistance gene sequence.
  • electroporation was carried out for 20 hours, and pluripotent stem cell medium containing hygromycin B (150 ⁇ g/mL) was added, and the cells were changed every day.
  • the cloned group was adhered to the MEF cell layer, and the solution was changed every day. After 3 days, the clone was digested with 0.25% trypsin and passaged into a 12-well plate. The cell morphology was as shown in Fig. 1(B), and the clone was in logarithmic growth. At the same time, the edge is neat and translucent and there is a clear boundary between the MEF cell layer and no differentiation occurs. Passage, amplification and cryopreservation are carried out according to cell state and growth density.
  • the iRunx1-p2a-Hoxa9 pluripotent stem cell total mRNA was extracted 24 hours after Dox treatment (no Dox group was used as a control group), and the expression levels of Runx1 and Hoxa9 mRNA were obtained by Q-PCR.
  • Figure 1 (C) shows that Dox was added. Expression of Runx1 and Hoxa9 can be induced.
  • a directional hematopoietic differentiation system as shown in Fig. 2(A) was used.
  • the formulation of each medium in the directional hematopoietic differentiation system is:
  • Basic differentiation medium BDM containing 15% fetal bovine serum, 200 ⁇ g / mL transferrin iron saturation, 4.5 ⁇ 10 -4 M thioglycerol, 2mM GlutaMAX TM -I additives, 0.5mM ascorbic acid IMDM medium;
  • D0 medium a basic differentiation medium containing 5 ng/mL bone morphogenetic protein 4;
  • D2.5 medium basic differentiation medium containing 5 ng/mL activin A and 5 ng/mL basic fibroblast growth factor;
  • D3 medium basal differentiation medium containing 5 ng/mL activin A, 5 ng/mL bone morphogenetic protein 4, and 5 ng/mL vascular endothelial growth factor;
  • D4 medium a basic differentiation medium containing 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelial growth factor;
  • D5 medium containing 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelial growth factor, 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng /mL recombinant human thrombopoietin and 20ng/mL hFlt3L basal differentiation medium;
  • D6 medium containing 5 ng/mL bone morphogenetic protein 4, 5 ng/mL vascular endothelial growth factor, 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng /mL basal differentiation medium recombinant human thrombopoietin, 20 ng/mL hFlt3L and 1 ⁇ g/mL doxycycline;
  • D7 medium containing 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin, 20 ng/mL hFlt3L and 1 ⁇ g/mL Basic differentiation medium for doxycycline.
  • the suspended cells were collected, centrifuged at 250 g for 5 min, and washed once with DPBS. The cells were resuspended using D0 medium and counted, and the cell concentration was adjusted to 1 ⁇ 10 5 /mL. 5-10 mL of the cell suspension was added to a tilted 10 cm dish, 20 ⁇ L of the cell suspension was aspirated, and the embryoid body (EB) was suspended in a 15 cm dish, and the single EB was 20 ⁇ L (about 2000 cells). The dish was then inverted and a 10 cm Petri dish lid was placed at the bottom of the Petri dish and 5-6 mL of cell culture water was added to the lid. Incubate for 2.5 days in a 37 ° C incubator.
  • the EB was collected into a centrifuge tube with a Pasteur pipette, and the bottom of the dish was washed with DPBS. After the EB was naturally settled, the supernatant was carefully aspirated. The supernatant was removed by centrifugation at 90 g for 5 min, and DPBS was added to the supernatant for further sedimentation or centrifugation. Go to the supernatant. After resuspending the EB in D2.5 medium, the cells were transferred to a low-adherence 24-well plate and cultured for 12 hours to observe whether the EB was contaminated.
  • the liquid was then changed every other day and the medium used was D7 medium.
  • the medium used was D7 medium.
  • Fig. 2(B) on the 11th day, obvious hematopoietic clusters were observed in the iRunx1-p2a-Hoxa9 differentiation group; the flow cytometry analysis shown in Fig. 2(C) showed that hematopoiesis on the 11th day of directional differentiation
  • the relevant cell population is CD41 + hematopoietic precursor cells and CD45 + blood cells.
  • hematopoietic progenitor cells differentiated from pluripotent stem cells have the ability to proliferate embryonic hematopoietic stem cell precursor cell populations, that is, the ability to form cep-like regions of high expansion potential on stromal cells
  • the inventors will make hematopoietic stem cells.
  • the precursor cells are co-cultured with mouse bone marrow stromal cells.
  • Co-cultivation medium containing 15% DFBS, 200 ⁇ g / mL transferrin iron saturation, 4.5 ⁇ 10 -4 M thioglycerol, 2mM GlutaMAX TM -I additives, 0.5mM ascorbic acid, 2% AFT024-mSCF conditioned medium, 2% AFT024-mIL3 conditional medium, 2% AFT024-hFlt3L conditional medium and 1 ⁇ g/mL Dox alpha-MEM medium.
  • the hematopoietic stem cell precursor cells were sorted by flow cytometry using the sorting strategy of Figure 3(A) (CD31 + CD41 low CD45 - c-Kit + CD201 high ) . Subsequently, the cobblestone-like region formation experiment (CAFC) was used to examine whether the hematopoietic stem cell precursor cells differentiated from the pluripotent stem cells have the same proliferative ability as the embryo-derived hematopoietic stem cell precursor cells. As shown in Fig.
  • CAFC cobblestone-like region formation experiment
  • FIG. 3(B) the sorted hematopoietic stem cell precursor cell population was re-plated onto OP9-DL1 stromal cells, and the number of cobblestone-forming regions formed per 100 hematopoietic stem cell precursor cells 10 days later.
  • Figure 3 (C) and Figure 3 (D) show that iRunx1-p2a-Hoxa9 pluripotent stem cell-derived hematopoietic stem cell precursor cells have strong cobblestone-like region forming ability, and pluripotent stem cell-derived hematopoietic stem cell precursor cells are Highly uniform small, round, bright blood cells are formed on stromal cells OP9-DL1.
  • hematopoietic stem cell precursor cells were induced by adding Dox on OP9-DL1 stromal cells for 10 days to obtain a T-lineage system. Progenitor cells.
  • the OP9-DL1 cell line was resuscitated 4 days in advance, and was passaged in time according to the cell growth state to prevent the cells from aging due to excessive growth. After passage for the day before use, 50,000 cells (12-well plates) were re-plated per well and used the next day.
  • Figure 4 (B) shows that the T-lineage progenitor cells obtained by co-culture of the iRunx1-p2a-Hoxa9 pluripotent stem cell-derived hematopoietic stem cell precursor cell population can form hematopoietic chimerism in the peripheral blood of recipient NOD/SCID mice. And CD3 + T cells (97.7%), the effect of effective reconstruction of T lymphoid system was achieved.
  • peripheral blood, bone marrow, spleen and thymus blood cell lineages were analyzed by flow cytometry after 5 weeks of sacrifice. .
  • Flow cytometry analysis showed that, as shown in Fig. 4(C), in the bone marrow, thymus and spleen, blood cells derived from pluripotent stem cells were also mainly T-lineage hematopoiesis.
  • This group of CD3 + T cells contains small amounts of CD4 + CD8 + double positive cells and CD4 - CD8 - double negative cells in the spleen, bone marrow and thymus. .
  • CD45.2 + hematopoietic cells mainly T cells
  • primers were designed for PCR amplification and sequencing.
  • the genomes of the bone marrow and spleen-derived CD45.2 + cells were flow-sorted, and the genome was extracted, and the specific primers of the knock-in gene sequences were used for PCR identification.
  • Figure 4 (D) shows that iRunx1-p2a-Hoxa9 plasmid-derived sequences were present in these cell genomes, confirming that CD45.2 + blood cells (primarily T cells) were derived from iRunx1-p2a-Hoxa9 pluripotent stem cells.
  • Fig. 5(A) the T cells in the recipient mice develop normally and can detect DN1, DN2. DN3 and DN4 cell populations. Detection of pluripotent T cell receptors derived from pluripotent stem cells in peripheral blood, spleen and lymphatic vessels, as shown in Fig. 5(B), there is a certain proportion of TCR ⁇ / ⁇ cells (0.37-1.71%) in T cells. Most of them are TCR ⁇ cells.
  • the mixed lymphocyte reaction was carried out using Balb/C mouse spleen cells and T cells obtained from the spleen of recipient mice by CD3 magnetic bead enrichment, and were detected on days 3 and 6, respectively, as shown in Fig. 5(C). It was shown that pluripotent stem cell-derived T cells were able to proliferate after activation, confirming that these T cells have proliferative ability after stimulation.
  • the culture supernatant was analyzed by ELISA. As shown in Fig. 6, the regenerated T cells stimulated proliferation can secrete a large amount of interleukin 10 (IL10), interferon gamma (IFN- ⁇ ), interleukin 2 (IL-2) and tumor necrosis.
  • IL-10 interleukin 10
  • IFN- ⁇ interferon gamma
  • IL-2 interleukin 2
  • TNF- ⁇ tumor necrosis.
  • TNF- ⁇ tumor necrosis.
  • the present invention introduces exogenous Runx1 and Hoxa9 co-expression vectors into pluripotent stem cells, and successfully constructs pluripotent stem cells that inducibly coexpress exogenous Runx1 and Hoxa9, and the pluripotent stem cells are differentiated into T-lineage progenitors. Cells and will develop into T cells.
  • the pluripotent stem cell-derived T cells obtained by the method of the present invention are not only functionally normal but also have no tumorigenic risk, and can be used for preparing a drug for enhancing an immune effect, preventing and/or treating immunodeficiency, and treating a tumor.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Transplantation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供了一种多能干细胞及其分化的T细胞和应用，所述多能干细胞包含有Runx1和Hoxa9串联共表达载体。将外源Runx1和Hoxa9共表达载体引入多能干细胞中，成功构建了诱导性共表达外源Runx1和Hoxa9的多能干细胞，所述多能干细胞定向分化为T谱系祖细胞，并将发育为T细胞。使用该方法获得的多能干细胞来源的T细胞，不仅功能正常，而且没有致瘤风险。

Description

一种多能干细胞及其分化的T细胞和应用 技术领域
本发明属于医药生物工程技术领域，涉及一种多能干细胞及其分化的T细胞和应用。
背景技术
多能干细胞(pluripotent stem cells，PSCs)是一类具有无限增殖潜能、分化形成不同谱系细胞组织、易于进行基因修饰的细胞，是当前干细胞研究的热点。诱导患者自体来源的多能干细胞分化形成不同的组织，不但可以规避伦理争议，而且降低了免疫排斥风险，是再生医学领域的应用热点。CAR-T作为一种新兴的免疫细胞疗法，具有特异性强、癌细胞清除效率高的特点，受到了广泛的关注。目前CAR-T疗法的免疫细胞主要来源于患者自身T细胞，然而一部分患者(例如婴儿、肿瘤晚期免疫缺陷病人和重度化疗病人)无法提供有效剂量的T细胞，且费用昂贵，极大地限制了该疗法的应用。通过多能干细胞获得功能性T细胞，可以解决上述问题。
已有基础研究通过在人多能干细胞来源的生血内皮中表达转录因子ERG、HOXA5、HOXA9、HOXA10、LCOR、RUNX1和SPI1获得了具有多谱系造血重建能力的造血干组细胞(hematopoietic stem and progenitor cells，HSPCs)，移植后可以产生多个造血谱系细胞(包括T细胞)(R.Sugimura et al.Haematopoietic stem and progenitor cells from human pluripotent stem cells.Nature，545，432-438(2017))，但上述研究需要使用多达七个转录因子进行干细胞诱导，操作复杂、稳定性差、效率较低。
还有研究报道称，在内皮细胞中表达转录因子FOSB、GFI1、RUNX1和SPI1可以获得具有部分谱系造血重建能力(无T细胞谱系造血重建能力)的人造血多能祖细胞(hematopoietic multipotent progenitors)和具有全谱系造血重建能力的小鼠造血干细胞(hematopoietic stem cell)(V.M.Sandler et al.Reprogramming human endothelial cells to haematopoietic cells requires vascular induction.Nature 511，213-318(2014)；R.Lis et al.Conversion of adult endothelium to immunocompetent haematopoietic stem cells.Nature 545，439-445(2017))，然而上述研究存在内皮细胞取材不便、基因编辑困难、技术方法繁琐、T谱系产生效率低等问题。因此，需要一种简单的诱导多能干细胞获得单一T谱系细胞的方法。
发明内容
针对现有技术的不足，本发明提供一种多能干细胞及其分化的T细胞和应用，得到的多能干细胞来源的T细胞不仅功能正常，而且没有致瘤风险。
第一方面，本发明提供了一种载体，所述载体包括Runx1和Hoxa9串联共表达。
本发明中，将Runx1和Hoxa9的cDNA序列串联表达于同一载体，用于感染宿主细胞，可以得到稳定表达Runx1和Hoxa9的宿主细胞，操作简便、效率较高，得到的宿主细胞具有分化为T细胞的能力。
第二方面，本发明提供了一种表达如第一方面所述载体的核酸。
第三方面，本发明提供了一种宿主细胞，所述宿主细胞包含如第一方面所述的载体；
优选地，所述宿主细胞为多能干细胞。
第四方面，本发明提供了一种多能干细胞定向分化T细胞的方法，包括以下步骤：
(1)将Runx1和Hoxa9串联的表达载体整合到多能干细胞中，并进行抗性筛选；
(2)将步骤(1)所述多能干细胞定向分化为造血干细胞前体细胞；
(3)将步骤(2)所述造血干细胞前体细胞与小鼠骨髓基质细胞共培养，得到T谱系祖细胞；
(4)诱导步骤(3)所述T谱系祖细胞分化为T细胞。
本发明中，通过对Runx1和Hoxa9共表达的多能干细胞系进行定向分化，获得的造血干细胞前体细胞与OP9-DL1细胞系共培养获得T谱系祖细胞，分化后得到功能正常的T细胞，无致瘤风险。
优选地，步骤(1)所述Runx1和Hoxa9串联的表达载体整合到多能干细胞的Rosa26位点。
优选地，步骤(1)所述多能干细胞为基因编辑后的诱导性多能干细胞和/或胚胎多能干细胞系。
优选地，步骤(1)所述整合的方法包括同源重组、CRISPR/Cas9、TALEN、转染或病毒感染中的任意一种或至少两种的组合，优选为同源重组。
优选地，步骤(1)所述抗性筛选采用潮霉素B。
优选地，步骤(2)所述定向分化的方法为依次采用D0培养基、D2.5培养基、D3培养基、D4培养基、D5培养基、D6培养基和D7培养基培养多能干细胞得到所述造血干细胞前体细胞。
优选地，所述D0培养基为含有3-8ng/mL骨形态发生蛋白4(BMP4)的基础分化培养基，所述骨形态发生蛋白4的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL。
优选地，所述D2.5培养基为含有3-8ng/mL激活素A(Activin A)和3-8ng/mL碱性成纤维细胞生长因子(bFGF)的基础分化培养基，所述激活素A的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述碱性成纤维细胞生长因子的浓度例如可以是3 ng/mL、5ng/mL或8ng/mL，优选为5ng/mL。
优选地，所述D3培养基为含有3-8ng/mL激活素A(Activin A)、3-8ng/mL骨形态发生蛋白4(BMP4)和3-8ng/mL血管内皮生长因子(VEGF)的基础分化培养基，所述激活素A的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述骨形态发生蛋白4的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述血管内皮生长因子的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL。
优选地，所述D4培养基为含有3-8ng/mL骨形态发生蛋白4(BMP4)和3-8ng/mL血管内皮生长因子(VEGF)的基础分化培养基，所述骨形态发生蛋白4的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述血管内皮生长因子的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL。
优选地，所述D5培养基为含有3-8ng/mL骨形态发生蛋白4(BMP4)、3-8ng/mL血管内皮生长因子(VEGF)、10-30ng/mL重组小鼠白介素3(mIL3)、10-30ng/mL重组小鼠白介素6(mIL6)、10-30ng/mL重组小鼠干细胞因子(mSCF)、10-30ng/mL重组人促血小板生成素(hTPO)和10-30ng/mL人Fms相关酪氨酸激酶3配体(hFlt3L)的基础分化培养基，所述骨形态发生蛋白4的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述血管内皮生长因子的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述重组小鼠白介素3的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组小鼠白介素6的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组小鼠干细胞因子的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组人促血小板生成素的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述hFlt3L的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL。
优选地，所述D6培养基为含有3-8ng/mL骨形态发生蛋白4(BMP4)、3-8ng/mL血管内皮生长因子(VEGF)、10-30ng/mL重组小鼠白介素3(mIL3)、10-30ng/mL重组小鼠白介素6(mIL6)、10-30ng/mL重组小鼠干细胞因子(mSCF)、10-30ng/mL重组人促血小板生成素(hTPO)、10-30ng/mL hFlt3L和1-2μg/mL强力霉素(Dox)的基础分化培养基，所述骨形态发生蛋白4的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述血管内皮生长因子的浓度例如可以是3ng/mL、5ng/mL或8ng/mL，优选为5ng/mL，所述重组小鼠白介素3的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组小鼠白介素6的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL， 所述重组小鼠干细胞因子的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组人促血小板生成素的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述hFlt3L的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述强力霉素的浓度例如可以是1μg/mL或2μg/mL，优选为1μg/mL。
优选地，所述D7培养基为含有10-30ng/mL重组小鼠白介素3(mIL3)、10-30ng/mL重组小鼠白介素6(mIL6)、10-30ng/mL重组小鼠干细胞因子(mSCF)、10-30ng/mL重组人促血小板生成素(hTPO)、10-30ng/mL hFlt3L和1-2μg/mL强力霉素(Dox)的基础分化培养基，所述重组小鼠白介素3的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组小鼠白介素6的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组小鼠干细胞因子的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述重组人促血小板生成素的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述hFlt3L的浓度例如可以是10ng/mL、20ng/mL或30ng/mL，优选为20ng/mL，所述强力霉素的浓度例如可以是1μg/mL或2μg/mL，优选为1μg/mL。
优选地，所述基础分化培养基为包含10-20％胎牛血清、180-220μg/mL铁饱和转铁蛋白(iron-saturated transferrin)、4.5×10 -4M硫代甘油、1-3mM GlutaMAX TM-I添加剂、0.4-0.6mM抗坏血酸的IMDM培养基，所述胎牛血清的浓度例如可以是10％、15％或20％，优选为15％，所述铁饱和转铁蛋白的浓度例如可以是180μg/mL、200μg/mL或220μg/mL，优选为200μg/mL，所述硫代甘油的浓度例如可以是4×10 -4M、4.5×10 -4M或5×10 -4M，优选为4.5×10 -4M，所述GlutaMAX TM-I添加剂的浓度例如可以是1mM、2mM或3mM，优选为2mM，所述抗坏血酸的浓度例如可以是0.4mM、0.5mM或0.6mM，优选为0.5mM。
本发明中，发明人通过改变培养基中的添加物质，设计优化了定向造血分化体系，诱导多能干细胞造血分化为造血干细胞前体细胞，所述造血干细胞前体细胞通过进一步与小鼠骨髓基质细胞共培养，得到T谱系祖细胞。
优选地，步骤(3)所述基质细胞为OP9-DL1细胞。
优选地，步骤(3)所述共培养过程中采用强力霉素进行诱导。
优选地，步骤(4)所述T细胞主要为CD3 +T细胞。
优选地，所述T细胞为TCRβ细胞和/或TCRγ/δ细胞。
作为优选技术方案，本发明提供了一种多能干细胞定向分化T细胞的方法，包括以下步骤：
(1)将Runx1和Hoxa9串联的表达载体通过基因重组整合到多能干细胞的Rosa26位点， 并采用潮霉素B进行抗性筛选；
(2)将步骤(1)所述多能干细胞依次采用D0培养基、D2.5培养基、D3培养基、D4培养基、D5培养基、D6培养基和D7培养基培养进行培养，在第11天定向分化为造血干细胞前体细胞；
(3)将步骤(2)所述造血干细胞前体细胞与OP9-DL1细胞共培养，并采用强力霉素进行诱导，得到T谱系祖细胞；
(4)诱导步骤(3)所述T谱系祖细胞分化为T细胞，所述T细胞为TCRβ细胞和/或TCRγ/δ细胞。
第五方面，本发明提供了一种如第一方面所述的方法制备得到的T谱系祖细胞和/或T细胞。
第六方面，本发明提供了一种药物组合物，包括如第一方面所述的载体、如第三方面所述的宿主细胞、如第五方面所述的T谱系祖细胞或T细胞中的任意一种或至少两种的组合。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第七方面，本发明提供了一种如第四方面所述的药物组合物用于制备增强免疫响应的药物，优选为用于制备增强T细胞免疫响应的药物。
本发明中，所述药物组合物可以用于增强免疫响应，特别是增强T细胞免疫响应。
第八方面，本发明提供了一种如第四方面所述的药物组合物用于制备预防和/或治疗免疫缺陷的药物，优选为用于制备预防和/或治疗T细胞免疫缺陷的药物。
本发明中，所述药物组合物可以用于预防和/或治疗免疫缺陷，特别是预防和/或治疗T细胞免疫缺陷。
第九方面，本发明提供了一种如第四方面所述的药物组合物用于制备T细胞免疫疗法治疗肿瘤的药物。
本发明中，所述药物组合物可以用于T细胞免疫疗法。
与现有技术相比，本发明具有如下有益效果：
(1)本发明将外源Runx1和Hoxa9共表达载体引入多能干细胞中，成功构建了诱导性共表达外源Runx1和Hoxa9的多能干细胞，所述多能干细胞具有分化为T细胞的能力，并能够用于制备增强免疫效应、预防和/或治疗免疫缺陷以及治疗肿瘤的药物；
(2)本发明采用定向分化体系和共培养方法，将所述多能干细胞定向分化为T谱系祖细胞，所述T谱系祖细胞可以诱导分化为T细胞，并能够用于制备增强免疫效应、预防和/ 或治疗免疫缺陷以及治疗肿瘤的药物；
(3)采用本发明的方法获得的多能干细胞来源的T细胞，功能正常，没有致瘤风险，可以用于制备增强免疫效应、预防和/或治疗免疫缺陷以及治疗肿瘤的药物。
附图说明
图1(A)为定点敲入多能干细胞Rosa26位点的可诱导表达系统示意图，所述表达系统采用p2a序列将Runx1与Hoxa9的cDNA序列串联，采用强力霉素诱导基因表达，图1(B)为通过潮霉素抗性筛选获得的iRunx1-p2a-Hoxa9多能干细胞光场图，图1(C)为使用强力霉素处理24小时后Runx1和Hoxa9的相对表达水平；
图2(A)为诱导iRunx1-p2a-Hoxa9多能干细胞定向分化为造血前体、造血干细胞前体细胞以及血液细胞的拟胚体-单层培养体系示意图，图2(B)为诱导iRunx1-p2a-Hoxa9多能干细胞定向分化至第11天的细胞形态图，图2(C)为流式细胞术分析定向分化第11天时造血相关细胞的组成及比例；
图3(A)为造血干细胞前体细胞的流式分选策略，图3(B)为分选造血干细胞前体细胞群体(CD31 +CD41 lowCD45 -c-Kit +CD201 high)与OP9-DL1细胞系共培养的示意图，图3(C)为造血干细胞前体细胞群体与OP9-DL1细胞系共培养10天后显微镜下观察的鹅卵石样形成区域的数量，图3(D)为造血干细胞前体细胞群体与OP9-DL1细胞系共培养10天后显微镜下观察的鹅卵石样形成区域的光场图。
图4(A)为造血干细胞前体细胞与OP9-DL1细胞系进行共培养后收获T谱系祖细胞移植到CD45.1 +NOD/SCID免疫缺陷小鼠中，图4(B)为移植4周后，使用流式细胞术鉴定多能干细胞来源的血液细胞，iRunx1-p2a-Hoxa9组可以检测到造血嵌合，图4(C)为5周后牺牲受体小鼠的外周血、骨髓、脾脏和胸腺中多能干细胞来源的造血细胞的谱系分布以及CD3 +T淋巴细胞的表型，图4(D)为多能干细胞来源的血液细胞基因组的PCR及测序鉴定。
图5(A)为4周后牺牲受体小鼠的胸腺中多能干细胞来源的DN细胞群体(DN1/DN2/DN3/DN4)分析，图5(B)为4周后牺牲受体小鼠的外周血、脾脏及淋巴结中多能干细胞来源CD3 +T细胞中的TCR-β和TCR-γ/δ群体分析，图5(C)为4周后牺牲受体小鼠的混合淋巴细胞反应实验(MLR)，PSC-T为Runx1-p2a-Hoxa9诱导多能干细胞系分化获得T谱系祖细胞移植入NOD-SCID受体鼠6周后，于脾脏中磁珠富集的CD3 +T细胞。
图6为ELISA法检测的体外刺激T细胞分泌的代表性细胞因子，其中，IL10-白介素10、IFN-γ-γ干扰素、IL-2-白介素2、TNF-α-肿瘤坏死因子α。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例通过电转化法结合基因重组在多能干细胞的Rosa26位点定点敲入可诱导表达序列，如图1(A)所示，敲入序列包含Runx1-p2a-Hoxa9串联序列和用于抗性筛选的潮霉素B抗性基因序列。为了成功获得同源重组的多能干细胞，电转化20小时后加入含有潮霉素B(150μg/mL)的多能干细胞培养基，每天换液。采用潮霉素B筛选10天后，在显微镜下挑取单个克隆至提前铺好MEF细胞的12孔板中，每孔放入一个多能干细胞克隆，采用无潮霉素的培养基进行培养。
待克隆团粘附在MEF细胞层中，每天换液，3天后采用0.25％胰酶消化克隆团，传代至12孔板中，细胞形态如图1(B)所示，克隆团处于对数生长期，边缘整齐透亮与MEF细胞层有明显分界，无分化发生。根据细胞状态和生长密度，进行传代、扩增和冻存。
提取Dox处理24小时后的iRunx1-p2a-Hoxa9多能干细胞总mRNA(未加Dox组作为对照组)，利用Q-PCR获得Runx1和Hoxa9的mRNA的表达水平，图1(C)表明，加入Dox可以诱导Runx1和Hoxa9的表达。
实施例2
为了诱导多能干细胞的造血分化，采用如图2(A)所示的定向造血分化体系。定向造血分化体系中各培养基的配方为：
基础分化培养基BDM：含有15％胎牛血清、200μg/mL铁饱和转铁蛋白、4.5×10 -4M硫代甘油、2mM GlutaMAX TM-I添加剂、0.5mM抗坏血酸的IMDM培养基；
D0培养基：含有5ng/mL骨形态发生蛋白4的基础分化培养基；
D2.5培养基：含有5ng/mL激活素A和5ng/mL碱性成纤维细胞生长因子的基础分化培养基；
D3培养基：含有5ng/mL激活素A、5ng/mL骨形态发生蛋白4和5ng/mL血管内皮生长因子的基础分化培养基；
D4培养基：含有5ng/mL骨形态发生蛋白4和5ng/mL血管内皮生长因子的基础分化培 养基；
D5培养基：含有5ng/mL骨形态发生蛋白4和5ng/mL血管内皮生长因子、20ng/mL重组小鼠白介素3、20ng/mL重组小鼠白介素6、20ng/mL重组小鼠干细胞因子、20ng/mL重组人促血小板生成素和20ng/mL hFlt3L的基础分化培养基；
D6培养基：含有5ng/mL骨形态发生蛋白4、5ng/mL血管内皮生长因子、20ng/mL重组小鼠白介素3、20ng/mL重组小鼠白介素6、20ng/mL重组小鼠干细胞因子、20ng/mL重组人促血小板生成素、20ng/mL hFlt3L和1μg/mL强力霉素的基础分化培养基；
D7培养基：含有20ng/mL重组小鼠白介素3、20ng/mL重组小鼠白介素6、20ng/mL重组小鼠干细胞因子、20ng/mL重组人促血小板生成素、20ng/mL hFlt3L和1μg/mL强力霉素的基础分化培养基。
具体步骤为：
提前40min在6孔板中铺1mL浓度为0.1％的明胶(gelatin)，待用。使用0.05％胰酶将多能干细胞消化为单细胞，离心后重悬多能干细胞。吸去0.1％gelatin，将多能干细胞悬液转移到包被有gelatin的孔中，培养箱中放置40min，以除去MEF细胞。
收集悬浮细胞，250g下离心5min，使用DPBS清洗一次。使用D0培养基重悬细胞并计数，调整细胞浓度至1×10 5个/mL。将5-10mL细胞悬液加入到倾斜的10cm盘中，吸取20μL细胞悬液，加入到15cm培养皿中悬浮拟胚体(EB)，单个EB为20μL(约2000个细胞)。随后将培养皿倒置，并在培养皿底部放置一个10cm培养皿盖子，盖子中加入5-6mL细胞培养用水。在37℃培养箱中培养2.5天。
用巴氏吸管将EB收集到离心管中，用DPBS清洗皿底，待EB自然沉降后小心吸去上清，亦可在90g低速离心5min去上清，加入DPBS润洗一遍，再次沉降或离心去上清。用D2.5培养基重悬EB后，转移至低粘附的24孔板中，培养12小时观察EB是否有污染。
收集EB到15mL离心管中，待其自然沉降后小心吸去上清，加入DPBS润洗一遍，加入400μL 0.05％胰酶，转移至24孔低粘附培养皿中，37℃消化3min后反复轻柔吹打EB，待EB呈单细胞状态时加入D3培养基终止消化，350g下离心5min。用D3培养基重悬计数活细胞，接种到0.1％gelatin提前包被的12孔板中，密度为2×10 5个/孔。
采用DPBS润洗一遍，更换D4培养基培养一天。
采用DPBS润洗一遍，更换D5培养基培养一天。
采用DPBS润洗一遍，更换D6培养基培养一天。
采用DPBS润洗一遍，更换D7培养基培养一天。
随后隔天换液，采用的培养基为D7培养基。如图2(B)所示，在第11天，iRunx1-p2a-Hoxa9分化组可见明显的造血簇；图2(C)所示的流式细胞术分析结果表明，定向分化第11天的造血相关细胞群体为CD41 +造血前体细胞和CD45 +血液细胞。
实施例3
为了验证从多能干细胞分化而来的造血前体细胞具有胚胎造血干细胞前体细胞群的增殖能力，即在基质细胞上可以形成高扩增潜能的鹅卵石样形成区域的能力，发明人将造血干细胞前体细胞与小鼠骨髓基质细胞共培养。共培养培养基为含有15％DFBS、200μg/mL铁饱和转铁蛋白、4.5×10 -4M硫代甘油、2mM GlutaMAX TM-I添加剂、0.5mM抗坏血酸、2％AFT024-mSCF条件性培养基、2％AFT024-mIL3条件性培养基、2％AFT024-hFlt3L条件性培养基和1μg/mL Dox的α-MEM培养基。
在拟胚体-单层培养的第11天，采用如图3(A)的分选策略进行流式细胞仪分选造血干细胞前体细胞(CD31 +CD41 lowCD45 -c-Kit +CD201 high)。随后，采用的鹅卵石样区域形成实验(CAFC)检验多能干细胞分化而来的造血干细胞前体细胞是否具有与胚胎来源的造血干细胞前体细胞相同的增殖能力。如图3(B)所示，将分选到的造血干细胞前体细胞群体重铺到OP9-DL1基质细胞上，10天后对每100个造血干细胞前体细胞所形成的鹅卵石样形成区域的数量进行计数。图3(C)和图3(D)结果表明：iRunx1-p2a-Hoxa9多能干细胞来源的造血干细胞前体细胞具有很强的鹅卵石样区域形成能力，多能干细胞来源的造血干细胞前体细胞在基质细胞OP9-DL1上形成高度均一的小、圆、亮的血液细胞。
实施例4
为了利用体内微环境获得T细胞，发明人进一步设计了共培养后移植策略，如图4(A)所示，将造血干细胞前体细胞在OP9-DL1基质细胞上添加Dox诱导10天后获得T谱系祖细胞。提前4天复苏OP9-DL1细胞系，根据细胞生长状态及时传代，避免细胞由于过度生长而老化。使用前一天传代，每孔重铺5万细胞(12孔板)，第二天使用。将多能干细胞来源的造血干细胞前体细胞共培养后获得T谱系祖细胞通过眼静脉移植到6-8周龄的CD45.1NOD/SCID小鼠中，4周后借助流式细胞术检测外周血造血嵌合情况。
图4(B)表明：iRunx1-p2a-Hoxa9多能干细胞来源的造血干细胞前体细胞群体经过共培养后获得的T谱系祖细胞，可以在受体NOD/SCID小鼠外周血中形成造血嵌合，并且以CD3 +T细胞为主(97.7％)，实现了有效重建T淋系的效果。
为了进一步明确iRunx1-p2a-Hoxa9多能干细胞来源的血液细胞在其他造血、淋巴组织中的分布，5周后牺牲小鼠后通过流式细胞术分析其外周血、骨髓、脾脏和胸腺血液细胞谱系。 流式细胞术分析发现，如图4(C)所示，在骨髓、胸腺和脾脏中，多能干细胞来源的血液细胞同样以T淋系造血为主。这群CD3 +T细胞，既包括CD4 +单阳性细胞，也包括CD8 +单阳性细胞，同时在脾脏、骨髓和胸腺中都包含少量的CD4 +CD8 +双阳性细胞和CD4 -CD8 -双阴性细胞。
为了从基因组水平上确认受体小鼠中CD45.2 +造血细胞(主要是T细胞)来源于iRunx1-p2a-Hoxa9多能干细胞，设计引物进行PCR扩增及测序鉴定。首先通过流式分选骨髓和脾脏来源的CD45.2 +细胞，提取基因组，利用敲入基因序列的特异性引物进行PCR鉴定。图4(D)显示，这些细胞基因组内有iRunx1-p2a-Hoxa9质粒来源序列，证实CD45.2 +血液细胞(主要是T细胞)来自于iRunx1-p2a-Hoxa9多能干细胞。
实施例5
为了进一步鉴定小鼠体内多能干细胞来源免疫细胞的类型，对胸腺DN细胞群体进行分析，从图5(A)中看到：受体小鼠体内T细胞正常发育，能够检测到DN1、DN2、DN3和DN4细胞群体。对外周血、脾脏和淋巴管中的多能干细胞来源的T细胞TCR受体进行检测，如图5(B)所示，T细胞中存在一定比例的TCRγ/δ细胞(0.37-1.71％)，而绝大部分为TCRβ细胞。使用Balb/C小鼠脾脏细胞与从受体小鼠脾脏通过CD3磁珠富集获得的T细胞进行混合淋巴细胞反应，分别在第3天和第6天进行检测，如图5(C)所示，多能干细胞来源的T细胞被激活后能够进行增殖，证实了这些T细胞具有刺激后增殖能力。
采用ELISA技术分析培养上清，如图6所示，刺激增殖后的再生T细胞能够分泌大量的白介素10(IL10)、γ干扰素(IFN-γ)、白介素2(IL-2)和肿瘤坏死因子α(TNF-α)。
综上所述，本发明将外源Runx1和Hoxa9共表达载体引入多能干细胞中，成功构建了诱导性共表达外源Runx1和Hoxa9的多能干细胞，所述多能干细胞定向分化为T谱系祖细胞，并将发育为T细胞。使用本发明的方法获得的多能干细胞来源的T细胞，不仅功能正常，而且没有致瘤风险，可以用于制备增强免疫效应、预防和/或治疗免疫缺陷以及治疗肿瘤的药物。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种载体，其特征在于，所述载体包括Runx1和Hoxa9串联共表达。
  2. 一种表达如权利要求1所述载体的核酸。
  3. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求1所述的载体；
    优选地，所述宿主细胞为多能干细胞。
  4. 一种采用如权利要求3所述的多能干细胞定向分化T细胞的方法，其特征在于，包括以下步骤：
    (1)将Runx1和Hoxa9串联的表达载体整合到多能干细胞中，并进行抗性筛选；
    (2)将步骤(1)所述多能干细胞定向分化为造血干细胞前体细胞；
    (3)将步骤(2)所述造血干细胞前体细胞与小鼠骨髓基质细胞共培养，得到T谱系祖细胞；
    (4)诱导步骤(3)所述T谱系祖细胞分化为T细胞。
  5. 根据权利要求4所述的方法，其特征在于，步骤(1)所述Runx1和Hoxa9串联的表达载体整合到多能干细胞的Rosa26位点；
    优选地，步骤(1)所述多能干细胞为基因编辑后的诱导性多能干细胞和/或胚胎多能干细胞系；
    优选地，步骤(1)所述整合的方法包括同源重组、CRISPR/Cas9、TALEN、转染或病毒感染中的任意一种或至少两种的组合，优选为同源重组；
    优选地，步骤(1)所述抗性筛选采用潮霉素B；
    优选地，步骤(2)所述定向分化的方法为依次采用D0培养基、D2.5培养基、D3培养基、D4培养基、D5培养基、D6培养基和D7培养基培养多能干细胞得到所述造血干细胞前体细胞；
    优选地，所述D0培养基为含有3-8ng/mL骨形态发生蛋白4的基础分化培养基，优选为含有5ng/mL骨形态发生蛋白4的基础分化培养基；
    优选地，所述D2.5培养基为含有3-8ng/mL激活素A和3-8ng/mL碱性成纤维细胞生长因子的基础分化培养基，优选为含有5ng/mL激活素A和5ng/mL碱性成纤维细胞生长因子的基础分化培养基；
    优选地，所述D3培养基为含有3-8ng/mL激活素A、3-8ng/mL骨形态发生蛋白4和3-8ng/mL血管内皮生长因子的基础分化培养基，优选为含有5ng/mL激活素A、5ng/mL骨形态发生蛋白4和5ng/mL血管内皮生长因子的基础分化培养基；
    优选地，所述D4培养基为含有3-8ng/mL骨形态发生蛋白4和3-8ng/mL血管内皮生长 因子的基础分化培养基，优选为含有5ng/mL骨形态发生蛋白4和5ng/mL血管内皮生长因子的基础分化培养基；
    优选地，所述D5培养基为含有3-8ng/mL骨形态发生蛋白4、3-8ng/mL血管内皮生长因子、10-30ng/mL重组小鼠白介素3、10-30ng/mL重组小鼠白介素6、10-30ng/mL重组小鼠干细胞因子、10-30ng/mL重组人促血小板生成素和10-30ng/mL人Fms相关酪氨酸激酶3配体的基础分化培养基，优选为含有5ng/mL骨形态发生蛋白4和5ng/mL血管内皮生长因子、20ng/mL重组小鼠白介素3、20ng/mL重组小鼠白介素6、20ng/mL重组小鼠干细胞因子、20ng/mL重组人促血小板生成素和20ng/mL人Fms相关酪氨酸激酶3配体的基础分化培养基；
    优选地，所述D6培养基为含有3-8ng/mL骨形态发生蛋白4、3-8ng/mL血管内皮生长因子、10-30ng/mL重组小鼠白介素3、10-30ng/mL重组小鼠白介素6、10-30ng/mL重组小鼠干细胞因子、10-30ng/mL重组人促血小板生成素、10-30ng/mL人Fms相关酪氨酸激酶3配体和1-2μg/mL强力霉素的基础分化培养基，优选为含有5ng/mL骨形态发生蛋白4、5ng/mL血管内皮生长因子、20ng/mL重组小鼠白介素3、20ng/mL重组小鼠白介素6、20ng/mL重组小鼠干细胞因子、20ng/mL重组人促血小板生成素、20ng/mL人Fms相关酪氨酸激酶3配体和1μg/mL强力霉素的基础分化培养基；
    优选地，所述D7培养基为含有10-30ng/mL重组小鼠白介素3、10-30ng/mL重组小鼠白介素6、10-30ng/mL重组小鼠干细胞因子、10-30ng/mL重组人促血小板生成素、10-30ng/mL人Fms相关酪氨酸激酶3配体和1-2μg/mL强力霉素的基础分化培养基，优选为含有20ng/mL重组小鼠白介素3、20ng/mL重组小鼠白介素6、20ng/mL重组小鼠干细胞因子、20ng/mL重组人促血小板生成素、20ng/mL人Fms相关酪氨酸激酶3配体和1μg/mL强力霉素的基础分化培养基；
    优选地，所述基础分化培养基为含有10-20％胎牛血清、180-220μg/mL铁饱和转铁蛋白、4.5×10 -4M硫代甘油、1-3mM GlutaMAXm-I添加剂、0.4-0.6mM抗坏血酸的IMDM培养基，优选为含有15％胎牛血清、200μg/mL铁饱和转铁蛋白、4.5×10 -4M硫代甘油、2mM GlutaMAX TM-I添加剂、0.5mM抗坏血酸的IMDM培养基；
    优选地，步骤(3)所述基质细胞为OP9-DL1细胞；
    优选地，步骤(3)所述共培养过程中采用强力霉素进行诱导；
    优选地，步骤(4)所述T细胞主要为CD3 +T细胞；
    优选地，所述T细胞为TCRβ细胞和/或TCRγ/δ细胞；
    优选地，所述多能干细胞定向分化T细胞的方法，包括以下步骤：
    (1)将Runx1和Hoxa9串联的表达载体通过基因重组整合到多能干细胞的Rosa26位点，并采用潮霉素B进行抗性筛选；
    (2)将步骤(1)所述多能干细胞依次采用D0培养基、D2.5培养基、D3培养基、D4培养基、D5培养基、D6培养基和D7培养基培养进行培养，在第11天定向分化为造血干细胞前体细胞；
    (3)将步骤(2)所述造血干细胞前体细胞与OP9-DL1细胞共培养，并采用强力霉素进行诱导，得到T谱系祖细胞；
    (4)诱导步骤(3)所述T谱系祖细胞分化为T细胞，所述T细胞为TCRβ细胞和/或TCRγ/δ细胞。
  6. 一种如权利要求4或5所述的方法制备得到的T谱系祖细胞和/或T细胞。
  7. 一种药物组合物，其特征在于，包括如权利要求1所述的载体、如权利要求3所述的宿主细胞、如权利要求6所述的T谱系祖细胞或T细胞中的任意一种或至少两种的组合；
    优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  8. 一种如权利要求7所述的药物组合物用于制备增强免疫响应的药物，优选为用于制备增强T细胞免疫响应的药物。
  9. 一种如权利要求7所述的药物组合物用于制备预防和/或治疗免疫缺陷的药物，优选为用于制备预防和/或治疗T细胞免疫缺陷的药物。
  10. 一种如权利要求7所述的药物组合物用于制备T细胞免疫疗法治疗肿瘤的药物。
PCT/CN2018/072254 2017-12-30 2018-01-11 一种多能干细胞及其分化的t细胞和应用 WO2019127664A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/312,794 US11299709B2 (en) 2017-12-30 2018-01-11 Pluripotent stem cell and T cell differentiated therefrom and application thereof
JP2020535615A JP7098187B2 (ja) 2017-12-30 2018-01-11 多能性幹細胞及びその分化したｔ細胞と使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711490483.8A CN108220243B (zh) 2017-12-30 2017-12-30 一种多能干细胞及其分化的t细胞和应用
CN201711490483.8 2017-12-30

Publications (1)

Publication Number Publication Date
WO2019127664A1 true WO2019127664A1 (zh) 2019-07-04

Family

ID=62642102

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/072254 WO2019127664A1 (zh) 2017-12-30 2018-01-11 一种多能干细胞及其分化的t细胞和应用

Country Status (4)

Country Link
US (1) US11299709B2 (zh)
JP (1) JP7098187B2 (zh)
CN (1) CN108220243B (zh)
WO (1) WO2019127664A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113046311B (zh) * 2021-03-03 2022-09-30 中山大学孙逸仙纪念医院 一种诱导多能干细胞定向分化为淋巴组织诱导细胞的方法
CN115247151B (zh) * 2022-09-21 2022-12-27 呈诺再生医学科技(北京)有限公司 制备造血内皮细胞的方法及制备造血干细胞或造血干祖细胞的方法
CN115948472B (zh) * 2022-12-06 2024-02-06 广州市天河诺亚生物工程有限公司 诱导过表达的runx1在构建耗竭t细胞模型中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017192708A1 (en) * 2016-05-03 2017-11-09 The Children's Medical Center Corporation Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2970881A4 (en) * 2013-03-14 2017-01-25 Children's Medical Center Corporation Compositions and methods for reprogramming hematopoietic stem cell lineages
CN104789529A (zh) * 2015-04-28 2015-07-22 济南劲牛生物科技有限公司 促进小鼠骨髓造血干细胞体外克隆形成及分化能力的方法
AU2016342183B2 (en) * 2015-10-20 2022-03-03 FUJIFILM Cellular Dynamics, Inc. Methods for directed differentiation of pluripotent stem cells to immune cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017192708A1 (en) * 2016-05-03 2017-11-09 The Children's Medical Center Corporation Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEN, B.: "Inducible overexpression of RUNXIb/c in human embryonic stem cells blocks early hematopoiesis from mesoderm", JOURNAL OF MOLECULAR CELL BIOLOGY., vol. 9, no. 4, 9 August 2017 (2017-08-09), pages 262 - 273, XP055580470 *
KUVARDINA, O. N.: "Hematopoietic transcription factors and differential cofactor binding regulate PRKACB isoform expression.", ONCOTARGET, vol. 8, no. 42, 24 April 2017 (2017-04-24), pages 71685 - 71698, XP055623944 *
LIS, R.: "Conversion of adult endothelium to immunocompetent haematopoietic stem cells.", NATURE, vol. 545, no. 7655, 25 May 2017 (2017-05-25), pages 439 - 445, XP055436955 *
NAVARRO-MONTERO, O.: "RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.", STEM CELLS., vol. 35, no. 11, 4 September 2017 (2017-09-04), pages 2253 - 2266, XP055623966 *
SANDLER, V. M.: "Reprogramming human endothelial cells to haematopoietic cells requires vascular induction.", NATURE, vol. 511, no. 7509, 17 July 2014 (2014-07-17), pages 312 - 318, XP055328630 *
SUGIMURA, R.: "Haematopoietic stem and progenitor cells from human pluri-potent stem cells.", NATURE ., vol. 545, no. 7655, 25 May 2017 (2017-05-25), pages 432 - 438, XP055436951 *
TSUZUKI, S.: "Expansion of functionally defined mouse hematopoietic stem and progenitor cells by a short isoform of RUNX1/AML1", BLOOD, vol. 119, no. 3, 19 January 2012 (2012-01-19), pages 727 - 35, XP055623935 *
WANG, ZHEN: "Construction and Expression of Key Transcription Factors Involved in Hematopoiesis in Lentivirus", CHINESE MASTER'S THESES, no. 8, 15 August 2013 (2013-08-15), pages 1 - 73 *

Also Published As

Publication number Publication date
JP7098187B2 (ja) 2022-07-11
CN108220243B (zh) 2019-05-28
US20200208107A1 (en) 2020-07-02
US20210238548A9 (en) 2021-08-05
US11299709B2 (en) 2022-04-12
JP2021509272A (ja) 2021-03-25
CN108220243A (zh) 2018-06-29

Similar Documents

Publication Publication Date Title
AU2014207627B2 (en) Reprogramming of human endothelium into hematopoietic multi-lineage progenitors by defined factors
EP2841563B1 (en) Method for developing natural killer cells from stem cells
US9255249B2 (en) Isolation and purification of hematopoietic stem cells from post-liposuction lipoaspirates
KR20180057641A (ko) 자연살해 세포의 증식방법
CN114774365A (zh) 一种诱导iPSC分化获得CD34+细胞和NK细胞的方法及其应用
WO2019127664A1 (zh) 一种多能干细胞及其分化的t细胞和应用
Guo et al. Generation and clinical potential of functional T lymphocytes from gene-edited pluripotent stem cells
KR20110024660A (ko) 인간 배아줄기세포 또는 인간 유도만능줄기세포의 혈관내피세포로의 혈청 및 이종 비함유 분화방법
CN114402065A (zh) 低密度细胞培养
US20230071538A1 (en) Cytotoxic t cells derived from human t cell-derived ips cells
WO2022242359A1 (zh) 一种体液免疫系统再生的方法及其应用
AU4419396A (en) System for the maintenance, growth and differentiation of human and non-human primate pluripotent stem, progenitor and mature bone marrow cells
CN116479041A (zh) 一种基因构建体以及产生多谱系造血干祖细胞的方法
CN115418373A (zh) 一种体液免疫系统再生的方法及其应用
JP2007238473A (ja) 造血幹細胞の維持増幅を促進する因子及び造血幹細胞の増幅方法
US20100209396A1 (en) Method of Enhancing Proliferation and/or Hematopoietic Differentiation of Stem Cells
WO2006085482A1 (ja) 造血幹細胞の自己複製因子及び増幅方法
WO2023135225A1 (en) Method of producing vdelta1+ t cells
CN117417959A (zh) 再生髓系细胞的方法及用途
CN117866110A (zh) 一种靶向flt-3的第四代嵌合抗原受体及其应用
Imamura A suitable conditioning regimen in allogeneic hematopoietic stem cell transplantation for adult patients with acute lymphoblastic leukemia
Lisini et al. Stem Cell Production: Processes, Practices, and Regulation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18897361

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020535615

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18897361

Country of ref document: EP

Kind code of ref document: A1