WO2022242359A1 - 一种体液免疫系统再生的方法及其应用 - Google Patents

一种体液免疫系统再生的方法及其应用 Download PDF

Info

Publication number
WO2022242359A1
WO2022242359A1 PCT/CN2022/085730 CN2022085730W WO2022242359A1 WO 2022242359 A1 WO2022242359 A1 WO 2022242359A1 CN 2022085730 W CN2022085730 W CN 2022085730W WO 2022242359 A1 WO2022242359 A1 WO 2022242359A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
pluripotent stem
gene
medium
Prior art date
Application number
PCT/CN2022/085730
Other languages
English (en)
French (fr)
Inventor
王金勇
张琪
吴冰燕
胡房晓
翁启童
彭欢
王瑶
刘丽娟
刘晓飞
夏成祥
耿阳
吴红玲
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
Priority claimed from CN202210234297.2A external-priority patent/CN115418373A/zh
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 JP2023572007A priority Critical patent/JP2024518632A/ja
Priority to US18/562,645 priority patent/US20240269181A1/en
Publication of WO2022242359A1 publication Critical patent/WO2022242359A1/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
    • 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
    • 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
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K39/4612—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C12N5/0635—B lymphocytes
    • 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

Definitions

  • the application belongs to the technical field of medical bioengineering and relates to directed differentiation of pluripotent stem cells, in particular to a method for regeneration of the humoral immune system, ie, directed differentiation of B cells from pluripotent stem cells and its application.
  • B cells are the core cell components of the humoral immune system. Defects in B cell function will lead to a decline in the patient's humoral immunity, and even serious infection by bacteria, viruses or other pathogenic microorganisms. Therefore, restoring or even enhancing the humoral immune system through regenerative means is expected to benefit many patients with abnormal humoral immune systems.
  • Pluripotent stem cells are a type of cells with unlimited proliferation potential, the potential to differentiate into different lineage cells, and convenient gene editing and modification. They are a hot spot in the research of cell therapy and regenerative medicine. Inducing pluripotent stem cells (induced pluripotent stem cells, iPSCs) derived from the reprogramming of patients' autologous somatic cells to differentiate into different lineage cells can not only avoid the ethical controversy of using embryonic stem cells, but also reduce the risk of allogeneic immune rejection. Ideal cell development material in the field.
  • Mouse induced pluripotent stem cells co-cultured with stromal cells in vitro can induce T cells, but it is difficult to induce B cells (see Wada, Haruka et al. Successful differentiation to T cells, but unsuccessful B-cell generation, from B- cell-derived induced pluripotent stem cells. International immunology vol.23,1(2011):65-74).
  • hematopoietic stem cells hematopoietic stem and progenitor cells, HSPCs
  • hematopoietic stem and progenitor cells hematopoietic stem and progenitor cells
  • HSPCs hematopoietic stem and progenitor cells
  • This application provides a method for directional differentiation of pluripotent stem cells into B cells and its application.
  • Genetically modified pluripotent stem cells are used to induce differentiation in vitro to efficiently obtain B cell seeds. After transplantation, they can be fully regenerated in animals with a lack of humoral immune system
  • the humoral immune system is an efficient method for the regeneration of the humoral immune system, which can realize antigen-specific antibody immune response, produce specific high-affinity antibodies against antigens, and generate immune memory.
  • the immune system rebuilt by this method is safe and has never been seen. Tumor risk.
  • the present application provides an expression vector, which contains nucleotide sequences encoding RUNX1, HOXA9 and LHX2 genes, and is used to realize the tandem co-expression of the three genes RUNX1, HOXA9 and LHX2.
  • the cDNA sequences of RUNX1, HOXA9 and LHX2 are expressed in tandem in the same vector and integrated into the genome of mammalian pluripotent stem cells to obtain host cells stably expressing RUNX1, HOXA9 and LHX2, which are easy to operate and highly efficient.
  • the resulting host cells have the ability to differentiate into B cells.
  • RUNX1 is one of the members of the RUNX transcription factor protein family and is the most common target site of leukemia chromosomal translocation.
  • RUNX1 is a key hematopoietic regulatory transcription factor, which plays an important role in endothelial hematopoietic transformation, primitive hematopoiesis, permanent hematopoiesis and lymphopoiesis;
  • the RUNX1 gene can be of various origins, such as human or mouse origin, wherein the RUNX1 gene of mouse origin can be ENSMUSG00000022952, and the RUNX1 gene of human origin can be ENSG00000159216.
  • HOXA9 gene is a member of the HOX gene family, which is a coding sequence-specific transcriptional regulator, and plays an important role in embryonic development and hematopoietic regulation. HOXA9 may play an important role in the enhancement and maintenance of HSC, endothelial hematopoietic transformation and promotion of lymphogenesis;
  • the HOXA9 gene can be derived from various sources, such as human or mouse, wherein the mouse-derived HOXA9 gene can be ENSMUSG00000038227, and the human-derived HOXA9 gene can be ENSG00000078399.
  • the LHX2 gene (Lim homeobox 2), also known as LH-2, is a member of the transcription factor family and plays an important role in the development of various organs, especially in the nervous system. It plays an important role in hematopoiesis and erythropoiesis, and can promote the immortality of hematopoietic stem and progenitor cells, and LHX2 was found to be expressed in pre-B cell lines.
  • the LHX2 gene can be of various origins, such as human or mouse, wherein the LHX2 gene of mouse origin can be ENSMUSG00000000247, and the LHX2 gene of human origin can be ENSG00000106689.
  • the combination of the three genes is mainly that RUNX1 can promote the differentiation of pluripotent stem cells to form hematopoietic endothelial cells, while RUNX1 and HOXA9 can promote the formation of lymphoid lineage, and LHX2 further promotes the differentiation to B lineage; compared with other differentiation-related genes , if RUNX1, LMO2 and MEIS1 are used at the same time, the combination cannot normally produce hematopoietic clones during the co-culture of hematopoietic endothelium and OP9-DL1 in the later stage.
  • the present application provides a gene-edited pluripotent stem cell host cell, the host cell comprising the expression vector as described in the first aspect.
  • the host cells are pluripotent stem cells, including induced pluripotent stem cells and/or embryonic pluripotent stem cell lines.
  • the pluripotent stem cells include gene-edited induced pluripotent stem cells and/or embryonic pluripotent stem cell lines.
  • the present application provides a method for regeneration of the humoral immune system, that is, a method for directional differentiation of pluripotent stem cells into B cells, comprising the following steps:
  • iHEC induced hematopoietic endothelium
  • step (3) Co-culture the induced hematopoietic endothelium and bone marrow stromal cells described in step (2) to obtain hematopoietic progenitor cells with B lineage differentiation potential, that is, B lineage seed cells; and
  • step (3) Transplanting the B lineage seed cells described in step (3), and in vivo differentiation to produce B cells.
  • the obtained induced hematopoietic endothelium was co-cultured with bone marrow stromal cells to obtain B lineage seed cells, and after differentiation, B cells with normal functions were obtained, including all Mature cell type without tumorigenic risk.
  • the RUNX1, HOXA9, and LHX2 tandem expression vectors described in step (1) can be integrated into any safe site, and the insertion site can enable the stable expression of the inserted gene.
  • the tandem expressed gene is preferably integrated To ROSA26 locus, AAVS1 locus, CCR5 locus, H11 locus, COL1A1 locus or TIGRE locus of pluripotent stem cells.
  • the pluripotent stem cells in step (1) are gene-edited induced pluripotent stem cells and/or embryonic pluripotent stem cell lines.
  • the integration method described in step (1) includes any one or a combination of at least two of homologous recombination, CRISPR/Cas9, TALEN, transfection or virus infection, preferably homologous recombination.
  • hygromycin B is used for the resistance screening in step (1) to obtain the main clone stem cell line; other resistance screening strategies such as chloramphenicol, geneticin (G-418), blasticide oryzae can also be used Obtain the master clone stem cell line with mycophenolic acid and mycophenolic acid.
  • the directed differentiation method in step (2) is to sequentially adopt D0 medium, D2.5 medium, and D6 medium to culture pluripotent stem cells to obtain the induced hematopoietic endothelium.
  • the D0 medium is a basal differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4); the concentration of the bone morphogenetic protein 4 can be, for example, 3 ng/mL, 4 ng/mL, 5 ng /mL, 6ng/mL, 7ng/mL or 8ng/mL, preferably 5ng/mL.
  • BMP4 bone morphogenetic protein 4
  • the D2.5 medium contains 3-8 ng/mL (such as 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL or 8 ng/mL, preferably 5 ng/mL ) BMP4 and 3 ⁇ 8ng/mL (such as 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL or 8ng/mL, preferably 5ng/mL) vascular endothelial growth factor (VEGF) Basal Differentiation Medium.
  • 3-8 ng/mL such as 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL or 8 ng/mL, preferably 5 ng/mL
  • BMP4 and 3 ⁇ 8ng/mL (such as 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL or 8ng
  • the D6 medium contains 10-30 ng/mL interleukin 3 (IL3), 10-30 ng/mL interleukin 6 (IL6), 10-30 ng/mL stem cell factor (SCF), 10-30 ng/mL FMS-like Basal differentiation medium with tyrosine kinase 3 ligand (Flt3L) and 1-2 ⁇ g/mL doxycycline (Dox).
  • IL3 interleukin 3
  • IL6 interleukin 6
  • SCF stem cell factor
  • FMS-like Basal differentiation medium with tyrosine kinase 3 ligand Flt3L
  • Dox 1-2 ⁇ g/mL doxycycline
  • the concentration of the IL3 can be, for example, 10ng/mL, 15ng/mL, 18ng/mL, 22ng/mL, 25ng/mL or 30ng/mL, preferably 20ng/mL;
  • the concentration of the IL6 can be, for example, 10ng/mL mL, 15ng/mL, 18ng/mL, 22ng/mL, 25ng/mL or 30ng/mL, preferably 20ng/mL;
  • the concentration of the SCF can be 10ng/mL, 15ng/mL, 18ng/mL, 22ng/mL, for example mL, 25ng/mL or 30ng/mL, preferably 20ng/mL;
  • the concentration of the Flt3L can be, for example, 10ng/mL, 15ng/mL, 18ng/mL, 22ng/mL, 25ng/mL or 30ng/mL, preferably 20ng/mL;
  • the concentration of doxycycline can be, for example
  • the components of the D0 medium, the D2.5 medium and the D6 medium are shown in Table 1 below:
  • the basal differentiation medium contains 10-20% fetal bovine serum (% represents volume fraction), 180-220 ⁇ g/mL iron-saturated transferrin (iron-saturated transferrin), 4 ⁇ 10 -4 ⁇ 5 ⁇ IMDM medium with 10 -4 M thioglycerol, 1-3 mM GlutaMAX TM -I supplement and 30-70 ⁇ g/mL ascorbic acid;
  • the concentration of the fetal bovine serum can be, for example, 10%, 12%, 14%, 16%, 18% or 20%, preferably 15%;
  • the concentration of the iron-saturated transferrin can be, for example, 180 ⁇ g/mL , 190 ⁇ g/mL, 210 ⁇ g/mL or 220 ⁇ g/mL, preferably 200 ⁇ g/mL;
  • the concentration of the thioglycerol can be, for example, 4 ⁇ 10 -4 M, 4.2 ⁇ 10 -4 M, 4.4 ⁇ 10 -4 M, 4.8 ⁇ 10 -4 M or 5 ⁇ 10 -4 M, preferably 4.5 ⁇ 10 -4 M;
  • the concentration of the GlutaMAX TM -I additive can be, for example, 1 mM, 1.4 mM, 1.8 mM, 2.2 mM, 2.4 mM, 2.6 mM, 2.8mM or 3mM, preferably 2mM;
  • the concentration of the ascorbic acid can be,
  • the inventors designed and optimized the directional hematopoietic differentiation system by changing the added substances in the medium, and induced pluripotent stem cells to differentiate into hematopoietic endothelium, and the induced hematopoietic endothelium was further co-cultured with mouse bone marrow stromal cells, Obtain B lineage seed cells.
  • the bone marrow stromal cells in step (3) include OP9-DL1 cells, OP9-DL4 cells, OP9 cells, MS5 cells, MS5-DL1 cells, MS5-DL4 cells, HS-5 cells, HS-5-DL1 cells , HS-5-DL4 cells, MSC cells, MSC-DL1 cells or MSC-DL4 cells, any one or a combination of at least two; other bone marrow, thymus, lymph node, liver, spleen tissue and other sources of matrix can also be selected Any one or a combination of at least two of the cells and modified bone marrow stromal cells expressing DL1 or DL4.
  • the above cell lines also have the alias DLL1 when carrying DL1, and have the alias DLL4 when carrying DL4, and they are all the same corresponding cell lines.
  • doxycycline is used for induction during the co-cultivation process described in step (3); expression elements designed by other induction principles can also be used for corresponding drug induction, such as tamoxifen (tamoxifen, 4-OHT) .
  • the method of co-cultivation in step (3) is co-cultivating induced hematopoietic endothelium and OP9-DL1 cells in D11 medium to obtain the B lineage seed cells.
  • the components of the D11 medium are shown in Table 2 below:
  • the concentration of the IL3 can be, for example, 10ng/mL, 15ng/mL, 18ng/mL, 22ng/mL, 25ng/mL or 30ng/mL, preferably 20ng/mL;
  • the concentration of the SCF can be, for example, 10ng/mL mL, 15ng/mL, 18ng/mL, 22ng/mL, 25ng/mL or 30ng/mL, preferably 20ng/mL; mL, 25ng/mL or 30ng/mL, preferably 20ng/mL;
  • the concentration of doxycycline can be, for example, 1 ⁇ g/mL, 1.2 ⁇ g/mL, 1.4 ⁇ g/mL, 1.5 ⁇ g/mL, 1.6 ⁇ g/mL, 1.8 ⁇ g/mL or 2 ⁇ g/mL, preferably 1 ⁇ g/mL;
  • the concentration of the fetal calf serum can be, for example, 10%, 12%, 14%
  • the main medium of the D11 medium can be ⁇ -MEM medium or IMDM medium; since the bone marrow stromal cells used in the experiment process of this application are OP9-DL1, the medium is preferably ⁇ -MEM medium or IMDM medium; MEM medium.
  • the B cells differentiated in step (4) include B220 + B cells and/or CD19 + B cells.
  • the B cells produced by differentiation include any one or a combination of at least two of pro-B cells, pre-B cells, B1 cells, B2 cells or plasma cells.
  • the B1 cells include B1a cells and/or B1b cells.
  • the B2 cells are follicular B cells (Follicular B, FO B) and/or marginal zone B cells (Marginal zone, MZ B).
  • the present application provides a method for directional differentiation of pluripotent stem cells into B cells, comprising the following steps:
  • step (2) Culture the pluripotent stem cells described in step (1) using D0 medium, D2.5 medium, and D6 medium in sequence, and differentiate into induced hematopoietic endothelium on day 8-12;
  • step (3) Co-culture the induced hematopoietic endothelium and OP9-DL1 cells in D11 medium for 8-21 days in step (2), and induce with doxycycline to obtain B lineage seed cells;
  • step (3) transfer the B lineage seed cells described in step (3) to an animal model, and differentiate to produce B cells, which include pro-B cells, pre-B cells, B1 cells, B2 cells or plasma cells Any one or a combination of at least two.
  • step (2) directs differentiation to induce hematopoietic endothelium on day 11.
  • the co-cultivation time in step (3) is 10 days.
  • the present application provides a B lineage seed cell or B cell prepared by the method described in the third aspect.
  • the present application provides a pharmaceutical composition, including the expression vector as described in the first aspect, the host cell as described in the second aspect, the B lineage seed cell or B cell as described in the fourth aspect Any one or a combination of at least two.
  • the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant;
  • the pharmaceutically acceptable adjuvant includes any one or a combination of at least two of a carrier, an excipient or a diluent.
  • the present application also provides a pharmaceutical composition as described in the fifth aspect in the preparation of drugs for enhancing immune response, drugs for preventing and/or treating diseases, drugs for treating tumors with B cell immunotherapy, and B cell vaccines Or the application of B cells in cell therapy drugs that secrete therapeutic proteins.
  • the drug for enhancing immune response includes a drug for enhancing B cell immune response and/or T cell immune response.
  • the drugs for preventing and/or treating diseases include drugs for preventing and/or treating B cell immunodeficiency, infectious diseases, and tumors.
  • the drugs for cell therapy of B cells secreting therapeutic proteins include drugs for preventing and/or treating autoimmune diseases and genetic diseases.
  • said B cells secrete therapeutic proteins including antibodies.
  • the genetic hereditary disease includes any one or a combination of at least two of hemophilia, lysosomal storage disease, hypophosphatasia or phenylketonuria.
  • the pharmaceutical composition can be used for: (1) enhancing immune response, especially enhancing B cell immune response and/or T cell immune response; (2) preventing and/or treating diseases, preferably for the preparation of Prevention and/or treatment of B cell immunodeficiency, infectious diseases, tumors, etc.; (3) research and development and preparation of B cell vaccines; and (4) cell therapy of B cells secreting therapeutic proteins, preferably for prevention and/or treatment Autoimmune diseases, genetic diseases, etc.
  • This application introduces exogenous RUNX1, HOXA9 and LHX2 co-expression vectors into pluripotent stem cells, and successfully constructs pluripotent stem cells that inducibly co-express exogenous RUNX1, HOXA9 and LHX2, and the pluripotent stem cells have the ability to differentiate into B
  • the ability of cells and can be used to prepare immune-enhancing effects, prevent and/or treat immunodeficiency, prevent and/or treat infectious diseases, prevent and/or treat tumors, prepare B cell vaccines, and prepare B cells to secrete therapeutic proteins Drugs for cell therapy;
  • This application uses a directed differentiation system and a co-cultivation method to direct the differentiation of the pluripotent stem cells into B-lineage seed cells.
  • the B-lineage seed cells can be differentiated in vivo to produce B cells, and can be used to prepare Drugs for enhancing immune effect, preventing and/or treating immunodeficiency, preventing and/or treating infectious diseases, preventing and/or treating tumors, preparing B-cell vaccines, and preparing B-cell-secreted therapeutic proteins;
  • the B cells derived from pluripotent stem cells obtained by the method of the present application have normal functions and no risk of tumorigenesis, can be used to prepare various drugs, and have broad application prospects.
  • Figure 1 (A) is a schematic diagram of an inducible expression system for site-directed knock-in of the ROSA26 locus in pluripotent stem cells.
  • Figure 1(B) is the bright field image (left) and fluorescence image (right) of iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells obtained through hygromycin B resistance screening (scale bar 200 ⁇ m).
  • Figure 1(C) shows the relative expression levels of RUNX1 (left), HOXA9 (middle) and LHX2 (right) after doxycycline treatment for 24 hours.
  • Fig. 2 (A) is a schematic diagram of the directed differentiation system for inducing the directed differentiation of iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells into induced hematopoietic endothelium (iHEC).
  • iHEC induced hematopoietic endothelium
  • Figure 2(B) is the differentiation pattern of EB embryoid somatic cells after induction of iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells to day 11 (left) and hematopoietic-related cells differentiated from EB embryoid bodies Colony map (right) (bar 400 ⁇ m).
  • Fig. 2(C) is the flow cytometry results of sorting induced hematopoietic endothelium using the flow cytometry sorting strategy (CD31 + , CD41 + , CD45 - , c-Kit + , CD201 + ).
  • Fig. 3 (A) is a schematic diagram of the co-culture of sorted induced hemogenic endothelium and OP9-DL1 cells.
  • Fig. 3(B) is the light field diagram of the cobblestone-like formation area of hematopoietic cells observed under the microscope after co-culture of induced hematopoietic endothelium and OP9-DL1 cells for 10 days (scale bar 400 ⁇ m).
  • Fig. 3(C) is a graph showing the results of flow cytometric detection of the immunophenotype of the hematopoietic progenitor cells after co-culturing the induced hematopoietic endothelium and OP9-DL1 cells for 10 days.
  • Fig. 4 (A) is a schematic diagram of transplantation strategy after co-culture to obtain B cells using in vivo microenvironment.
  • Fig. 4(B) is a graph showing the results of flow cytometry detection of blood cells in peripheral blood, bone marrow, spleen and lymph nodes of recipient mice 6 weeks after transplantation.
  • Figure 4(C) is a schematic diagram of the PCR amplification position of the blood cell genome derived from pluripotent stem cells.
  • Figure 4(D) is the PCR amplification electrophoresis detection map of blood cell genome derived from pluripotent stem cells; wherein, lane M represents DNA Marker, lane 1 represents plasmid, lane 2 represents mouse lymph node (LN) cells, lane 3 represents mouse spleen (Spleen, SP) cells, swimming lane 4 represents mouse bone marrow (Bone marrow, BM) cells, and swimming lane 5 represents blank control.
  • LN mouse lymph node
  • lane 3 represents mouse spleen (Spleen, SP) cells
  • swimming lane 4 represents mouse bone marrow (Bone marrow, BM) cells
  • swimming lane 5 represents blank control.
  • FIG. 4(E) is a diagram showing the sequencing and identification results of the blood cell genome derived from pluripotent stem cells.
  • Fig. 4(F) is the ELISA detection of the serum immunoglobulin content of unimmunized recipient mice (iB mice) after transplantation.
  • Figure 5(A) shows the flow of B progenitor cells (pro/pre-B), immature B cells and mature B cells derived from pluripotent stem cells in the bone marrow of recipient mice 2 weeks after transplantation Formula analysis result graph.
  • Figure 5(B) shows the results of flow cytometric analysis of B1 (including B1a and B1b) and B2 (including FO B and MZ B) cell populations derived from pluripotent stem cells in the spleen, lymph nodes and peritoneal cavity of recipient mice 4 weeks after transplantation picture.
  • Figure 5(C) shows the heavy chain and light chain of the B cell receptor (BCR) of naive follicular B cells (naive FO B) derived from pluripotent stem cells in the spleen 4 weeks after recipient mice (iB mice) transplantation Diversity analysis.
  • BCR B cell receptor
  • Figure 6(A) is the expression of antigen-specific (anti-NP) IgM (left) and IgG3 (right) in the serum of recipient mice (iB mice) after immunization with T cell-independent type I antigen (NP-LPS) Diagram of ELISA detection results.
  • Figure 6(B) shows the antigen-specific (anti-NP) IgM (left) and IgG3 (right) in the serum of recipient mice (iB mice) immunized with T-cell-independent type II antigen (NP-AECM-FICOLL) ) ELISA detection result chart.
  • Figure 6 (C) is the antigen-specific (anti-NP) IgM (I diagram) and IgG1 (II diagram and III diagram) in the serum of T cell-dependent antigen (NP-CGG) primary immune recipient mice (iB mice) ), and the antigen-specific (anti-NP) IgG1 (anti-NP) IgG1 in recipient mouse serum (iB mice) after re-immunization (re-antigen stimulation on the 111th day after the initial immunization) (Figure IV and V Figure) is the result of ELISA detection.
  • Figure 7(A) shows the pluripotent stem cell-derived plasma cells and antigen-specific germinal centers in the spleen on the 14th day after T cell-dependent antigen (NP-CGG) immune recipient mice (iB mice) Flow cytometry results of B cells (NP-specific GC B).
  • Figure 7(B) shows the flow cytometric detection of IgM + memory B cells (IgM + memory B) and IgG1 + memory B cells in the spleen on the 14th day after T cell-dependent antigen (NP-CGG) immune recipient mice (iB mice) Flow cytometry results of B cells (IgG1 + memory B).
  • Figure 7(C) shows the long-lived plasma cells in the bone marrow of recipient mice (iB mice) on the 21st day after the initial antigen stimulation and on the 17th day after the second antigen stimulation (re-antigen stimulation was performed on the 111th day after the initial antigen stimulation). Long lived plasma cell) flow detection results.
  • an inducible expression sequence was knocked in at the ROSA26 site of pluripotent stem cells by electroporation combined with gene recombination.
  • the cDNA sequences of LHX2 (CCDS16008.1) were concatenated, and doxycycline (Dox) was used to induce gene expression.
  • the knock-in sequence contained the iRUNX1-p2a-HOXA9-t2a-LHX2 tandem sequence and the hygromycin B resistance gene (HygroR) sequence used for resistance screening.
  • HygroR hygromycin B resistance gene
  • pluripotent stem cell medium containing hygromycin B 150 ⁇ g/mL was added, and the medium was changed every day. After 10 days of screening with hygromycin B, a single clone was picked under a microscope and placed in a 12-well plate with mouse embryonic fibroblast (MEF) in advance, and one pluripotent stem cell clone was placed in each well. cultured in hygromycin-free medium.
  • the medium was changed every day. After 3 days, the cloning group was digested with 0.25% trypsin and passed to a 12-well plate. The cell morphology was shown in Figure 1(B), and the cloning group was in logarithmic growth In the early stage, the edges were neat and bright, and there was a clear boundary with the MEF cell layer, and no differentiation occurred. According to the cell state and growth density, passage, expansion and cryopreservation are carried out.
  • each medium in the directional induction differentiation system is:
  • Basal differentiation medium IMDM medium containing 15% fetal bovine serum, 200 ⁇ g/mL iron-saturated transferrin, 4.5 ⁇ 10 -4 M thioglycerol, 2 mM GlutaMAX TM -I supplement and 50 ⁇ g/mL ascorbic acid;
  • D0 medium basic differentiation medium containing 5ng/mL bone morphogenetic protein 4;
  • D2.5 medium basic differentiation medium containing 5ng/mL bone morphogenetic protein 4 and 5ng/mL vascular endothelial growth factor;
  • D6 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 human FMS-like tyrosine kinase 3 ligand and 1 ⁇ g/mL potentiogenic Mycin basal differentiation medium.
  • the sorting strategy (CD31 + , CD41 + , CD45 - , c-Kit + and CD201 + ) as shown in Figure 2 (C) was used to sort by flow cytometry to induce hematopoiesis inner skin.
  • the sorted induced hematopoietic endothelium was co-cultured with OP9-DL1 stromal cells.
  • the co-culture medium is D11 medium, which contains 20ng/mL recombinant mouse interleukin 3, 20ng/mL recombinant mouse stem cell factor, 20ng/mL human FMS-like tyrosine kinase 3 ligand, 1 ⁇ g/mL Dox, 15% Fetal bovine serum, 200 ⁇ g/mL iron-saturated transferrin, 4.5 ⁇ 10 -4 M thioglycerol, 2 mM GlutaMAX TM -I supplement and 50 ⁇ g/mL ascorbic acid ⁇ -MEM medium;
  • Figure 3(B) shows that the induced hematopoietic endothelium derived from iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells and stromal cells OP9-DL1 co-cultured for 10 days, formed highly uniform small, round, bright spots on the stromal cells OP9-DL1 of hematopoietic cells.
  • Figure 3(C) shows that after 10 days of co-culture of induced hematopoietic endothelium derived from iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells and stromal cells OP9-DL1, the generated hematopoietic cells exhibited the immunophenotype of hematopoietic progenitor cells: LSK( Lin - c-Kit + Sca1 + ).
  • the transplantation strategy after co-cultivation is shown in Figure 4(A).
  • the induced hematopoietic endothelium was induced by adding Dox on OP9-DL1 stromal cells for 10 days to obtain B lineage seed cells; subsequently, the B lineage seed cells derived from pluripotent stem cells were In vivo B-lineage regeneration was performed by ocular vein transplantation into 8- to 12-week-old B-cell-deficient mice ( ⁇ MT mice).
  • Figure 4(B) shows that the B-lineage seed cells obtained after co-culture of induced hematopoietic endothelium derived from iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells can form hematopoietic cells in various hematopoietic tissues and organs of recipient ⁇ MT mice Chimeric.
  • GFP + hematopoietic cells mainly B cells
  • primers were designed for PCR amplification and sequencing identification .
  • the GFP + cells derived from bone marrow, lymph nodes and spleen were sorted by flow cytometry, the genome was extracted, and PCR identification was performed using specific primers for the knock-in gene sequence (as shown in Figure 4(C)).
  • Figure 4(D) shows that there are iRUNX1-p2a-HOXA9-t2a-LHX2 plasmid-derived sequences in the genomes of these cells, confirming that GFP + blood cells (mainly B cells) are derived from iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells , and the sequencing results (as shown in Figure 4(E)) also proved this result.
  • ELISA was performed to detect the content of immunoglobulin in the serum of unimmunized recipient mice 4 to 6 weeks after transplantation.
  • various immunoglobulin types including IgM, IgG1, IgG2b can be detected in the serum of ⁇ MT recipient mice (iB mice) after transplantation of B lineage seeded cells , IgG2c, IgG3 and IgA, achieved the effect of rebuilding the functional B lymphoid system.
  • Example 5 The occurrence process of regenerated B lineage
  • B lineage seed cells can regenerate pro/pre in bone marrow - B progenitor cells, which further develop into immature B cells and mature B cells;
  • Figure 5(B) shows that there are mature B1 cells (including B1a and B1b) and mature B2 cells (including FO B and MZ B) derived from pluripotent stem cells in the spleen, lymph nodes and peritoneal cavity;
  • the initial follicular B cells (naive FO B) in the spleen of recipient mice (iB mice) were sorted for B cell receptor (BCR) sequencing, as shown in Figure 5(C), the initial follicular B cells derived from pluripotent stem cells
  • the cells (naive FO B) have the diversity rearrangement of heavy chain and light chain, and the BCR diversity of naive FO B derived from pluripotent stem cells is similar to the BCR diversity of naive FO B of C57BL/6 mice in the positive control group;
  • the examples demonstrate the normal development of the pluripotent stem cell-derived B lineage in recipient mice.
  • This example further verifies whether B cells derived from iRUNX1-p2a-HOXA9-t2a-LHX2 pluripotent stem cells can produce antigen-specific antibodies.
  • B cells derived from pluripotent stem cells can secrete antigen-specific (anti-NP) IgM and IgG3.
  • FIG. 6(B) shows that pluripotent stem cell-derived B cells can secrete antigen-specific (anti-NP) IgM after immunization with recipient mice (iB mice) with T-independent type II antigen (NP-AECM-FICOLL) and IgG3.
  • anti-NP antigen-specific
  • iB mice recipient mice
  • T-independent type II antigen NP-AECM-FICOLL
  • Figure 6(C) shows that T cell-dependent antigen (NP-CGG) after primary immunization (Panels I, II and III) and re-immunization (Panels IV and V) of recipient mice (iB mice), the multipotent Stem cell-derived B cells can secrete antigen-specific antigen-specific (anti-NP) IgM and IgG1.
  • NP-CGG T cell-dependent antigen
  • this example demonstrates that B cells derived from pluripotent stem cells can produce specific antibodies against specific antigens.
  • Figure 7(B) shows that on the 14th day after immunization, IgM + memory B cells can be detected in the spleen of recipient mice, and B cells derived from pluripotent stem cells can undergo class switching to produce IgG1 + memory B cells;
  • Figure 7(C) shows that longevity can be detected by flow cytometry in recipient mouse bone marrow at day 21 after primary antigen challenge and at day 17 after second antigen challenge (re-antigen challenge was performed at day 111 after primary antigen challenge) Plasma cells (long lived plasma cells);
  • this example confirms that B cells derived from recipient mouse pluripotent stem cells can normally form germinal center B cells (Germinal center B, GC B), memory B cells (memory B) and long-lived plasma cells after antigen stimulation, Can effectively participate in the adaptive immune response.
  • the present application introduces exogenous RUNX1, HOXA9 and LHX2 co-expression vectors into pluripotent stem cells, and successfully constructs pluripotent stem cells that inducibly co-express exogenous RUNX1, HOXA9 and LHX2, and the pluripotent stem cells are directed to differentiate Seed cells for the B lineage and will develop into B cells.
  • the B cells derived from pluripotent stem cells obtained using the method of the present application not only function normally, but also have no risk of tumorigenesis, and can be used to prepare immune enhancement effects, prevent and/or treat immunodeficiency, prevent and/or treat infectious diseases, As well as drugs for preventing and/or treating tumors, preparing B cell vaccines and cell therapy for B cells secreting therapeutic proteins.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

一种体液免疫系统再生的方法，使用多能干细胞表达RUNX1基因、HOXA9基因和LHX2基因，在体外诱导分化后高效获得B细胞种子，移植后可以在体液免疫系统缺失的动物体内重建完备的体液免疫系统。该方法能够实现抗原特异性抗体免疫应答，针对抗原产生特异性高亲和力抗体，并产生免疫记忆。同时，重建的免疫系统安全，未见致瘤性风险。

Description

一种体液免疫系统再生的方法及其应用 技术领域
本申请属于医药生物工程技术领域，涉及多能干细胞的定向分化，尤其涉及一种体液免疫系统再生，即多能干细胞定向分化B细胞的方法及其应用。
背景技术
B细胞是体液免疫系统的核心细胞成分，B细胞功能缺陷会导致病人体液免疫下降，甚至出现严重的细菌、病毒或其他病原微生物的感染。所以，通过再生手段恢复甚至增强体液免疫系统，有望造福众多体液免疫系统异常的患者。
多能干细胞(pluripotent stem cells，PSCs)是一类具有无限增殖潜能、能分化产生不同谱系细胞潜力、方便进行基因编辑修饰的细胞，是细胞疗法再生医学研究的热点。诱导患者自体体细胞重编程衍生的多能干细胞(induced pluripotent stem cells，iPSC)分化形成不同的谱系细胞，不但可以规避使用胚胎干细胞的伦理争议，而且降低了异基因免疫排斥的风险，成为再生医学领域的理想细胞开发材料。
如何诱导多能干细胞分化产生B谱系种子，移植后重建体液免疫系统，是全世界研究的热点和难点，迄今没有实质性突破，更没有临床转化的案例。
研究表明，人胚胎多能干细胞(embryonic stem cells，ESCs)在体外与基质细胞共培养后更容易诱导产生NK细胞而不是B细胞(参见Martin,Colin H et al.Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.Blood vol.112,7(2008):2730-7)。
小鼠诱导多能干细胞在体外与基质细胞共培养可以诱导出T细胞，却很难诱导出B细胞(参见Wada,Haruka et al.Successful differentiation to T cells,but unsuccessful B-cell generation,from B-cell-derived induced pluripotent stem cells.International immunology vol.23,1(2011):65-74)。
同时，关于体内B细胞重建的研究方法也很少，其中有研究表明体外ESC诱导的pro/pre-B祖细胞移植入免疫缺陷鼠后可以产生B1和B2细胞，但是上述研究产生的B细胞在体内存在的时间很短暂，同时在移植后6-8周就检测不到分泌的抗体(参见Potocnik,A J et al.Reconstitution of B cell subsets in Rag deficient mice by transplantation of in vitro differentiated embryonic stem cells.Immunology letters vol.57,1-3(1997):131-7)。还有研究报道ESC在体外可以诱导出B1细胞的祖细胞，移植入免疫缺陷小鼠的体内可以长期重建B1细胞(参见Lin,Yang et al.Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.Stem cell reports vol.12,3(2019):572-583)，但是此方法却不能获得对适应性体液免疫应答更重要的B2细胞。
此外，还有研究通过在多能干细胞中表达特定的转录因子获得具有多谱系造血重建能力 的造血干组细胞(hematopoietic stem and progenitor cells，HSPCs)，移植后可以产生包括B细胞在内的多个造血谱系细胞(参见Lu,Yi-Fen et al.Engineered Murine HSCs Reconstitute Multi-lineage Hematopoiesis and Adaptive Immunity.Cell reports vol.17,12(2016):3178-3192；和Sugimura,Ryohichi et al.Haematopoietic stem and progenitor cells from human pluripotent stem cells.Nature vol.545,7655(2017):432-438)，但上述研究体系存在稳定性差、效率较低的问题。
因此，本领域亟需一种高效的诱导多能干细胞获得单一B谱系种子细胞的方法。
发明内容
本申请提供了一种多能干细胞定向分化B细胞的方法及其应用，采用基因修饰的多能干细胞，在体外诱导分化高效获得B细胞种子，移植后可以在体液免疫系统缺失的动物体内再生完备的体液免疫系统，是一种高效的体液免疫系统再生的方法，能够实现抗原特异性抗体免疫应答，针对抗原产生特异性高亲和力抗体，并产生免疫记忆，该方法重建的免疫系统安全，未见致瘤性风险。
第一方面，本申请提供一种表达载体，所述表达载体含有编码RUNX1、HOXA9和LHX2基因的核苷酸序列，用于实现RUNX1、HOXA9以及LHX2三个基因的串联共表达。
本申请中，将RUNX1、HOXA9和LHX2的cDNA序列串联表达于同一载体，并整合入哺乳动物多能干细胞基因组中，可以得到稳定表达RUNX1、HOXA9和LHX2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B细胞的能力。
其中，RUNX1基因又称为AML1，是RUNX转录因子蛋白家族中的成员之一，是白血病染色体易位最常见的靶位点。RUNX1是十分关键的造血调控转录因子，其在内皮生血转化、原始造血、永久造血以及淋巴细胞生成中起着重要的作用；
所述RUNX1基因可以为多种来源，如人源或鼠源，其中，小鼠来源的RUNX1基因可以是ENSMUSG00000022952，人源来源的RUNX1基因可以是ENSG00000159216。
HOXA9基因是HOX基因家族的一员，是编码序列特异性转录调控因子，在胚胎发育以及造血调控方面起着重要作用。HOXA9可以在HSC的增强与维持、内皮生血转化以及促进淋系生成方面起着重要作用；
所述HOXA9基因可以为多种来源，如人源或鼠源，其中小鼠来源的HOXA9基因可以是ENSMUSG00000038227，人源来源的HOXA9基因可以是ENSG00000078399。
LHX2基因(Lim homeobox 2)又称为LH-2，作为转录因子家族成员之一，在多种器官的发育过程中都起着较为重要的作用，尤其在神经系统中高水平表达；同时LHX2在胚胎造血、红系生成中起着重要作用，此外可以促进造血干祖细胞的永生，且LHX2被发现在pre-B细胞系中表达。
所述LHX2基因可以为多种来源，如人源或鼠源，其中小鼠来源的LHX2基因可以是ENSMUSG00000000247，人源来源的LHX2基因可以是ENSG00000106689。
三个基因的联用主要是RUNX1可以促进多能干细胞分化形成生血内皮细胞，同时 RUNX1以及HOXA9可以促进淋系的生成，而LHX2进一步促进了向B谱系的分化；相比于其他分化相关的基因，如RUNX1、LMO2和MEIS1同时使用，该组合在后期诱导生血内皮与OP9-DL1共培养的过程中无法正常产生造血克隆。
第二方面，本申请提供了一种基因编辑的多能干细胞宿主细胞，所述宿主细胞包含如第一方面所述的表达载体。
优选地，所述宿主细胞为多能干细胞，包括诱导性多能干细胞和/或胚胎多能干细胞系。
优选地，所述多能干细胞包括基因编辑后的诱导性多能干细胞和/或胚胎多能干细胞系。
第三方面，本申请提供了一种体液免疫系统再生，即多能干细胞定向分化B细胞的方法，包括以下步骤：
(1)将如第一方面所述的表达载体，即RUNX1，HOXA9和LHX2三个基因串联的表达载体整合到多能干细胞中，并进行抗性克隆化筛选；
(2)将步骤(1)所得的多能干细胞定向分化为诱导生血内皮(iHEC)；
(3)将步骤(2)所述诱导生血内皮与骨髓基质细胞共培养，得到具备B谱系分化潜能的造血祖细胞，也就是B谱系种子细胞；以及
(4)将步骤(3)所述B谱系种子细胞移植，体内分化产生B细胞。
本申请中，通过对RUNX1、HOXA9和LHX2共表达的多能干细胞系进行定向分化，获得的诱导生血内皮与骨髓基质细胞共培养获得B谱系种子细胞，分化后得到功能正常的B细胞，包括所有成熟细胞类型，且无致瘤风险。
优选地，步骤(1)所述RUNX1、HOXA9和LHX2串联的表达载体可整合到任意安全位点，插入位点能够使得插入的基因稳定表达即可，本申请中优选地将串联表达的基因整合到多能干细胞的ROSA26位点、AAVS1位点、CCR5位点、H11位点、COL1A1位点或TIGRE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，获得主克隆干细胞系；也可以采用其他抗性筛选策略诸如氯霉素、遗传霉素(G-418)、杀稻瘟菌素、霉酚酸等获得主克隆干细胞系。
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、D6培养基培养多能干细胞得到所述诱导生血内皮。
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、4ng/mL、5ng/mL、6ng/mL、7ng/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(例如可以是3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL或8ng/mL，优选为5ng/mL)BMP4和3～8ng/mL(例如可以是3ng/mL、 4ng/mL、5ng/mL、6ng/mL、7ng/mL或8ng/mL，优选为5ng/mL)血管内皮生长因子(VEGF)的基础分化培养基。
优选地，所述D6培养基为含有10～30ng/mL白介素3(IL3)、10～30ng/mL白介素6(IL6)、10～30ng/mL干细胞因子(SCF)、10～30ng/mL FMS样酪氨酸激酶3配体(Flt3L)和1～2μg/mL强力霉素(Dox)的基础分化培养基。
其中，所述IL3的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/mL或30ng/mL，优选为20ng/mL；所述IL6的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/mL或30ng/mL，优选为20ng/mL；所述SCF的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/mL或30ng/mL，优选为20ng/mL；所述Flt3L的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/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、1.2μg/mL、1.4μg/mL、1.5μg/mL、1.6μg/mL、1.8μg/mL或2μg/mL，优选为1μg/mL。
所述D0培养基、所述D2.5培养基和所述D6培养基的组分如下表1所示：
表1
Figure PCTCN2022085730-appb-000001
优选地，所述基础分化培养基为包含10～20％胎牛血清(％表示体积分数)、180～220μg/mL铁饱和转铁蛋白(iron-saturated transferrin)、4×10 -4～5×10 -4M硫代甘油、1～3mMGlutaMAX TM-I添加剂和30～70μg/mL抗坏血酸的IMDM培养基；
其中，所述胎牛血清的浓度例如可以是10％、12％、14％、16％、18％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、190μg/mL、210μg/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、4.2×10 -4M、4.4×10 -4M、4.8×10 -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、1.4mM、1.8mM、2.2mM、2.4mM、2.6mM、2.8mM或3mM，优选为2mM；所述抗坏血酸的浓度例如可以是30μg/mL、35μg/mL、40μg/mL、45μg/mL、55μg/mL、60μg/mL、65μg/mL或70μg/mL，优选为50μg/mL。
本申请中，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诱导生血内皮，所述诱导生血内皮通过进一步与小鼠骨髓基质细胞共 培养，得到B谱系种子细胞。
优选地，步骤(3)所述骨髓基质细胞包括OP9-DL1细胞、OP9-DL4细胞、OP9细胞、MS5细胞、MS5-DL1细胞、MS5-DL4细胞、HS-5细胞、HS-5-DL1细胞、HS-5-DL4细胞、MSC细胞、MSC-DL1细胞或MSC-DL4细胞中的任意一种或至少两种的组合；也可选择其他骨髓、胸腺、淋巴结、肝脏、脾脏组织等来源的基质细胞以及修饰表达DL1或DL4的骨髓基质细胞中的任意一种或至少两种的组合。
上述细胞系带有DL1的时候也有别名DLL1，带有DL4的时候也有别名DLL4，均是相同的对应细胞系。
优选地，步骤(3)所述共培养过程中采用强力霉素(Dox)进行诱导；也可以采用其他诱导原理设计的表达元件进行相应药物诱导，例如他莫昔芬(tamoxifen、4-OHT)。
优选地，步骤(3)所述共培养的方法为将诱导生血内皮与OP9-DL1细胞采用D11培养基进行共培养，得到所述B谱系种子细胞。
优选地，所述D11培养基的组分如下表2所示：
表2
组分 D11培养基
α-MEM培养基 主体成分
白介素3 10～30ng/mL
干细胞因子 10～30ng/mL
FMS样酪氨酸激酶3配体 10～30ng/mL
强力霉素 1～2μg/mL
胎牛血清 10～20％
铁饱和转铁蛋白 180～220μg/mL
硫代甘油 4.5×10 -4M
GlutaMAX TM-I添加剂 1～3mM
抗坏血酸 30～70μg/mL
其中，所述IL3的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/mL或30ng/mL，优选为20ng/mL；所述SCF的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/mL或30ng/mL，优选为20ng/mL；所述Flt3L的浓度例如可以是10ng/mL、15ng/mL、18ng/mL、22ng/mL、25ng/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、1.2μg/mL、1.4μg/mL、1.5μg/mL、1.6μg/mL、1.8μg/mL或2μg/mL，优选为1μg/mL；所述胎牛血清的浓度例如可以是10％、12％、14％、16％、18％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、190μg/mL、210μg/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、4.2×10 -4M、4.4×10 -4M、4.8×10 -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、1.4mM、1.8mM、2.2mM、2.4mM、2.6mM、2.8mM或3mM，优选为2mM；所述 抗坏血酸的浓度例如可以是30μg/mL、35μg/mL、40μg/mL、45μg/mL、55μg/mL、60μg/mL、65μg/mL或70μg/mL，优选为50μg/mL。
需要说明的是，所述D11培养基的主体培养基可以是α-MEM培养基或IMDM培养基；由于本申请在实验过程中所用的骨髓基质细胞为OP9-DL1，所以培养基优选为α-MEM培养基。
优选地，步骤(4)所述分化产生的B细胞包括B220 +B细胞和/或CD19 +B细胞。
优选地，所述分化产生的B细胞包括pro-B细胞、pre-B细胞、B1细胞、B2细胞或浆细胞(plasma cell)中的任意一种或至少两种的组合。
优选地，所述B1细胞包括B1a细胞和/或B1b细胞。
优选地，所述B2细胞为滤泡B细胞(Follicular B，FO B)和/或边缘区B细胞(Marginal zone，MZ B)。
作为本申请优选的技术方案，本申请提供了一种多能干细胞定向分化B细胞的方法，包括以下步骤：
(1)将RUNX1、HOXA9和LHX2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、D6培养基进行培养，在第8-12天定向分化为诱导生血内皮；
(3)将步骤(2)所述诱导生血内皮与OP9-DL1细胞采用D11培养基进行共培养8-21天，并采用强力霉素进行诱导，得到B谱系种子细胞；以及
(4)将步骤(3)所述B谱系种子细胞转至动物模型中，分化产生B细胞，所述B细胞包括pro-B细胞、pre-B细胞、B1细胞、B2细胞或浆细胞中的任意一种或至少两种的组合。
优选地，步骤(2)在第11天定向分化为诱导生血内皮。
优选地，步骤(3)所述共培养的时间为10天。
第四方面，本申请提供一种如第三方面所述的方法制备得到的B谱系种子细胞或B细胞。
第五方面，本申请提供了一种药物组合物，包括如第一方面所述的表达载体、如第二方面所述的宿主细胞、如第四方面所述的B谱系种子细胞或B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辅料；所述药学上可接受的辅料包括载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第六方面，本申请还提供了一种如第五方面所述的药物组合物在制备增强免疫响应的药物、预防和/或治疗疾病的药物、B细胞免疫疗法治疗肿瘤的药物、B细胞疫苗或B细胞分泌治疗性蛋白的细胞疗法的药物中的应用。
优选地，所述增强免疫响应的药物包括增强B细胞免疫响应和/或T细胞免疫响应的药物。
优选地，所述预防和/或治疗疾病的药物包括预防和/或治疗B细胞免疫缺陷、感染性疾病、肿瘤的药物。
优选地，所述B细胞分泌治疗性蛋白的细胞疗法的药物包括预防和/或治疗自身免疫病、基因遗传性疾病的药物。
优选地，所述B细胞分泌治疗性蛋白包括抗体。
优选地，所述基因遗传性疾病包括血友病、溶酶体贮积症、低磷酸酯酶症或苯丙酮尿症中的任意一种或至少两种的组合。
本申请中，所述药物组合物可以用于：(1)增强免疫响应，特别是增强B细胞免疫响应和/或T细胞免疫响应；(2)预防和/或治疗疾病，优选为用于制备预防和/或治疗B细胞免疫缺陷、感染性疾病、肿瘤等；(3)B细胞疫苗研发及制备；以及(4)B细胞分泌治疗性蛋白的细胞疗法，优选为用于预防和/或治疗自身免疫病、基因遗传性疾病等。
与现有技术相比，本申请的有益效果为：
(1)本申请将外源RUNX1、HOXA9和LHX2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、HOXA9和LHX2的多能干细胞，所述多能干细胞具有分化为B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、预防和/或治疗感染性疾病、预防和/或治疗肿瘤、制备B细胞疫苗以及制备B细胞分泌治疗性蛋白的细胞疗法的药物；
(2)本申请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B谱系种子细胞，所述B谱系种子细胞移植后，可以在体内分化产生B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、预防和/或治疗感染性疾病、预防和/或治疗肿瘤、制备B细胞疫苗以及制备B细胞分泌治疗性蛋白的细胞疗法的药物；以及
(3)采用本申请的方法获得的多能干细胞来源的B细胞，功能正常，没有致瘤风险，可以用于制备多种药物，具有广阔的应用前景。
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。
图1(B)为通过潮霉素B抗性筛选获得的iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞明场图(左)以及荧光图(右)(标尺200μm)。
图1(C)为使用强力霉素处理24小时后RUNX1(左)，HOXA9(中)以及LHX2(右)的相对表达水平。
图2(A)为诱导iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞定向分化为诱导生血内皮(iHEC)的拟胚体定向诱导分化体系示意图。
图2(B)为诱导iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞定向分化至第11天的EB拟胚体细胞分化形态图(左)以及由EB拟胚体分化而来的造血相关细胞集落图(右)(标尺400μm)。
图2(C)为采用流式分选策略(CD31 +、CD41 +、CD45 -、c-Kit +、CD201 +)分选诱导生血内皮的流式结果图。
图3(A)为分选的诱导生血内皮与OP9-DL1细胞共培养的示意图。
图3(B)为诱导生血内皮与OP9-DL1细胞共培养10天后显微镜下观察的造血细胞鹅卵石样形成区域的光场图(标尺400μm)。
图3(C)为诱导生血内皮与OP9-DL1细胞共培养10天后造血祖细胞免疫表型的流式检测结果图。
图4(A)为利用体内微环境获得B细胞的共培养后移植策略示意图。
图4(B)为移植6周后，受体小鼠外周血、骨髓、脾脏和淋巴结中血液细胞的流式检测结果图。
图4(C)为多能干细胞来源的血液细胞基因组的PCR扩增位置示意图。
图4(D)为多能干细胞来源的血液细胞基因组的PCR扩增电泳检测图；其中，泳道M表示DNA Marker，泳道1表示质粒，泳道2表示小鼠淋巴结(Lymph node,LN)细胞，泳道3表示小鼠脾脏(Spleen,SP)细胞，泳道4表示小鼠骨髓(Bone marrow,BM)细胞，泳道5表示空白对照。
图4(E)为多能干细胞来源的血液细胞基因组的测序鉴定结果图。
图4(F)为移植后ELISA检测未免疫受体鼠(iB小鼠)血清中免疫球蛋白的含量。
图5(A)为受体鼠移植后2周，骨髓中多能干细胞来源的B祖细胞(pro/pre-B)，非成熟B(immature B)细胞以及成熟B(mature B)细胞的流式分析结果图。
图5(B)为受体鼠移植后4周，脾脏、淋巴结以及腹膜腔中多能干细胞来源的B1(包括B1a和B1b)以及B2(包括FO B和MZ B)细胞群体的流式分析结果图。
图5(C)为受体鼠(iB小鼠)移植后4周，脾脏中多能干细胞来源的初始滤泡B细胞(naive FO B)的B细胞受体(BCR)的重链和轻链多样性分析。
图6(A)为非T细胞依赖性Ⅰ型抗原(NP-LPS)免疫受体鼠(iB小鼠)后血清中的抗原特异性(anti-NP)IgM(左)以及IgG3(右)的ELISA法检测结果图。
图6(B)为非T细胞依赖性Ⅱ型抗原(NP-AECM-FICOLL)免疫受体鼠(iB小鼠)后血清中的抗原特异性(anti-NP)IgM(左)以及IgG3(右)的ELISA法检测结果图。
图6(C)为T细胞依赖性抗原(NP-CGG)初次免疫受体鼠(iB小鼠)血清中的抗原特异性(anti-NP)IgM(I图)以及IgG1(II图和III图)的ELISA法检测结果图，以及再次免疫(在初次免疫后第111天进行再次抗原刺激)后受体鼠血清(iB小鼠)中的抗原特异性(anti-NP)IgG1(IV图和V图)的ELISA法检测结果图。
图7(A)为T细胞依赖性抗原(NP-CGG)免疫受体鼠(iB小鼠)后第14天，脾脏中多能干细胞来源的浆细胞(plasma cell)以及抗原特异性的生发中心B细胞(NP-specific GC B)的流式检测结果图。
图7(B)为T细胞依赖性抗原(NP-CGG)免疫受体鼠(iB小鼠)后第14天，流式检测脾脏中IgM +记忆B细胞(IgM +memory B)以及IgG1 +记忆B细胞(IgG1 +memory B)的流式检测结果图。
图7(C)为在初次抗原刺激后第21天、在再次抗原刺激(初次抗原刺激后第111天进行再次抗原刺激)后第17天受体鼠(iB小鼠)骨髓中长寿浆细胞(long lived plasma cell)的流式检测结果图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本申请的技术方案，但下述的实例仅仅是本申请的简易例子，并不代表或限制本申请的权利保护范围，本申请的保护范围以权利要求书为准。
以下实施例中，若无特殊说明，所用试剂及耗材均购自本领域常规试剂厂商；若无特殊说明，所用实验方法和技术手段均为本领域常规的方法和手段。
实施例1 制备表达RUNX1、HOXA9和LHX2基因的载体和多能干细胞
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，所述表达系统采用p2a以及t2a序列将RUNX1(CCDS28339.1)、HOXA9(CCDS20146.1)和LHX2(CCDS16008.1)的cDNA序列串联，采用强力霉素(Dox)诱导基因表达。
如图1(A)所示，敲入序列包含iRUNX1-p2a-HOXA9-t2a-LHX2串联序列和用于抗性筛选的潮霉素B抗性基因(HygroR)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小鼠胚胎成纤维细胞(Mouse embryonic fibroblast,MEF)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-t2a-LHX2多能干细胞总mRNA(未加Dox组作为对照组)，利用Q-PCR检测RUNX1，HOXA9和LHX2的mRNA的表达水平，图1(C)表明，加入Dox可以诱导RUNX1，HOXA9和LHX2的表达。
实施例2 诱导多能干细胞向诱导生血内皮分化
为了诱导多能干细胞向诱导生血内皮分化，采用如图2(A)所示的拟胚体定向诱导分化体系。
定向诱导分化体系中各培养基的配方为：
基础分化培养基：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添加剂和50μg/mL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；
D6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人FMS样酪氨酸激酶3配体和1μg/mL强力霉素的基础分化培养基。
具体步骤为：
(1)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。吸去多余的明胶，将多能干细胞悬液转移到包被有明胶的孔中，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。
(2)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。
(3)提前40min在6孔板中铺1mL浓度为0.1％的明胶(gelatin)，待用。用巴氏吸管将EB收集到离心管中，用DPBS清洗培养皿底，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。用D2.5培养基重悬EB后，吸去多余的明胶，将EB转移至包被有明胶的6孔板中，培养12小时观察EB是否有污染。
(4)随后在D4进行换液继续培养两天，采用的培养基为D2.5培养基。
(5)更换D6培养基进行培养一天。随后隔天换液，采用的培养基为D6培养基。
拟胚体在培养过程中逐渐向外围扩散迁移形成中胚层细胞，如图2(B)所示，在第11天，iRUNX1-p2a-HOXA9-t2a-LHX2拟胚体中心的外围可见明显的一圈分化细胞(左)，并且在拟胚体周围可见明显的造血簇(右)。
在拟胚体诱导分化培养的第11天，采用如图2(C)的分选策略(CD31 +、CD41 +、CD45 -、c-Kit +以及CD201 +)进行流式细胞仪分选诱导生血内皮。
实施例3 共培养诱导生血内皮与OP9-DL1基质细胞
为了进一步从诱导生血内皮诱导分化得到B谱系种子细胞，如图3(A)所示，本实施例中将分选获得的诱导生血内皮与OP9-DL1基质细胞进行共培养。
具体步骤如下：
(1)提前4天复苏OP9-DL1细胞，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；
(2)使用前一天传代，每孔重铺2万细胞(12孔板)，第二天使用；
共培养培养基为D11培养基，是含有20ng/mL重组小鼠白介素3、20ng/mL重组小鼠干细胞因子、20ng/mL人FMS样酪氨酸激酶3配体、1μg/mL Dox、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添加剂和50μg/mL抗坏血酸的α-MEM培养基；
图3(B)显示iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞来源的诱导生血内皮与基质细 胞OP9-DL1共培养10天后，在基质细胞OP9-DL1上形成高度均一的小、圆、亮的造血细胞。
图3(C)显示iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞来源的诱导生血内皮与基质细胞OP9-DL1共培养10天后，生成的造血细胞呈现出造血祖细胞的免疫表型：LSK(Lin -c-Kit +Sca1 +)。
实施例4 共培养后移植进行体内B谱系再生
为了利用体内微环境获得B细胞，本实施例中进一步设计了共培养后移植策略。
所述共培养后移植策略如图4(A)所示，将诱导生血内皮在OP9-DL1基质细胞上添加Dox诱导10天后获得B谱系种子细胞；随后，将多能干细胞来源的B谱系种子细胞通过眼静脉移植到8～12周龄的B细胞缺陷小鼠(μMT小鼠)中，进行体内B谱系再生。
图4(B)表明iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞来源的诱导生血内皮经过共培养后获得的B谱系种子细胞，可以在受体μMT小鼠各种造血组织以及器官中形成造血嵌合。
流式分析移植后6周的受体鼠，结果显示在外周血、骨髓、脾脏和淋巴结中，多能干细胞来源的血液细胞以CD19 +B细胞为主，实现了有效重建B淋系的效果。
本实施例中，为了从基因组水平上确认受体小鼠中GFP +造血细胞(主要是B细胞)来源于iRUNX1-p2a-HOXA9-t2a-LHX2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、淋巴结和脾脏来源的GFP +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(如图4(C)所示)。
图4(D)显示，这些细胞基因组内有iRUNX1-p2a-HOXA9-t2a-LHX2质粒来源序列，证实GFP +血液细胞(主要是B细胞)来自于iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞，同时测序结果(如图4(E)显示)也证明了这一结果。
同时，为了验证多能干细胞来源的B细胞是否具备抗体分泌的功能，移植4～6周后，进行ELISA检测未免疫受体鼠血清中免疫球蛋白的含量。
如图4(F)显示，相比于阴性对照μMT小鼠，B谱系种子细胞移植后的μMT受体鼠(iB小鼠)血清中可以检测到各种免疫球蛋白类型包括IgM、IgG1、IgG2b、IgG2c、IgG3以及IgA，实现了重建功能性B淋系的效果。
实施例5 再生B谱系的发生过程
为了进一步明确iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞来源的B谱系发生过程，流式细胞术发现，如图5(A)所示，B谱系种子细胞在骨髓中可以再生出pro/pre-B祖细胞，进一步发育为非成熟B细胞和成熟B细胞；
图5(B)显示，在脾脏、淋巴结以及腹膜腔中均存在有多能干细胞来源的成熟B1细胞(包括B1a以及B1b)和成熟B2细胞(包括FO B和MZ B)；
分选受体鼠(iB小鼠)脾脏中的初始滤泡B细胞(naive FO B)进行B细胞受体(BCR)测序，如图5(C)显示，多能干细胞来源的初始滤泡B细胞(naive FO B)具备重链以及轻链的多样性重排，多能干细胞来源的naive FO B的BCR多样性类似于阳性对照组C57BL/6小鼠的naive FO B的BCR多样性；本实施例证实了受体鼠体内多能干细胞来源B谱系的正 常发育。
实施例6 B细胞免疫功能验证
本实施例进一步验证iRUNX1-p2a-HOXA9-t2a-LHX2多能干细胞来源的B细胞是否能够产生抗原特异性的抗体。
如图6(A)显示，用非T细胞依赖性Ⅰ型抗原(NP-LPS)免疫受体鼠(iB小鼠)后，多能干细胞来源的B细胞可以分泌抗原特异性(anti-NP)IgM以及IgG3。
图6(B)显示非T细胞依赖性Ⅱ型抗原(NP-AECM-FICOLL)免疫受体鼠(iB小鼠)后，多能干细胞来源的B细胞可以分泌抗原特异性(anti-NP)IgM以及IgG3。
图6(C)显示T细胞依赖性抗原(NP-CGG)初次免疫(I图、II图和III图)以及再次免疫(IV图和V图)受体鼠(iB小鼠)后，多能干细胞来源的B细胞可以分泌抗原特异性抗原特异性(anti-NP)IgM以及IgG1。
因此，本实施例证实了多能干细胞来源的B细胞可以针对特定抗原产生特异性的抗体。
实施例7 适应性免疫应答
T细胞依赖性抗原(NP-CGG)免疫受体鼠(iB小鼠)后，如图7(A)所示，在免疫后第14天，流式检测结果显示脾脏中多能干细胞来源的B细胞可以形成浆细胞(plasma cell)以及产生抗原特异性的生发中心B细胞(NP-specific GC B)；
图7(B)显示，在免疫后第14天，受体鼠脾脏中可以检测到IgM +记忆B细胞，同时多能干细胞来源的B细胞可以进行类别转换，产生IgG1 +记忆B细胞；
图7(C)显示，受体鼠骨髓中在初次抗原刺激后第21天以及在再次抗原刺激(初次抗原刺激后第111天进行再次抗原刺激)后第17天通过流式均可检测到长寿浆细胞(long lived plasma cell)；
因此，本实施例证实了受体鼠多能干细胞来源的B细胞经过抗原刺激后可以正常的形成生发中心B细胞(Germinal center B,GC B)，记忆B细胞(memory B)以及长寿浆细胞，可有效地参与适应性免疫应答。
综上所述，本申请将外源RUNX1、HOXA9和LHX2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、HOXA9和LHX2的多能干细胞，所述多能干细胞定向分化为B谱系种子细胞，并将发育为B细胞。使用本申请的方法获得的多能干细胞来源的B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、预防和/或治疗感染性疾病、以及预防和/或治疗肿瘤、制备B细胞疫苗以及制备B细胞分泌治疗性蛋白的细胞疗法的药物。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (12)

  1. 一种表达载体，其含有编码RUNX1基因、HOXA9基因和LHX2基因的核苷酸序列。
  2. 根据权利要求1所述的表达载体，其中，所述编码RUNX1基因、HOXA9基因和LHX2基因的核苷酸序列采用编码2A肽的核苷酸序列进行串联连接。
  3. 根据权利要求2所述的表达载体，其中，所述2A肽包括T2A、P2A、E2A或F2A中的任意一种或至少两种的组合。
  4. 根据权利要求1～3任一项所述的表达载体，其中，所述表达载体上，编码RUNX1基因的核苷酸序列、编码HOXA9基因的核苷酸序列和编码LHX2基因的核苷酸序列顺次连接，且所述编码RUNX1基因的核苷酸序列和编码HOXA9基因的核苷酸序列之间采用P2A核苷酸序列进行连接，所述编码HOXA9基因的核苷酸序列和编码LHX2基因的核苷酸序列之间采用T2A核苷酸序列进行连接。
  5. 一种宿主细胞，其包含如权利要求1～4任一项所述的表达载体；
    优选地，所述宿主细胞为多能干细胞，包括诱导性多能干细胞和/或胚胎多能干细胞系；
    优选地，所述多能干细胞包括基因编辑后的诱导性多能干细胞和/或胚胎多能干细胞系。
  6. 一种体液免疫系统再生的方法，其包括如下步骤：
    (1)将如权利要求1～4任一项所述的表达载体整合到多能干细胞中，并进行抗性克隆化筛选；
    (2)将步骤(1)所得的多能干细胞定向分化为诱导生血内皮；
    (3)将步骤(2)所述诱导生血内皮与骨髓基质细胞共培养，得到B谱系种子细胞；以及
    (4)将步骤(3)所述B谱系种子细胞转至动物模型中，分化产生B细胞。
  7. 根据权利要求6所述的方法，其中，步骤(1)中将所述表达载体整合到多能干细胞的位点包括ROSA26位点、AAVS1位点、CCR5位点、H11位点、COL1A1位点或TIGRE位点；
    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培养基和D6培养基培养所述多能干细胞，得到所述诱导生血内皮；
    优选地，步骤(3)所述骨髓基质细胞包括OP9-DL1细胞、OP9-DL4细胞、OP9细胞、MS5细胞、MS5-DL1细胞、MS5-DL4细胞、HS-5细胞、HS-5-DL1细胞、HS-5-DL4细胞、MSC细胞、MSC-DL1细胞、MSC-DL4细胞中的任意一种或至少两种的组合；
    优选地，步骤(3)所述共培养的过程中采用强力霉素进行诱导；
    优选地，步骤(3)所述共培养的方法为：将所述诱导生血内皮与OP9-DL1细胞采用D11培养基进行共培养，得到所述B谱系种子细胞。
  8. 根据权利要求7所述的方法，其中，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；
    优选地，所述D2.5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；
    优选地，所述D6培养基为含有10～30ng/mL白介素3、10～30ng/mL白介素6、10～30ng/mL干细胞因子、10～30ng/mL FMS样酪氨酸激酶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、4×10 -4～5×10 -4M硫代甘油、1～3mM GlutaMAX TM-I添加剂和30～70μg/mL抗坏血酸的IMDM培养基；
    优选地，所述D11培养基为含有10～30ng/mL白介素3、10～30ng/mL干细胞因子、10～30ng/mL FMS样酪氨酸激酶3配体、1～2μg/mL强力霉素、10～20％胎牛血清、180～220μg/mL铁饱和转铁蛋白、4×10 -4～5×10 -4M硫代甘油、1～3mM GlutaMAX TM-I添加剂和30～70μg/mL抗坏血酸的α-MEM培养基。
  9. 根据权利要求6～8任一项所述的方法，其中，步骤(4)所述分化产生的B细胞包括B220 +B细胞和/或CD19 +B细胞；
    优选地，所述分化产生的B细胞包括pro-B细胞、pre-B细胞、B1细胞、B2细胞或浆细胞中的任意一种或至少两种的组合；
    优选地，所述B1细胞包括B1a细胞和/或B1b细胞；
    优选地，所述B2细胞为滤泡B细胞和/或边缘区B细胞。
  10. 如权利要求6～9任一项所述的方法制备得到的B谱系种子细胞或B细胞。
  11. 一种药物组合物，其包括如权利要求1～4任一项所述的表达载体、权利要求5所述的宿主细胞、权利要求10所述的B谱系种子细胞或B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辅料。
  12. 如权利要求11所述的药物组合物在制备增强免疫响应的药物、预防和/或治疗疾病的药物、B细胞免疫疗法治疗肿瘤的药物、B细胞疫苗或B细胞分泌治疗性蛋白的细胞疗法的药物中的应用；
    优选地，所述增强免疫响应的药物包括增强B细胞免疫响应和/或T细胞免疫响应的药物；
    优选地，所述预防和/或治疗疾病的药物包括预防和/或治疗B细胞免疫缺陷、感染性疾病、肿瘤的药物；
    优选地，所述B细胞分泌治疗性蛋白的细胞疗法的药物包括预防和/或治疗自身免疫病、基因遗传性疾病的药物；
    优选地，所述B细胞分泌治疗性蛋白包括抗体；
    优选地，所述基因遗传性疾病包括血友病、溶酶体贮积症、低磷酸酯酶症或苯丙酮尿症中的任意一种或至少两种的组合。
PCT/CN2022/085730 2021-05-20 2022-04-08 一种体液免疫系统再生的方法及其应用 WO2022242359A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2023572007A JP2024518632A (ja) 2021-05-20 2022-04-08 体液免疫系の再生方法およびその使用
US18/562,645 US20240269181A1 (en) 2021-05-20 2022-04-08 Method for regenerating humoral immunity system and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110552087.3 2021-05-20
CN202110552087 2021-05-20
CN202210234297.2A CN115418373A (zh) 2021-05-20 2022-03-10 一种体液免疫系统再生的方法及其应用
CN202210234297.2 2022-03-10

Publications (1)

Publication Number Publication Date
WO2022242359A1 true WO2022242359A1 (zh) 2022-11-24

Family

ID=84141073

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/085730 WO2022242359A1 (zh) 2021-05-20 2022-04-08 一种体液免疫系统再生的方法及其应用

Country Status (3)

Country Link
US (1) US20240269181A1 (zh)
JP (1) JP2024518632A (zh)
WO (1) WO2022242359A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140037600A1 (en) * 2011-02-08 2014-02-06 Cellular Dynamics International, Inc. Hematopoietic precursor cell production by programming
CN108431211A (zh) * 2015-10-20 2018-08-21 富士胶片细胞动力公司 通过遗传编程的多谱系造血前体细胞产生
CN110637083A (zh) * 2017-03-20 2019-12-31 华盛顿大学 细胞及其使用和制备方法
CN112119086A (zh) * 2018-03-01 2020-12-22 堪萨斯大学 用于使用重组t细胞受体基因生成基于细胞的治疗剂的技术
CN114181968A (zh) * 2022-02-11 2022-03-15 北京干细胞与再生医学研究院 一种具备b谱系分化潜能的人造血祖细胞的制备方法及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140037600A1 (en) * 2011-02-08 2014-02-06 Cellular Dynamics International, Inc. Hematopoietic precursor cell production by programming
CN108431211A (zh) * 2015-10-20 2018-08-21 富士胶片细胞动力公司 通过遗传编程的多谱系造血前体细胞产生
CN110637083A (zh) * 2017-03-20 2019-12-31 华盛顿大学 细胞及其使用和制备方法
CN112119086A (zh) * 2018-03-01 2020-12-22 堪萨斯大学 用于使用重组t细胞受体基因生成基于细胞的治疗剂的技术
CN114181968A (zh) * 2022-02-11 2022-03-15 北京干细胞与再生医学研究院 一种具备b谱系分化潜能的人造血祖细胞的制备方法及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HOU TENG, NA ZHANG: "The role of Breg in tumor immunotherapy", CHEMISTRY OF LIFE : COMMUNICATIONS OF THE CHINESE BIOCHEMICAL SOCIETY, SHANGHAI : ZHONGGUO SHENGWU HUAXUEHUI, CN, vol. 40, no. 2, 30 March 2020 (2020-03-30), CN , pages 153 - 159, XP093006535, ISSN: 1000-1336, DOI: 10.13488/j.smhx.20190294 *
ZHANG, QI ET AL.: "Regeneration of Immunocompetent B Lymphopoiesis from Pluripotent Stem Cells Guided by Transcription Factors", CELLULAR & MOLECULAR IMMUNOLOGY, vol. 19, no. 4, 31 December 2021 (2021-12-31), XP037788971, ISSN: 2042-0226, DOI: 10.1038/s41423-021-00805-6 *
ZHU YAOYAO, ZHANG SHUO; WANG ZHENGYU: "Using the Ｒunx1c-mNeongreen Ｒeporter Cell Line Derived from the Human Embryonic Stem Cells to Track the Definitive Hematopoiesis", CURRENT BIOTECHNOLOGY, KEY LABORATORY OF FEED BIOTECHNOLOGY，MINISTRY OF AGRICULTURE; FEED RESEARCH INSTITUTE, CHINESE ACADEMY OF AGRICULTURAL SCIENCES, CN, vol. 10, no. 5, 25 September 2020 (2020-09-25), CN , pages 524 - 533, XP093006538, ISSN: 2095-2341, DOI: 10.19586/j.2095-2341.2020.0080 *

Also Published As

Publication number Publication date
US20240269181A1 (en) 2024-08-15
JP2024518632A (ja) 2024-05-01

Similar Documents

Publication Publication Date Title
JP2022141641A (ja) 血管コロニー形成細胞
JP6339998B2 (ja) 幹細胞よりナチュラルキラー細胞を発生させる方法
KR101803562B1 (ko) 다능성 줄기세포로부터 유도된 탈핵 적혈구계 세포를 생산하는 방법
ES2685969T3 (es) Células formadoras de colonias hemangioblásticas y células hemangioblásticas no injertables
KR102292843B1 (ko) 역분화줄기세포(iPSC) 유래 자연 살해 세포 및 이의 용도
CN114181968B (zh) 一种具备b谱系分化潜能的人造血祖细胞的制备方法及其应用
JP6592551B2 (ja) 免疫調節活性を有する細胞集団、その調製方法、及び、その使用
WO2012133948A1 (ja) 生体組織から単離できるssea-3陽性の多能性幹細胞を含む他家移植用細胞治療用組成物
JP2017531448A (ja) Ｔ細胞前駆細胞を生成するための方法
JP7098187B2 (ja) 多能性幹細胞及びその分化したｔ細胞と使用
WO2022242359A1 (zh) 一种体液免疫系统再生的方法及其应用
CN115418373A (zh) 一种体液免疫系统再生的方法及其应用
WO2006085482A1 (ja) 造血幹細胞の自己複製因子及び増幅方法
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
Imamura A suitable conditioning regimen in allogeneic hematopoietic stem cell transplantation for adult patients with acute lymphoblastic leukemia

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: 22803684

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18562645

Country of ref document: US

Ref document number: 2023572007

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22803684

Country of ref document: EP

Kind code of ref document: A1