WO2020156387A1 - 干细胞发生器及其构建方法 - Google Patents

干细胞发生器及其构建方法 Download PDF

Info

Publication number
WO2020156387A1
WO2020156387A1 PCT/CN2020/073591 CN2020073591W WO2020156387A1 WO 2020156387 A1 WO2020156387 A1 WO 2020156387A1 CN 2020073591 W CN2020073591 W CN 2020073591W WO 2020156387 A1 WO2020156387 A1 WO 2020156387A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
stem cell
cell generator
stem cells
stem
Prior art date
Application number
PCT/CN2020/073591
Other languages
English (en)
French (fr)
Inventor
刘昌胜
戴凯
王靖
张庆昊
邓顺书
李贵龙
Original Assignee
华东理工大学
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 华东理工大学 filed Critical 华东理工大学
Priority to US17/427,737 priority Critical patent/US20220106568A1/en
Publication of WO2020156387A1 publication Critical patent/WO2020156387A1/zh

Links

Images

Classifications

    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • 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
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • 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
    • C12N2513/00—3D culture
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/72—Chitin, chitosan
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/80—Hyaluronan
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the invention belongs to the cross field of materials, life and medicine, and relates to a stem cell generator and a construction method thereof.
  • the bone marrow tissue contained in it provides a suitable microenvironment for various pluripotent stem cells such as hematopoietic stem/progenitor cells and mesenchymal stem cells, ensuring the normal function of stem cells, such as hematopoiesis Development, bone regeneration, etc.
  • stem cells such as hematopoietic stem/progenitor cells and mesenchymal stem cells
  • mesenchymal stem cells There are two typical stem cells in bone marrow tissue, namely hematopoietic stem cells and mesenchymal stem cells.
  • Hematopoietic stem cells are a type of pluripotent stem cells with self-renewal and multi-lineage differentiation capabilities, and are the most widely used type of stem cells in clinical applications so far.
  • Hematopoietic stem cell transplantation (HSCT) therapy is a treatment method for patients with hematopoietic system damage, such as leukemia patients and patients with hematopoietic disorders after receiving chemotherapy and radiotherapy, to infuse healthy hematopoietic stem cells (HSC) to rebuild the patient's hematopoietic and immune system.
  • HSC healthy hematopoietic stem cells
  • Many clinical treatment results show that hematopoietic stem cell transplantation has a good effect on the treatment of various malignant hematological diseases, tumors, hematopoietic failure, severe radiation sickness, genetic diseases and other diseases.
  • Mesenchymal stem cells are a type of fibroblast-like pluripotent stem cells that can grow adherently. They are cultured in vitro and exhibit the ability to differentiate into osteoblasts, cartilage and adipogenesis. Because of its easy isolation and culture, strong plasticity and wide sources, it can be used to treat diseases such as premature aging, spinal cord injury, insomnia, ovarian injury, Alzheimer's disease, chronic wounds, liver cirrhosis, autoimmune diseases, etc. It is the current stem cell therapy The most commonly used type of stem cells.
  • stem cells Existing methods for obtaining stem cells are usually in vitro methods, such as embryonic stem cells, cord blood stem cells, adult stem cells in vitro transformation to induce induced pluripotent stem cells (IPS), and in vitro culture and expansion of stem cells, etc., and the amount of obtained is small.
  • Bone marrow is the habitat of many kinds of stem cells.
  • in vitro methods cannot be imitated.
  • the purpose of the present invention is to provide a stem cell generator, which is implanted in the body to produce functionalized bone-like organs through the development process, which contains a variety of stem cells including hematopoietic stem/ Pluripotent stem cells including progenitor cells and mesenchymal stem cells.
  • a stem cell generator which is produced by implanting biological materials loaded with active substances and/or cells, or biological materials with osteogenic induction ability into animals or humans. Organoids are formed.
  • the active substance is bone morphogenetic protein-2 (Bone Morphogenetic Protein-2, BMP-2), bone morphogenetic protein-7 (Bone Morphogenetic Protein-7, BMP-7), osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis, such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • BMP-2 bone morphogenetic Protein-2
  • BMP-7 bone morphogenetic protein-7
  • osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • the bone morphogenetic protein-2 is recombinant bone morphogenetic protein-2.
  • the bone morphogenetic protein-7 is recombinant bone morphogenetic protein-7.
  • the cells are mesenchymal stem cells
  • the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or mesenchymal stem cells from other sources; other types have osteogenic differentiation Competent cells; cells that assist mesenchymal stem cells in osteogenic differentiation, such as vascular endothelial cells.
  • the biological material is selected from: collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, polyhydroxy fatty acid ester , Polycarbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate , Calcium silicate, bioglass, decalcified bone matrix, etc. or their copolymerization/blending combination.
  • the biological material is autologous bone or allogeneic bone.
  • the animal or human body refers to the muscle pocket, muscle space, intramuscular, subcutaneous, or dorsal muscle of the abdominal cavity of the animal or human.
  • the stem cell generator contains T cells (CD3 + ), B cells (B220 + ), myeloid cells (CD11b + ), red blood cells (Ter119 + ), hematopoietic progenitor cells (LKS-), Hematopoietic stem cells (LKS+).
  • the organoids have structures and functions similar to in situ bones, including complete bone tissues, bone marrow-like tissues and various functional stem cells.
  • the organoid contains stem cells, and the stem cells are hematopoietic stem/progenitor cells, mesenchymal stem cells, endothelial progenitor cells or other types of pluripotent stem cells.
  • biomaterials loaded with active substances and/or osteoinductive capacity or biomaterials with osteoinductive capacity are used to implant the animal/human body to create a special local microenvironment. After a certain period of time The body develops in the body to form organoids with specific functions, which have the function of producing stem cells and produce functional stem cells in the organoids within a certain period of time.
  • Osteogenic activity growth factors represented by bone morphogenetic protein (Bone Morphogenetic Protein, BMP) have the effect of ectopic osteogenic induction. Under the combined action of the microenvironment in the body, they induce the development of specific organoids, which contain fully functional Bone marrow, as well as a variety of pluripotent stem cells, become the generator of stem cells.
  • BMP bone morphogenetic Protein
  • the formed cells include complete hematopoietic precursor cells such as erythroid, myeloid and giant cells, and hematopoietic stem cells with long-term reconstruction ability, which can rebuild the hematopoietic system of mice irradiated with a lethal dose; at the same time, the stem cell generator can also Produce a large number of mesenchymal stem cells, the content of which is higher than that of normal bone marrow.
  • the mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1.
  • the number of cells used is 1 ⁇ 10 5 -5 ⁇ 10 8 cells per 100-150 mm 3 of biological material.
  • the second aspect of the present invention provides the method for constructing the stem cell generator described in the first aspect, including the following steps:
  • the biological material is a biological material loaded with active substances and/or cells, or a biological material with osteoinductive ability.
  • the active substance is bone morphogenetic protein-2 (Bone Morphogenetic Protein-2, BMP-2), bone morphogenetic protein-7 (Bone Morphogenetic Protein-7, BMP-7), osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis, such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • BMP-2 bone morphogenetic Protein-2
  • BMP-7 bone morphogenetic protein-7
  • osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • the bone morphogenetic protein-2 is recombinant bone morphogenetic protein-2.
  • the bone morphogenetic protein-7 is recombinant bone morphogenetic protein-7.
  • the cells are mesenchymal stem cells
  • the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or mesenchymal stem cells from other sources; other types have osteogenic differentiation Competent cells; cells that assist mesenchymal stem cells in osteogenic differentiation, such as vascular endothelial cells.
  • the biological material is selected from: collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, polyhydroxy fatty acid ester , Polycarbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate , Calcium silicate, bioglass, decalcified bone matrix, etc. or their copolymerization/blending combination.
  • the biological material is autologous bone or allogeneic bone.
  • the animal or human body refers to the muscle pocket, muscle space, intramuscular, subcutaneous, or dorsal muscle of the abdominal cavity of the animal or human.
  • the organoids have structures and functions similar to in situ bones, including complete bone tissues, bone marrow-like tissues and various functional stem cells.
  • the organoid contains stem cells, and the stem cells are hematopoietic stem/progenitor cells, mesenchymal stem cells, endothelial progenitor cells or other types of pluripotent stem cells.
  • the third aspect of the present invention provides a method for enriching stem cells, the method comprising the following steps:
  • the biological material is a biological material loaded with active substances and/or cells, or a biological material with osteoinductive ability.
  • the active substance is bone morphogenetic protein-2 (Bone Morphogenetic Protein-2, BMP-2), bone morphogenetic protein-7 (Bone Morphogenetic Protein-7, BMP-7), osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis, such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • BMP-2 bone morphogenetic Protein-2
  • BMP-7 bone morphogenetic protein-7
  • osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • the bone morphogenetic protein-2 is recombinant bone morphogenetic protein-2.
  • the bone morphogenetic protein-7 is recombinant bone morphogenetic protein-7.
  • the cells are mesenchymal stem cells
  • the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or mesenchymal stem cells from other sources; other types have osteogenic differentiation Competent cells; cells that assist mesenchymal stem cells in osteogenic differentiation, such as vascular endothelial cells.
  • the biological material is selected from: collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, polyhydroxy fatty acid ester , Polycarbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate , Calcium silicate, bioglass, decalcified bone matrix, etc. or their copolymerization/blending combination.
  • the biological material is autologous bone or allogeneic bone.
  • the animal or human body refers to the muscle pocket, muscle space, intramuscular, subcutaneous, or dorsal muscle of the abdominal cavity of the animal or human.
  • the organoids have structures and functions similar to in situ bones, including complete bone tissues, bone marrow-like tissues and various functional stem cells.
  • the organoid contains stem cells, and the stem cells are hematopoietic stem/progenitor cells, mesenchymal stem cells, endothelial progenitor cells or other types of pluripotent stem cells.
  • the fourth aspect of the present invention provides the use of the stem cell generator described in the first aspect to prepare materials for the prevention and/or treatment of graft-versus-host disease and hematopoietic injury or for the preparation of bone marrow transplantation materials.
  • the stem cells are used to prepare medicines for treating hematopoietic injury.
  • the hematopoietic injury is hematopoietic injury caused by radiotherapy or chemotherapy.
  • the treatment is the transplantation of bone marrow cells produced in a stem cell generator.
  • bone marrow cells are a single cell suspension made from cells in a stem cell generator.
  • the bone marrow cells are derived from biomaterials loaded with growth factors and/or cells, or biomaterials with osteoinductive ability, implanted in animal/human muscle pockets or subcutaneous parts, and develop over a period of time.
  • the formed organoid stem cell generator.
  • the cells used are adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or other cells with osteogenic differentiation ability or a combination thereof.
  • the cells produced are hematopoietic stem/hidrosis cells (HSC/HPC), mesenchymal stem cells (MSC) or other types of pluripotent stem cells.
  • HSC/HPC hematopoietic stem/hidrosis cells
  • MSC mesenchymal stem cells
  • stem cells are also used to prepare medicines for promoting the recovery of blood cells and hematopoietic progenitor/stem cells after bone marrow failure caused by radiotherapy and chemotherapy.
  • stem cells is also used to prepare bone marrow transplantation materials, to prepare medicines for treating low hematopoietic function, to prepare medicines for treating leukopenia, or to prepare medicines for treating acute or chronic leukemia.
  • the stem cell generator can be used for the following occasions or disease treatment:
  • bone marrow cells are used before, during, or after radiotherapy or chemotherapy.
  • the said hematopoietic hypofunction is hypohematopoietic hypofunction caused by radiotherapy or chemotherapy injury drugs or hypohematopoietic hypofunction caused by bone marrow suppression.
  • the preparation includes grinding the stem cell generator in a buffer, crushing, and passing through a cell sieve to obtain a single cell suspension.
  • the method for producing stem cells of the present invention is completely different from the existing methods for obtaining stem cells, such as embryonic stem cells, umbilical cord blood stem cells, adult stem cells transformed in vitro to induce induced pluripotent stem cells (IPS), and in vitro culture and expansion of stem cells.
  • the stem cell generator of the present invention induces a functionalized bone-like organ in the body from a biological material loaded with an active substance or a biological material that is itself active, which contains a variety of hematopoietic stem/progenitor cells, mesenchymal stem cells, etc. Pluripotent stem cells inside.
  • the research results of the present invention show that the stem cell generator can induce or highly enrich pluripotent stem cells such as hematopoietic progenitor/stem cells and mesenchymal stem cells, and the induced or highly enriched pluripotent stem cells have complete functions and can be used for Scientific research or clinical treatment of related stem cells is needed.
  • the method of the present invention is a brand-new method for generating/obtaining stem cells, opens up a brand-new way for obtaining stem cells, and has important scientific significance and broad application prospects.
  • Figure 1 shows the organoids induced by different doses of rhBMP-2 at 1 week and 3 weeks, which are stem cell generators.
  • Figure 2 shows the H&E section pictures of stem cell generators induced by 30 ⁇ g rhBMP-2 at 1 week and 3 weeks.
  • Figure 3 shows the stem cell generator produced when the composite collagen sponge loaded with human bone marrow mesenchymal stem cells is implanted under the skin of NCG mice for 8 weeks.
  • Figure 4 shows the H&E section of the stem cell generator produced when the human bone marrow mesenchymal stem cell composite collagen sponge is implanted under the skin of NCG mice for 8 weeks.
  • Figure 5 and Figure 6 respectively show a typical flow cytometric diagram and a scale diagram of each line of blood cells in the stem cell generator induced by loading 30 ⁇ g rhBMP-2 at 3 weeks.
  • Figures 7 and 8 respectively show a typical flow cytometric diagram and a scale diagram of hematopoietic progenitor/stem cells in a stem cell generator induced by loading 30 ⁇ g rhBMP-2 at 3 weeks.
  • Figure 9 and Figure 10 respectively show a typical flow cytometric diagram and a scale diagram of hematopoietic progenitor/stem cells in a stem cell generator induced by loading 10 ⁇ g rhBMP-7 at 3 weeks.
  • Figure 11 shows typical flow cytometry diagrams at different time points when the hematopoietic stem cells produced in the stem cell generator are used for long-term competitive reconstruction.
  • Figure 12 shows the ratio of CD45.1 cell reconstitution at different time points when hematopoietic stem cells produced in the stem cell generator are used for long-term competitive reconstitution.
  • Figure 13, Figure 14, and Figure 15 respectively show the B cells (B220+ cells), T cells (CD3+ cells), and myeloid cells (CD11b+) at different time points when the hematopoietic stem cells produced in the stem cell generator are used for long-term competitive reconstruction.
  • Cell reconstruction scale map.
  • Figure 16 and Figure 17 respectively show the typical flow diagram and scale diagram of mesenchymal stem cells in the stem cell generator induced by the biomaterial loaded with 30 ⁇ g rhBMP-2 for 1 week and 3 weeks.
  • Fig. 18, Fig. 19, Fig. 20 and Fig. 21 respectively show the macroscopic picture, H&E slice picture, flow typogram, and flow statistic picture of the stem cell generator produced after material implantation.
  • Figure 22, Figure 23 and Figure 24 respectively show the changes in the weight of mice after tail vein injection of cells at 6.0Gy, 7.0Gy, and 8.0Gy radiation doses.
  • Figure 25, Figure 26, and Figure 27 respectively show the changes in the number of white blood cells in mice after tail vein injection of cells at a dose of 6.0Gy, 7.0Gy, and 8.0Gy.
  • Figure 28 and Figure 30 respectively show the changes in the number of red blood cells in mice after tail vein injection of cells at 6.0Gy, 7.0Gy, and 8.0Gy radiation doses.
  • Figure 31, Figure 32 and Figure 33 respectively show 6.0Gy, 7.0Gy, 8.0Gy showing the changes in the number of platelets in mice after tail vein injection of cells at a dose of 6.0Gy.
  • Figures 34, 35, 36 and 37 respectively show the macroscopic image, H&E slice image, flow cytometric diagram and flow statistic diagram of the stem cell generator produced 8 weeks after the material of Example 10 was implanted.
  • biomaterials such as autologous bone, allogeneic bone, etc.
  • active substances and/or cells such as BMP-2, BMP-7 or interfacial
  • organoids can be formed after development to form a stem cell generator, which contains various lines of hematopoietic cells and hematopoietic progenitor/stem cells, and a high proportion of mesenchymal stem cells.
  • stem cells have complete functions and can be further used for scientific research and clinical applications of stem cells.
  • Corresponding stem cell therapies can be developed based on various pluripotent stem cells such as hematopoietic stem/progenitor cells or mesenchymal stem cells in the stem cell generator. On this basis, the present invention has been completed.
  • Stem cell generator is a biological material loaded with active substances and/or cells, or a biological material with osteogenic induction ability, implanted in the body of an animal/human, forming a special local microenvironment, and developing in vivo after a certain period of time , To form organoids with specific functions, in which various functionalized stem cells can be produced.
  • the organoids have structures and functions similar to in situ bones, including complete bone tissues, bone marrow-like tissues and various functional stem cells.
  • Organoids are usually constructed using in vitro methods. Bone marrow is the habitat of many kinds of stem cells. However, due to the various types of cells, multiple growth factors/cytokines and complex microenvironments involved in bone marrow, in vitro methods cannot be imitated.
  • the present invention adopts the method of in vivo construction, uses materials and active molecules to form organoids, studies the components therein, and proposes a method for enriching stem cells of the present invention.
  • rhBMP-2 recombinant human bone morphogenetic protein-2
  • rhBMP-2 Optimized DNA sequences encoding recombinant human bone morphogenetic protein-2 (rhBMP-2), preparation method and the uses thereof.Patent Authorization No.US 7947821 B2; Liu Changsheng et al., ZL 200610118006.4; ZL200910045832.4;).
  • recombinant human bone morphogenetic protein-2 was added to 5 ⁇ 5 ⁇ 5mm gelatin sponge (mass of 10mg), and the active material containing growth factors was formed after lyophilization.
  • rhBMP-7 human bone morphogenetic protein-7
  • hMSCs third-generation human mesenchymal stem cells
  • the active material containing rhBMP-2 of Example 1 was implanted into the body to develop a stem cell generator.
  • Different doses (10 ⁇ g, 30 ⁇ g, 80 ⁇ g, 200 ⁇ g) of rhBMP-2 were loaded into the gelatin sponge material and implanted into the muscle space of the thigh of C57BL/6 male mice.
  • the organoids formed were taken out, namely stem cells
  • One part of the generator is used for macro-photographs and histological evaluation ( Figure 1 and Figure 2), and the other part is used to remove the muscles attached to the surface, add a little PBS buffer to the mortar, and then use the pestle to make the organs (stem cells)
  • the generator) is crushed and passed through a cell sieve to obtain a single cell suspension. The resulting single cell suspension can be used in subsequent experiments.
  • Figure 1 shows the stem cell generators produced when different doses of rhBMP-2 are implanted for 1 or 3 weeks. It can be seen from general observation that it is bone-like tissue.
  • Figure 2 is the H&E section of the stem cell generator induced by 30 ⁇ g rhBMP-2 at 1 week and 3 weeks. It can be seen that at 1 week, chondrocytes appeared in the organoid (stem cell generator), and at 3 weeks, the organoid ( Obvious bone marrow-like tissue appears in the stem cell generator.
  • the active material containing rhBMP-7 of Example 1 was used to generate a stem cell generator in vivo.
  • the material was implanted into the thigh muscle of male NCG immunodeficient mice. After 3 weeks of rearing, the stem cell generator was taken out, one part was used for macro-photographing and histological evaluation ( Figure 3 and Figure 4), and the other part was used to remove the muscles attached to the surface. Add a little buffer to the mortar, and then use the pestle to remove The stem cell generator is crushed and passed through a cell sieve to obtain a single cell suspension. The resulting single cell suspension can be used in subsequent experiments.
  • the purpose of this example is to detect each blood line cell and hematopoietic progenitor/stem cell ratio in the stem cell generator, and compare the content with the corresponding cells of normal bone marrow to prove that the stem cell generator has a fully functional hematopoietic system containing cells of each blood line And hematopoietic progenitor/stem cells can provide treatment for abnormal hematopoietic function.
  • the active material containing rhBMP-2 is the gelatin sponge scaffold with 30 ⁇ g rhBMP-2 prepared in Example 1;
  • the single cell suspension is a single cell suspension prepared according to the method of Example 2
  • Fig. 5 and Fig. 6 are typical diagrams of flow cytometry of each blood line cell in the stem cell generator induced by rhBMP-2 and the corresponding diagram of the proportion of each blood line cell.
  • the typical flow cytometric diagram shown in Figure 5 shows that there are complete blood line cells in the stem cell generator, which appear to contain T cells (CD3 + ), B cells (B220 + ), myeloid cells (CD11b + ), and red blood cells (Ter119 + ) .
  • Figure 6 shows that the proportion of B cells, red blood cells, and T cells in the stem cell generator is significantly higher than that in the in situ bone marrow group, while the proportion of myeloid cells is significantly lower than that in the in situ bone marrow group, indicating that the stem cell generator has complete blood Cell line, but the proportion is not completely consistent with that of the orthotopic bone marrow group.
  • Figures 7 and 8 are typical diagrams of flow cytometric detection of hematopoietic progenitors/stem cells and the ratio of hematopoietic progenitors/stem cells in the stem cell generator induced by rhBMP-2.
  • the flow cytometric diagram shown in Figure 7 shows that there are complete hematopoietic progenitor/stem cells in the stem cell generator, which appears to contain hematopoietic progenitor cells (LKS-) and hematopoietic stem cells (LKS+).
  • Figure 8 shows that there is no significant difference between the hematopoietic progenitor/stem cells in the stem cell generator and the in situ bone marrow group. It shows that the stem cell generator has complete hematopoietic progenitor/stem cells, which can provide cells for the treatment of bone marrow injury after radiotherapy and chemotherapy.
  • the purpose of this example is to detect the hematopoietic progenitor/stem cell ratio in the stem cell generator, and compare the content with the corresponding cells of normal bone marrow, to prove that the stem cell generator bone marrow has a fully functional hematopoietic system, containing hematopoietic progenitor/stem cells, and can be used for hematopoiesis Treatment of dysfunction.
  • the active material containing rhBMP-7 is the gelatin sponge scaffold with 10 ⁇ g rhBMP-7 prepared by the method in Example 1;
  • the single cell suspension is a single cell suspension prepared according to the method of Example 3.
  • Figures 9 and 10 are typical graphs of flow cytometric detection of hematopoietic progenitors/stem cells in the stem cell generator induced by rhBMP-7 and graphs of the ratio of hematopoietic progenitors/stem cells.
  • the typical flow diagram shown in Fig. 9 shows that there are complete hematopoietic progenitor/stem cells in the stem cell generator, which appear to contain hematopoietic progenitor cells (LKS-) and hematopoietic stem cells (LKS+).
  • Figure 10 shows that the content of hematopoietic progenitor/stem cells in the stem cell generator is significantly higher than that in the in situ bone marrow group, indicating that the stem cell generator is rich in hematopoietic progenitor/stem cells and can provide cells for the treatment of abnormal hematopoietic function.
  • Example 7 Evaluation of the pluripotency of hematopoietic stem/progenitor cells in stem cell generators induced by rhBMP-2 active materials.
  • the purpose of this example is to evaluate the pluripotency of hematopoietic stem/progenitor cells in the stem cell generator induced by rhBMP-2, using the cells in the stem cell generator (CD45.1) and in situ bone marrow cells (CD45.2) After mixing, long-term competitive reconstruction of the mouse (CD45.2) hematopoietic system destroyed by 10Gy X-ray irradiation, where cells from the stem cell generator account for more than 0.1% of the blood cells, it is regarded as the hematopoietic stem in the stem cell generator Progenitor cells are pluripotent and provide a realistic basis for the treatment of abnormal hematopoietic function.
  • the active material containing rhBMP-2 is the gelatin sponge scaffold with 30 ⁇ g rhBMP-2 prepared by the method in Example 1;
  • Figure 11-15 is a long-term competitive hematopoietic reconstitution experiment, at different time points in the stem cell generator induced by rhBMP-2, the typical flow cytometric diagram of each blood line cell and the corresponding blood line cell ratio diagram.
  • Fig. 12 is a graph showing changes in the proportion of CD45.1 cells at different time points. It can be seen that the proportion of stem cell generator-derived cells at all time points is greater than 0.1%, that is, the hematopoietic stem/progenitor cells derived from the stem cell generator have long-term hematopoietic reconstitution ability.
  • Figures 13-14 have the same trend as Figure 12, and only the stem cell generator-derived myeloid cells in Figure 15 have some cell reconstitution ratios below 0.1% at 12 and 20 weeks.
  • the hematopoietic stem/progenitor cells in the stem cell generator have long-term hematopoietic reconstruction capabilities, that is, pluripotent stem cells.
  • Example 8 Detection of the content of bone marrow mesenchymal stem cells in the stem cell generator induced by rhBMP-2.
  • the purpose of this example is to detect the proportion of bone marrow mesenchymal stem cells in the stem cell generator induced by biomaterials containing rhBMP-2, in order to use mesenchymal stem cells to treat bone and cartilage defect repair, graft versus host disease (GVHD), etc. disease.
  • GVHD graft versus host disease
  • the active material containing rhBMP-2 is the gelatin sponge scaffold containing 30 ⁇ g rhBMP-2 prepared in Example 1;
  • the single cell suspension is the single cell suspension prepared in Example 2 from the stem cell generator induced in the scaffold containing 30 ⁇ g rhBMP-2.
  • Figures 16 and 17 are typical diagrams of flow cytometric detection of bone marrow mesenchymal stem cells and a diagram of the ratio of mesenchymal stem cells in a stem cell generator induced by rhBMP-2 active materials. It can be seen from Figure 17 that at 1 week, the content of mesenchymal stem cells in the stem cell generator was significantly higher than that in the bone marrow in situ. At 3 weeks, the content of mesenchymal cells in the stem cell generator approached that in the bone marrow in situ.
  • mesenchymal stem cells are enriched in the stem cell generator, and the use of the enriched mesenchymal stem cells has great potential utilization value for the treatment of bone and cartilage defect repair, graft versus host disease (GVHD) and other diseases.
  • GVHD graft versus host disease
  • rhBMP-2 recombinant human bone morphogenetic protein-2 (rhBMP-2) synthesized by eukaryotic or prokaryotic expression system was added to gelatin sponge (10 mg), and the active material containing growth factors was formed after lyophilization.
  • the prepared material was implanted into the thigh muscle bag of an 8-week-old C57BL/6 male mouse. After feeding for 3 weeks, the stem cell generator and the in-situ bone were taken out, and a part of the stem cell generator or the muscle attached to the surface of the in-situ bone was removed. Add a little PBS buffer to the mortar, then use the pestle to crush the stem cell generator or the bone in situ. After passing through the cell sieve, a single cell suspension is obtained. One part is made into 200 ⁇ L single cell suspension for bone marrow transplantation; the other part Stem cell generator and bone in situ are used to take macro pictures and make H&E section.
  • mice were given one-time cobalt-60 irradiation according to the irradiation doses given in the grouping table, namely 0Gy irradiation, 6Gy irradiation, 7Gy irradiation, 8Gy irradiation.
  • Intervention treatment 24 hours after irradiation, the irradiated mice of the corresponding group were given intervention treatment, namely, tail vein injection of 200 ⁇ L PBS solution, 200 ⁇ L in situ bone marrow cell suspension, 200 ⁇ L stem cell generator cell suspension, among which the injected original
  • the bone marrow cell suspension or the stem cell generator cell suspension is the single cell suspension prepared by the method described in Example 4.
  • the peripheral blood of each group of mice was collected by orbital blood sampling according to the set sampling point for blood phase detection to observe the treatment effect.
  • the blood test indicators are as follows:
  • Figure 18 shows a digital photo of the stem cell generator 8 weeks after implantation of the muscle bag. It can be seen that the color of the stem cell generator is similar to that of the bone in situ, which implies that it contains a large number of red blood cells and has a bone-like morphology, but the volume is compared with the original. Bone is bigger.
  • Figure 19 H&E section of the stem cell generator further confirms that the microstructure of the stem cell generator is similar to that of bone in situ, and the bone marrow cavity is filled with bone marrow cells and blood vessels.
  • Figures 20 and 21 are related analysis of stem cell generator flow cytometry. It can be seen that the stem cell generator and in situ bone have similar cell composition, and the stem cell generator contains LKS - cells, LSK + cells and hematopoietic stem cells (HSCs). There is no significant difference between the ratio of) and the corresponding cell ratio in bone marrow in situ.
  • LKS - cells LSK + cells
  • HSCs hematopoietic stem cells
  • the examples illustrate that the constructed stem cell generator has a structure and function similar to in situ bone marrow, and the hematopoietic stem/progenitor cells contained therein have the potential to treat abnormal hematopoietic function.
  • mice were given one-time cobalt-60 irradiation (0Gy, 6Gy, 7Gy, 8Gy).
  • Figure 22-24 shows the changes in the body weight of the mouse model after treatment under different irradiation doses. It can be seen from Figure 22 that after mice were irradiated with cobalt 60 (6.0 Gy), they were injected with 200 ⁇ L of bone marrow single cell suspension of the same species produced by the stem cell generator immediately after the tail vein. In the control group, the body weight did not change much from 0 to 9 days, but dropped sharply after 9 days, until death.
  • Figure 25-27 shows the changes in the number of white blood cells in injured mice under different irradiation doses. It can be seen from Figure 25 that the number of white blood cells in the irradiated control group and the treatment group dropped sharply to 0 after the irradiation, but the white blood cell number in the treatment group increased steadily with time, and was equal to the normal control group after 30 days, while the irradiation control group remained unchanged Is 0. It shows that after treatment in the irradiated mice, hematopoietic function is restored and the number of white blood cells increases steadily.
  • the change trends of white blood cell counts in Figures 26 and 27 are almost the same as those in Figures 23 and 24.
  • Figure 28-30 shows the changes in the number of red blood cells in the mouse model under different irradiation doses after treatment. It can be seen from Figure 28 that after the tail vein injection treatment after irradiation, the number of red blood cells in the treatment group and the normal control group maintained the same change, with little difference in values. In the irradiated control group, the number of red blood cells quickly dropped to a minimum within 9 days, until death. This shows that the injection of bone marrow cell suspension in the bone organ (stem cell reactor) in the irradiated group promoted hematopoietic differentiation in the body, restored hematopoietic function, and promoted the number of red blood cells to be roughly the same as the normal group.
  • the changing trends of red blood cell counts in Figures 29 and 30 are almost the same as those in Figure 1.
  • Figures 31-33 are the changes in the number of platelets in the mouse model after treatment under different irradiation doses. It can be seen from Figure 31 that both the treatment group and the irradiated control group decreased sharply with time after the irradiation, and reached the lowest point at 9 days. After that, the irradiated group remained unchanged until death, while the treatment group increased reversely. It gradually rises to the level of the normal control group and returns to the normal level.
  • the obvious difference between Fig. 32 and Fig. 33 is that although the recovery range and speed of bone marrow cell treatment using the stem cell generator in the irradiation treatment group are lower than that of the orthotopic bone group, the overall trend is the same as that of the normal control group. This is consistent with the weight change trend.
  • the bone marrow cells in the stem cell generator produced by loading rhBMP-2 biomaterial have an effective therapeutic effect on hematopoietic injury caused by radiotherapy and chemotherapy. Its hematopoietic effect is mainly that bone marrow cells enter the hematopoietic system and improve the hematopoietic microenvironment, and the various progenitor/stem cells contained therein can normally differentiate into various functional cells, thereby rebuilding the blood system.
  • rhBMP-2 human bone morphogenetic protein-2
  • mMSCs mouse mesenchymal stem cells
  • the stem cell generator was taken out, and after removing a part of the muscles attached to the surface of the stem cell generator, a little PBS buffer was added to the mortar Then use a pestle to crush it and pass through a cell sieve to obtain a single cell suspension.
  • the resulting single cell suspension can be used for flow cytometry; another part of the stem cell generator is used to take macro pictures and make H&E sections.
  • the experiment is grouped as follows.
  • Figure 34 shows a digital photo of the stem cell generator generated after 8 weeks of subcutaneous implantation on the back. It can be seen that the stem cell generator is similar in color to the bone in situ, suggesting that it contains a large number of red blood cells and has a bone-like shape.
  • Figure 35 The H&E section of the stem cell generator further confirms that the microstructure of the stem cell generator is similar to that of in situ bone, with the same cancellous bone and cortical bone structure, and the bone marrow cavity is filled with bone marrow cells and blood vessels.
  • Figures 36 and 37 are related analysis of stem cell generator flow cytometry. It can be seen that the stem cell generator and the bone in situ have similar cell composition, and the stem cell generator contains LKS- cells, LSK+ cells and hematopoietic stem cells (HSCs) There is no significant difference between the ratio and the corresponding cell ratio in the bone marrow in situ.
  • LKS- cells LSK+ cells
  • HSCs hematopoietic stem cells
  • This example illustrates that the constructed stem cell generator has a structure and function similar to in situ bone marrow, and the hematopoietic stem/progenitor cells contained therein have the potential to treat abnormal hematopoietic function.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Transplantation (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Materials For Medical Uses (AREA)

Abstract

一种干细胞发生器及其构建方法和用途，该干细胞发生器由负载活性物质和/或细胞的生物材料，或本身具有成骨诱导能力的生物材料植入动物或人体内经发育后产生类器官而形成。

Description

干细胞发生器及其构建方法 技术领域
本发明属于材料、生命与医学的交叉领域，涉及一种干细胞发生器及其构建方法。
背景技术
人体骨骼系统除承担力学支撑作用外，所含有的骨髓组织为存在于其中的造血干/祖细胞，间充质干细胞等各种多能干细胞提供适宜的微环境，保证干细胞的正常功能，如造血发育，骨组织再生等。骨髓组织中存在两种典型的干细胞，即造血干细胞与间充质干细胞。
造血干细胞是一类具有自我更新，多系分化能力的多能干细胞，是迄今为止临床应用最为广泛的一类干细胞。造血干细胞移植(HSCT)疗法是在针对造血系统损伤患者，如白血病患者、接受放化疗后造血受到障碍患者，为其输入健康的造血干细胞(HSC)，重建患者造血和免疫系统的治疗方法。许多临床治疗结果表明造血干细胞移植对于各种恶性血液病、肿瘤、造血功能衰竭、重度放射病、遗传性疾病等疾病的治疗有良好的效果。
间充质干细胞是一类能够贴壁生长的、成纤维细胞样的多能干细胞，体外培养表现出向成骨、成软骨及成脂分化能力。由于其易于分离培养，且可塑性极强，来源广泛，可用于治疗早衰症、脊髓损伤、失眠症、卵巢损伤、老年痴呆症、慢性创面、肝硬化、自体免疫性疾病等疾病，是当今干细胞治疗中最常用的一类干细胞。
现有获得干细胞的方法，通常为体外方法，如胚胎干细胞、脐带血干细胞、成体干细胞在体外转化诱导形成诱导性多能干细胞(IPS)，以及体外培养扩增干细胞等，获得量少。骨髓是多种干细胞栖息的场所。但由于骨髓中涉及各类细胞、多种生长因子/细胞因子及复杂的微环境，体外方法无法仿制。
发明内容
本发明的目的在于提供一种干细胞发生器，由负载活性物质的生物材料或本身具有活性的生物材料，植入体内后经发育过程产生功能化的类骨器官，其中含有多种包括造血干/祖细胞、间充质干细胞等在内的多能干细胞。
本发明的第一方面，提供一种干细胞发生器，所述干细胞发生器由负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料植入动物或人体内经发育后产生类器官而形成。
在另一优选例中，所述活性物质为骨形态发生蛋白-2(Bone Morphogenetic Protein-2，BMP-2)、骨形态发生蛋白-7(Bone Morphogenetic Protein-7，BMP-7)、成骨多肽(Osteogenic peptides)或其他具有诱导骨再生、血管生成能力的生长因子如VEGF，PDG、多肽或生长因子/多肽组合。
在另一优选例中，所述骨形态发生蛋白-2为重组骨形态发生蛋白-2。
在另一优选例中，所述骨形态发生蛋白-7为重组骨形态发生蛋白-7。
在另一优选例中，所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
在另一优选例中，所述生物材料选自：胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙 二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质等中的一种或其共聚/共混组合。
在另一优选例中，所述生物材料为自体骨或同种异体骨。
在另一优选例中，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
在另一优选例中，所述干细胞发生器中含有T细胞(CD3 +)，B细胞(B220 +)，髓系细胞(CD11b +)，红细胞(Ter119 +)、造血祖细胞(LKS-)，造血干细胞(LKS+)。
本发明中，所述的类器官具有与原位骨相似的结构与功能，包括完整的骨组织、骨髓样组织及各种功能干细胞。
在另一优选例中，所述类器官中包含干细胞，所述干细胞为造血干/祖细胞、间充质干细胞、内皮祖细胞或其他种类多能干细胞。
本发明中，利用负载活性物质和/或具有成骨诱导能力的生物材料，或本身具有成骨诱导能力的生物材料，植入动物/人的体内，创建一个特殊的局部微环境，经过一定时间的体内发育，形成特定功能的类器官，该类器官具有产生干细胞的功能，在特定时间内在类器官内产生具有功能的干细胞。
以骨形态发生蛋白(Bone Morphogenetic Protein，BMP)为代表的成骨活性生长因子具有异位诱导成骨的作用，其在体内微环境共同作用下，诱导发育产生特定类器官，其中含有功能完备的骨髓，以及多种多能干细胞，成为干细胞发生器。所形成的细胞包括红系、髓系及巨细胞等完整的造血前体细胞和具有长期重建能力的造血干细胞，能够重建受致死剂量辐照小鼠的造血系统；同时，干细胞发生器中还可产生大量间充质干细胞，其含量高于正常骨骨髓中的含量。
在另一优选例中，所述活性物质与生物材料的质量比范围为0.0001-1:1。
在另一优选例中，所用细胞的接种数量为每100-150mm 3生物材料接种1×10 5-5×10 8个细胞。
本发明的第二方面，提供第一方面所述的干细胞发生器的构建方法，包括以下步骤：
(1)将生物材料植入动物或人体内；
(2)经体内发育后产生类器官从而形成所述干细胞发生器，其中，
所述生物材料为负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料。
在另一优选例中，所述活性物质为骨形态发生蛋白-2(Bone Morphogenetic Protein-2，BMP-2)、骨形态发生蛋白-7(Bone Morphogenetic Protein-7，BMP-7)、成骨多肽(Osteogenic peptides)或其他具有诱导骨再生、血管生成能力的生长因子如VEGF，PDG、多肽或生长因子/多肽组合。
在另一优选例中，所述骨形态发生蛋白-2为重组骨形态发生蛋白-2。
在另一优选例中，所述骨形态发生蛋白-7为重组骨形态发生蛋白-7。
在另一优选例中，所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
在另一优选例中，所述生物材料选自：胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙 二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质等中的一种或其共聚/共混组合。
在另一优选例中，所述生物材料为自体骨或同种异体骨。
在另一优选例中，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
本发明中，所述的类器官具有与原位骨相似的结构与功能，包括完整的骨组织、骨髓样组织及各种功能干细胞。
在另一优选例中，所述类器官中包含干细胞，所述干细胞为造血干/祖细胞、间充质干细胞、内皮祖细胞或其他种类多能干细胞。
本发明的第三方面，提供一种富集干细胞的方法，所述方法包括以下步骤：
(1)将生物材料植入动物或人体内；
(2)经体内发育后产生类器官并富集所述干细胞，其中，
所述生物材料为负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料。
在另一优选例中，所述活性物质为骨形态发生蛋白-2(Bone Morphogenetic Protein-2，BMP-2)、骨形态发生蛋白-7(Bone Morphogenetic Protein-7，BMP-7)、成骨多肽(Osteogenic peptides)或其他具有诱导骨再生、血管生成能力的生长因子如VEGF，PDG、多肽或生长因子/多肽组合。
在另一优选例中，所述骨形态发生蛋白-2为重组骨形态发生蛋白-2。
在另一优选例中，所述骨形态发生蛋白-7为重组骨形态发生蛋白-7。
在另一优选例中，所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
在另一优选例中，所述生物材料选自：胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质等中的一种或其共聚/共混组合。
在另一优选例中，所述生物材料为自体骨或同种异体骨。
在另一优选例中，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
本发明中，所述的类器官具有与原位骨相似的结构与功能，包括完整的骨组织、骨髓样组织及各种功能干细胞。
在另一优选例中，所述类器官中包含干细胞，所述干细胞为造血干/祖细胞、间充质干细胞、内皮祖细胞或其他种类多能干细胞。
本发明的第四方面，提供第一方面所述的干细胞发生器的用途，用于制备预防和/或治疗移植物抗宿主病、造血损伤的材料或制备骨髓移植的材料。
本发明中，干细胞的用途，用于制备治疗造血损伤的药物。
在另一优选例中，所述造血损伤为放、化疗引起的造血损伤。
在另一优选例中，所述治疗是用干细胞发生器中产生的骨髓细胞移植治疗。在另一优 选例中，骨髓细胞是由干细胞发生器中的细胞制成的单细胞悬液。
在另一优选例中，所述骨髓细胞来自于由负载生长因子和/或细胞的生物材料、或具有成骨诱导能力的生物材料植入动物/人肌袋或皮下等部位，经一段时间发育形成的类器官(干细胞发生器)内。
在另一优选例中，所用的细胞为脂肪来源间充质干细胞、骨髓来源间充质干细胞或其他具有成骨分化能力的细胞或其组合。
在另一优选例中，所产生的细胞为造血干/组细胞(HSC/HPC)、间充质干细胞(MSC)或其他种类多能干细胞。
本发明中，干细胞的用途，还用于制备促进放化疗造成的骨髓衰竭后的血细胞及造血祖/干细胞的恢复的药物。
本发明中，干细胞的用途，还用于制备骨髓移植材料、用于制备治疗造血功能低下的药物、用于制备治疗白细胞减少症的药物、或用于制备治疗急性或慢性白血病的药物。
在另一优选例中，所述干细胞发生器，可用于以下场合或病症治疗：
(1)用于骨髓移植；
(2)促进放/化疗后造血系统的恢复；
(3)治疗白血病等血液系统异常症。
在另一优选例中，骨髓细胞在放疗、化疗的之前、之中、或之后使用。
在另一优选例中，所述造血功能低下为放、化疗损伤药物引起的造血功能低下或骨髓抑制引起的造血功能低下。
在另一优选例中，所述制备包括将所述干细胞发生器在缓冲液中研磨、压碎、过细胞筛后得到单细胞悬液。
本发明产生干细胞的方法，完全不同于现有获得干细胞的方法，如胚胎干细胞、脐带血干细胞、成体干细胞在体外转化诱导形成诱导性多能干细胞(IPS)，以及体外培养扩增干细胞等。本发明的干细胞发生器，由负载活性物质的生物材料或本身具有活性的生物材料，在体内诱导产生功能化的类骨器官，其中含有多种包括造血干/祖细胞、间充质干细胞等在内的多能干细胞。
本发明研究结果表明，该干细胞发生器能够对造血祖/干细胞、间充质干细胞等多能干细胞诱导产生或进行高度富集，并且其诱导产生或高度富集的多能干细胞功能完备，可用于需要相关干细胞的科学研究或临床治疗。本发明方法是一个全新的产生/获取干细胞的方法，为干细胞的获取开辟了一条全新的途径，具有重要的科学意义和广阔的应用前景。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一赘述。
附图说明
图1显示了1周、3周时，不同剂量rhBMP-2诱导产生的类器官，是干细胞发生器。
图2显示了1周、3周时，30μg rhBMP-2诱导产生的干细胞发生器的H&E切片图片。
图3显示了负载人骨髓间充质干细胞的复合胶原海绵植入NCG小鼠皮下8周时产生的干细胞发生器。
图4显示了人骨髓间充质干细胞复合胶原海绵植入NCG小鼠皮下8周时产生的干细胞 发生器H&E切片图。
图5、图6分别显示了3周时由负载30μg rhBMP-2诱导产生的干细胞发生器中各系血细胞的流式典型图、比例图。
图7、图8分别显示了3周时由负载30μg rhBMP-2诱导产生的干细胞发生器中造血祖/干细胞的流式典型图、比例图。
图9、图10分别显示了3周时由负载10μg rhBMP-7诱导产生的干细胞发生器中造血祖/干细胞的流式典型图、比例图。
图11显示了利用干细胞发生器中所产生的造血干细胞进行长期竞争性重建时不同时间点的流式典型图。
图12显示了利用干细胞发生器中所产生的造血干细胞进行长期竞争性重建时不同时间点的CD45.1细胞重建比例图。
图13、图14和图15分别显示了利用干细胞发生器中所产生的造血干细胞进行长期竞争性重建时不同时间点的B细胞(B220+细胞)、T细胞(CD3+细胞)、髓系细胞(CD11b+细胞)重建比例图。
图16、图17分别显示了由负载30μg rhBMP-2的生物材料诱导产生的干细胞发生器中1周、3周时间充质干细胞的流式典型图、比例图。
图18、图19、图20和图21分别示出材料植入后产生的干细胞发生器的宏观图、H&E切片图、流式典型图、流式统计图。
图22、图23和图24分别显示了6.0Gy、7.0Gy、8.0Gy辐照剂量时尾静脉注射细胞后小鼠体重数变化。
图25、图26和图27分别显示了6.0Gy、7.0Gy、8.0Gy辐照剂量时尾静脉注射细胞后小鼠白细胞数变化。
图28、图29和图30分别显示了6.0Gy、7.0Gy、8.0Gy辐照剂量时尾静脉注射细胞后小鼠红细胞数变化。
图31、图32和图33分别显示了6.0Gy、7.0Gy、8.0Gy显示了6.0Gy辐照剂量时尾静脉注射细胞后小鼠血小板数变化。
图34、35、36和图37分别显示了实施例10材料植入8周后产生的干细胞发生器的宏观图、H&E切片图、流式典型图和流式统计图。
具体实施方式
本发明人基于长期而深入的研究，发现本身具有成骨诱导能力的生物材料(如自体骨、同种异体骨等)或负载活性物质和/或细胞(如BMP-2、BMP-7或间充质干细胞)的生物材料植入体内后，经发育后产生类器官可形成干细胞发生器，其中含有各系造血细胞及造血祖/干细胞，以及高比例的间充质干细胞。这些干细胞功能完备，可进一步用于干细胞的科学研究及临床应用。可基于干细胞发生器中的造血干/祖细胞或间充质干细胞等多种多能干细胞开发出相应的干细胞疗法。在此基础上，完成了本发明。
术语
“干细胞发生器”为负载活性物质和/或细胞的生物材料，或本身具有成骨诱导能力的生物材料，植入动物/人的体内，形成一个特殊的局部微环境，经过一定时间的体内发育，形成特定功能的类器官，在该类器官内可产生富含各种功能化的干细胞。
本发明中，所述的类器官具有与原位骨相似的结构与功能，包括完整的骨组织、骨髓 样组织及各种功能干细胞。
类器官通常是采用体外方法构建的。骨髓是多种干细胞栖息的场所。但由于骨髓中涉及各类细胞、多种生长因子/细胞因子及复杂的微环境，体外方法无法仿制。本发明采用体内构建的方法，利用材料和活性分子形成类器官，研究其中的成分，并提出本发明富集干细胞的方法。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1植入材料的制备
用真核或原核表达系统合成重组人骨形态发生蛋白-2(rhBMP-2)(Optimized DNA sequences encoding recombinant human bone morphogenetic protein-2(rhBMP-2),preparation method and the uses thereof.Patent Authorization No.US 7947821 B2；刘昌胜等，ZL 200610118006.4；ZL200910045832.4；)。
按不同剂量(10μg，30μg，80μg，200μg)将重组人骨形态发生蛋白-2加入5×5×5mm明胶海绵(质量为10mg)，经冻干后形成含有生长因子的活性材料。
将10μg真核或原核表达系统合成重组人骨形态发生蛋白-7(rhBMP-7)加入5×5×5mm明胶海绵(质量为10mg)，经冻干后形成含有生长因子的活性材料。
将含1×10 6个第三代人间充质干细胞(hMSCs)的浓缩液接种在5×5×5mm胶原海绵材料(质量为10mg)上，放置于37℃培养箱中孵育2h，形成含细胞的活性材料。
实施例2
使用实施例1的含rhBMP-2活性材料植入体内，发育形成干细胞发生器。将不同剂量(10μg，30μg，80μg，200μg)的rhBMP-2负载于明胶海绵材料中，植入至C57BL/6雄性小鼠大腿肌肉间隙内，饲养3周后取出形成的类器官，即为干细胞发生器，一部分用于拍摄宏观照片及组织学评价(图1和图2)，另一部分去除表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将该类器官(干细胞发生器)压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于后续实验。
图1为不同剂量rhBMP-2植入1周或3周时产生的干细胞发生器。大体观察可见外观为类骨样组织。
图2为30μg rhBMP-2诱导生成的干细胞发生器1周及3周时H&E切片图，从中可以看出1周时，类器官(干细胞发生器)中出现软骨细胞，3周时，类器官(干细胞发生器)中出现明显的骨髓样组织。
实施例3
使用实施例1的含rhBMP-7活性材料体内产生干细胞发生器。
将10μg rhBMP-7负载于明胶海绵材料中，植入至C57BL/6雄性小鼠大腿肌肉内，饲养3周后取出干细胞发生器，去除表面附着的肌肉后，在研钵中加入少许Hank's平衡盐溶液 (HBSS)，再使用研杵将干细胞发生器压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于后续实验。
实施例4
使用实施例1的负载人骨髓间充质干细胞的胶原海绵，将材料植入至雄性NCG免疫缺陷小鼠大腿肌肉内。饲养3周后取出干细胞发生器，一部分用于拍摄宏观照片及组织学评价(图3和图4)，另一部分去除表面附着的肌肉后，在研钵中加入少许缓冲液，再使用研杵将干细胞发生器压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于后续实验。
实施例5
rhBMP-2诱导产生的干细胞发生器所含各血系细胞及造血祖/干细胞含量检测。
该实施例的目的是检测干细胞发生器中各血系细胞及造血祖/干细胞比例，并与正常骨髓的相应细胞进行含量比较，证明干细胞发生器中具有功能完备的造血系统，含有各血系细胞及造血祖/干细胞，可提供用于造血功能异常的治疗。
含rhBMP-2活性材料为实施例1中制得的负载30μg rhBMP-2的明胶海绵支架；
单细胞悬液为按照实施例2方法制得的单细胞悬液
方法：采用SPF级C57BL/6小鼠，雄性，8周龄，随机分组，随后于大腿肌肉中植入实施例1中所制得的含30μg rhBMP-2的材料。饲养3周后取出形成的类器官，即为干细胞发生器。将得到的干细胞发生器去除表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将该类器官(干细胞发生器)压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于后续流式检测试验。实验分组(列表)如下：
组别 原位骨髓组 干细胞发生器组
数量 5 5
图5和图6为rhBMP-2诱导产生的干细胞发生器中各血系细胞流式检测典型图及对应各血系细胞比例图。
图5所示流式典型图显示干细胞发生器中有完整的血系细胞，表现为含有T细胞(CD3 +)，B细胞(B220 +)，髓系细胞(CD11b +)，红细胞(Ter119 +)。
图6显示干细胞发生器中B细胞、红细胞、T细胞比例显著高于原位骨髓组中相应细胞比例，而髓系细胞比例则明显低于原位骨髓组比例，表明干细胞发生器具有完整的血系细胞，但与原位骨髓组比例不完全一致。
图7和图8为rhBMP-2诱导产生的干细胞发生器中造血祖/干细胞流式检测典型图及造血祖/干细胞比例图。图7所示流式典型图显示干细胞发生器中有完整的造血祖/干细胞，表现为含有造血祖细胞(LKS-)，造血干细胞(LKS+)。图8显示干细胞发生器中造血祖/干细胞相较于原位骨髓组无显著性差异。表明干细胞发生器具有完整的造血祖/干细胞，可为治疗放、化疗后骨髓损伤造血功能低下的治疗提供细胞。
实施例6
含10μg rhBMP-7活性材料诱导产生的干细胞发生器中所含造血祖/干细胞含量检测。
该实施例的目的是检测干细胞发生器中造血祖/干细胞比例，并与正常骨髓的相应细胞进行含量比较，证明干细胞发生器骨髓中具有功能完备的造血系统，含有造血祖/干细胞，可用于造血功能异常的治疗。
含rhBMP-7活性材料为实施例1方法制得的负载10μg rhBMP-7的明胶海绵支架；
单细胞悬液为按照实施例3方法制得的单细胞悬液
方法：采用SPF级C57BL/6小鼠，雄性，8周龄，随机分组，随后于大腿肌肉中植入所制得的含10μg rhBMP-7的材料。饲养3周后取出形成的类器官，即为干细胞发生器。将得到的干细胞发生器去除表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将该类器官(干细胞发生器)压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于后续流式检测试验。实验分组(列表)如下：
组别 原位骨髓组 干细胞发生器组
数量 5 5
图9和图10为rhBMP-7诱导产生的干细胞发生器中造血祖/干细胞流式检测典型图及造血祖/干细胞比例图。图9所示流式典型图显示干细胞发生器中有完整的造血祖/干细胞，表现为含有造血祖细胞(LKS-)，造血干细胞(LKS+)。图10显示干细胞发生器中造血祖/干细胞含量显著高于原位骨髓组，表明干细胞发生器具有丰富的造血祖/干细胞，可为造血功能异常的治疗提供细胞。
实施例7含rhBMP-2活性材料诱导产生的干细胞发生器中造血干/祖细胞多能性评价。
该实施例的目的是评价rhBMP-2诱导产生的干细胞发生器中造血干/祖细胞的多能性，利用干细胞发生器中的细胞(CD45.1)与原位骨髓细胞(CD45.2)共混后，长期竞争性重建经10Gy X射线辐照摧毁的小鼠(CD45.2)造血系统，其中若来自干细胞发生器的细胞占血细胞比例超过0.1％，即视为干细胞发生器中的造血干/祖细胞具有多能性，为造血功能异常的治疗提供现实依据。
含rhBMP-2活性材料为实施例1方法制得的负载30μg rhBMP-2的明胶海绵支架；
方法：采用SPF级C57BL/6的CD45.1雄性小鼠，8周龄，随机分组，随后于大腿肌肉中植入实施例1中所制得的含30μg rhBMP-2的材料。饲养3周后取出形成的类器官，即为干细胞发生器。将得到的干细胞发生器及股骨或髂骨分别去除表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将该类器官(干细胞发生器)及股骨或髂骨压碎，过细胞筛后得到干细胞发生器CD45.1单细胞悬液和原位骨髓CD45.1单细胞悬液。另采用同等周龄的SPF级C57BL/6 CD45.2小鼠，取其股骨及髂骨骨髓制CD45.2单细胞悬液。将1×10 6个干细胞发生器或原位骨髓CD45.1细胞与2×10 5个CD45.2细胞分别混合制成两组200μL单细胞悬液后移植到经过10Gy X射线照射的CD45.2受体小鼠体内。得所得到的单细胞悬液用于后续的干细胞移植试验。之后在6周、12周和20周取各组受体小鼠外周血做流式检测评价干细胞发生器中所含造血干细胞的多能性。实验分组(列表)如下：
组别 原位骨髓组 干细胞发生器组
数量 5 8
图11-图15是长期竞争性造血重建实验，在不同时间点rhBMP-2诱导产生的干细胞发生器中各血系细胞流式检测典型图及对应各血系细胞比例图。图12是不同时间点CD45.1细胞比例变化图，可见所有时间点干细胞发生器来源细胞比例皆大于0.1％，即干细胞发生器来源的造血干/祖细胞具有长期造血重建能力。图13-14与图12趋势相同，仅图15的干细胞发生器来源髓系细胞在12周与20周有部分细胞重建比例低于0.1％。总体来说，干细胞发生器中的造血干/祖细胞具有长期造血重建能力，亦即为多能干细胞。
实施例8 rhBMP-2诱导产生的干细胞发生器中骨髓间充质干细胞含量检测。
该实施例的目的是检测含rhBMP-2生物材料诱导产生的干细胞发生器中骨髓中间充 质干细胞的比例，以期利用间充质干细胞治疗骨、软骨缺损修复，移植物抗宿主病(GVHD)等疾病。
含rhBMP-2活性材料为实施例1中制得的含30μg rhBMP-2的明胶海绵支架；
单细胞悬液为实施例2由含30μg rhBMP-2的支架体内诱导产生的干细胞发生器制得的单细胞悬液。
方法：采用SPF级C57BL/6雄性小鼠，8周龄，随机分组，随后于大腿肌肉中植入实施例1中所制得的含30μg rhBMP-2的材料。饲养3周后取出形成的类器官，即为干细胞发生器。将得到的干细胞发生器去除表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将该类器官(干细胞发生器)压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于后续流式检测试验。实验分组(列表)如下：
组别 原位骨髓组 干细胞发生器组
数量 5 5
图16和图17为含rhBMP-2活性材料诱导产生的干细胞发生器中，骨髓间充质干细胞流式检测典型图及间充质干细胞比例图。由图17可知，1周时，干细胞发生器中间充质干细胞含量显著高于原位骨髓中的含量。3周时，干细胞发生器中间充质细胞含量趋近原位骨髓中的含量。可见干细胞发生器中富集了大量的间充质干细胞，利用富集的间充质干细胞对治疗骨、软骨缺损修复，移植物抗宿主病(GVHD)等疾病具有重大的潜在利用价值。
实施例9
考察干细胞发生器中骨髓细胞在促进辐照损伤小鼠的造血恢复中的作用。
方法：将30μg真核或原核表达系统合成的重组人骨形态发生蛋白-2(rhBMP-2)加入明胶海绵(10mg)，经冻干后形成含有生长因子的活性材料。将制好的材料植入8周龄C57BL/6雄性小鼠大腿肌袋内，饲养3周后取出干细胞发生器及原位骨，去除一部分干细胞发生器或原位骨表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将干细胞发生器或原位骨压碎，过细胞筛后得到单细胞悬液，一部分制成200μL单细胞悬液，用于骨髓移植；另一部分干细胞发生器及原位骨用于拍摄宏观照片并做H&E切片。
骨髓移植部分：采用SPF级C57BL/6小鼠，雌性，8周龄，随机分组。进一步将上一步制得的单细胞悬液经尾静脉移植入不同分组小鼠体内
实验分组如下：
Figure PCTCN2020073591-appb-000001
小鼠放疗损伤模型：对小鼠按照分组表给定的辐照剂量分别给予一次性钴-60辐照，即0Gy辐照，6Gy辐照，7Gy辐照，8Gy辐照。
干预治疗：辐照24小时后对相应组别辐照小鼠分别给予干预治疗，即尾静脉注射200μL PBS溶液，200μL原位骨髓细胞悬液，200μL干细胞发生器细胞悬液，其中所注射的原位骨髓细胞悬液或干细胞发生器细胞悬液为实施例4所介绍的方法制得的单细胞悬液。之后按设定采样点利用眼眶取血方式采集各组小鼠外周血进行血相检测以观察治疗效果。血相检测指标如下：
(1)各组分别于第3天，第6天，……(每间隔3天，连续30天)连续检测外周血白细胞(WBC)数；
(2)各组分别于第3天，第6天，……(每间隔3天，连续30天)连续检测外周血红细胞(RBC)数；
(3)各组分别于第3天，第6天，……(每间隔3天，连续30天)连续检测外周血血小板(PLT)数；
(4)各组分别于第3天，第6天，……(每间隔3天，连续30天)连续检测体重数。
图18所示为肌袋植入8周后产生的干细胞发生器数码照片，由图可见干细胞发生器与原位骨颜色相近，暗示其中含有大量红细胞，同时具有类骨形态，但体积相较原位骨更大。图19干细胞发生器的H&E切片图进一步证实干细胞发生器与原位骨的微结构类似，骨髓腔中充满骨髓细胞及血管。
图20、21为干细胞发生器流式检测相关分析，由图可见干细胞发生器与原位骨具有相似的细胞组成，且干细胞发生器中所含的LKS -细胞，LSK +细胞及造血干细胞(HSCs)比例与原位骨髓中相应细胞比例没有显著性差异。
实施例说明，所构建的干细胞发生器具有与原位骨髓类似的结构与功能，其中所含的造血干/祖细胞具有治疗造血功能异常的潜能。
为了进一步验证干细胞发生器中所含的造血干细胞对于放疗所致造血损伤的治疗效果，对小鼠按照分组表给定的辐照剂量分别给予一次性钴-60辐照(0Gy，6Gy，7Gy，8Gy)。图22-24是不同辐照剂量下小鼠模型经过治疗后体重的变化情况。由图22可以看出，小鼠经钴60辐照(6.0Gy)后，立即一次性尾静脉注射200μL由干细胞发生器产生的同物种骨髓单细胞悬液后，辐照对照组相比于正常对照组，体重在0至9天内变化不大，而在9天后急剧降低，直至死亡。反之辐照治疗组体重变化与正常对照组保持大致相同的变化趋势。图23、24体重(钴60辐照剂量7.0Gy和8.0Gy)的变化趋势与图26、27大抵相同。图24特别需要指出的是，由于辐照剂量过重，辐照对照组在9天内死亡率已达到100％，而治疗组虽稳步上升，但与正常对照组仍有差距。
图25-27是不同辐照剂量下损伤小鼠经过治疗后白细胞数的变化情况。由图25可以看出，辐照后辐照对照组和治疗组白细胞数急剧将至0，但治疗组随时间而白细胞数稳步上升，30天后与正常对照组相平，而辐照对照组仍旧为0。表明辐照小鼠体内经过治疗后，恢复了造血功能，促使白细胞数稳步上升。图26、27白细胞数(钴60辐照剂量7.0Gy和8.0Gy)的变化趋势与图23、24体重变化趋势大抵相同。
图28-30是不同辐照剂量下小鼠模型经过治疗后红细胞数的变化情况。由图28可以看出，辐照后经过尾静脉注射治疗，治疗组和正常对照组的红细胞数保持着相同的变化态势，数值差距不大。而辐照对照组在9天内红细胞数快速降至最低值，直至死亡。这说明，辐照组通过注射的骨类器官(干细胞反应器)内骨髓细胞悬液，促进了体内造血分化，恢复了造血功能，促使红细胞数与正常组大抵持平。图29、30红细胞数(钴60辐照剂量7.0Gy和 8.0Gy)的变化趋势与图1体重变化趋势大抵相同。
图31-33是不同辐照剂量下小鼠模型经过治疗后血小板数的变化情况。由图31可以看出，辐照后治疗组和辐照对照组都随着时间而急剧降低，9天时降至最低点，之后辐照组保持不变直至死亡，而治疗组逆向上升，随时间逐渐上升至与正常对照组相平，恢复至正常水平。而图32和图33明显不同的是辐照治疗组中使用干细胞发生器内骨髓细胞治疗的恢复幅度和速度虽然低于原位骨组，但总体趋势与正常对照组相同。这与体重变化趋势相吻合。
由此可见，负载rhBMP-2生物材料产生的干细胞发生器中的骨髓细胞对放、化疗引起的造血损伤有有效的治疗作用。其促造血作用，主要是骨髓细胞进入造血系统，改善造血微环境，且其中所含的各类祖/干细胞能够正常分化为各类功能细胞，从而重建造血系统。
实施例10
体内制造的干细胞发生器中含有的造血干细胞含量评价
方法：将5μg真核或原核表达系统合成的重组人骨形态发生蛋白-2(rhBMP-2)与1×10 6个小鼠间充质干细胞(mMSCs)加入含有磷酸三钙(TCP)的胶原凝胶(20mg)，经冻干后形成含有生长因子的活性材料。将制好的活性材料植入8周龄SPF级C57BL/6雄性小鼠背部皮下，饲养8周后取出干细胞发生器，去除一部分干细胞发生器表面附着的肌肉后，在研钵中加入少许PBS缓冲液，再使用研杵将其压碎，过细胞筛后得到单细胞悬液。所得到的单细胞悬液可用于流式细胞术检测；另一部分干细胞发生器用于拍摄宏观照片并做H&E切片。实验分组如下。
组别 原位骨 干细胞发生器
数量 6 6
图34所示为背部皮下植入8周后产生的干细胞发生器数码照片，由图可见干细胞发生器与原位骨颜色相近，暗示其中含有大量红细胞，同时具有类骨形态。图35干细胞发生器的H&E切片图进一步证实干细胞发生器与原位骨的微结构类似，具有相同的松质骨及皮质骨结构，骨髓腔中充满骨髓细胞及血管。
图36、37为干细胞发生器流式检测相关分析，由图可见干细胞发生器与原位骨具有相似的细胞组成，且干细胞发生器中所含的LKS-细胞，LSK+细胞及造血干细胞(HSCs)比例与原位骨髓中相应细胞比例没有显著性差异。
本实施例说明所构建的干细胞发生器具有与原位骨髓类似的结构与功能，其中所含的造血干/祖细胞具有治疗造血功能异常的潜能。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种干细胞发生器，其特征在于，所述干细胞发生器由负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料植入动物或人体内经发育后产生类器官而形成。
  2. 如权利要求1所述的干细胞发生器的构建方法，其特征在于，所述构建方法包括以下步骤：
    (1)将生物材料植入动物或人体内；
    (2)经体内发育后产生类器官从而形成所述干细胞发生器，其中，
    所述生物材料为负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料。
  3. 一种富集干细胞的方法，其特征在于，所述方法包括以下步骤：
    (1)将生物材料植入动物或人体内；
    (2)经体内发育后产生类器官并富集所述干细胞，其中，
    所述生物材料为负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料。
  4. 如权利要求1所述的干细胞发生器或权利要求2或3所述的方法，其特征在于，所述活性物质为骨形态发生蛋白-2(Bone Morphogenetic Protein-2，BMP-2)、骨形态发生蛋白-7(Bone Morphogenetic Protein-7，BMP-7)、成骨多肽(Osteogenic peptides)或其他具有诱导骨再生、血管生成能力的生长因子如VEGF，PDG、多肽或生长因子/多肽组合。
  5. 如权利要求1所述的干细胞发生器或权利要求2或3所述的方法，其特征在于，所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
  6. 如权利要求1所述的干细胞发生器或权利要求2或3所述的方法，其特征在于，所述生物材料选自：胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质等中的一种或其共聚/共混组合。
  7. 如权利要求1所述的干细胞发生器或权利要求2或3所述的方法，其特征在于，所述生物材料为自体骨或同种异体骨。
  8. 如权利要求1所述的干细胞发生器或权利要求2或3所述的方法，其特征在于，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
  9. 如权利要求1所述的干细胞发生器或权利要求2或3所述的方法，其特征在于，所述类器官中包含干细胞，所述干细胞为造血干/祖细胞、间充质干细胞、内皮祖细胞或其他种类多能干细胞。
  10. 如权利要求1所述的干细胞发生器的用途，其特征在于，用于制备预防和/或治疗移植物抗宿主病、造血损伤的材料或制备骨髓移植的材料。
PCT/CN2020/073591 2019-01-31 2020-01-21 干细胞发生器及其构建方法 WO2020156387A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/427,737 US20220106568A1 (en) 2019-01-31 2020-01-21 Stem cell generator and construction method therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910099941.8 2019-01-31
CN201910099941.8A CN111500533B (zh) 2019-01-31 2019-01-31 干细胞发生器及其构建方法

Publications (1)

Publication Number Publication Date
WO2020156387A1 true WO2020156387A1 (zh) 2020-08-06

Family

ID=71841998

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/073591 WO2020156387A1 (zh) 2019-01-31 2020-01-21 干细胞发生器及其构建方法

Country Status (3)

Country Link
US (1) US20220106568A1 (zh)
CN (1) CN111500533B (zh)
WO (1) WO2020156387A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115089762B (zh) * 2022-07-20 2023-06-06 中南大学湘雅医院 镁预处理脱细胞组织工程骨支架的制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102089426A (zh) * 2008-06-24 2011-06-08 亨利庞加莱南锡第一大学 细胞分化方法及其在血管建立中的应用
CN103476440A (zh) * 2011-02-25 2013-12-25 新特斯有限责任公司 促进成骨的植入物以及诱导骨生长的方法
CN104411318A (zh) * 2011-12-23 2015-03-11 人类起源公司 包含脱细胞并再群体化的胎盘血管支架的类器官
CN106456669A (zh) * 2014-02-11 2017-02-22 人类起源公司 微类器官以及制造和使用它们的方法
CN107427537A (zh) * 2015-03-03 2017-12-01 哈佛学院院长及董事 产生功能性人体组织的方法
CN109072179A (zh) * 2016-02-18 2018-12-21 学校法人庆应义塾 细胞培养基、培养方法以及类器官

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5226914A (en) * 1990-11-16 1993-07-13 Caplan Arnold I Method for treating connective tissue disorders
US5197985A (en) * 1990-11-16 1993-03-30 Caplan Arnold I Method for enhancing the implantation and differentiation of marrow-derived mesenchymal cells
CN1159063C (zh) * 2000-06-21 2004-07-28 中国医学科学院血液学研究所 骨形态发生蛋白（bmp）作为治疗急性再生障碍性贫血药物的应用
JP2007252393A (ja) * 2004-04-28 2007-10-04 Nippon Medical School 脂肪組織由来幹細胞から骨髄を形成するための組成物およびその方法
WO2007015546A1 (ja) * 2005-08-04 2007-02-08 Genomix Co., Ltd 間葉系幹細胞誘導剤及び組織再生促進剤並びに間葉系幹細胞の調製方法
US10098333B2 (en) * 2008-12-09 2018-10-16 University Of Southern California Method for treating an SLE-like autoimmune disease in a human subject consisting of administering stem cells from human exfoliated deciduous teeth (SHED) and erythropoietin (EPO) to said human subject
US20150065425A1 (en) * 2013-09-02 2015-03-05 Muffin Incorporated Products comprising an extracellular matrix material and osteogenic protein

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102089426A (zh) * 2008-06-24 2011-06-08 亨利庞加莱南锡第一大学 细胞分化方法及其在血管建立中的应用
CN103476440A (zh) * 2011-02-25 2013-12-25 新特斯有限责任公司 促进成骨的植入物以及诱导骨生长的方法
CN104411318A (zh) * 2011-12-23 2015-03-11 人类起源公司 包含脱细胞并再群体化的胎盘血管支架的类器官
CN106456669A (zh) * 2014-02-11 2017-02-22 人类起源公司 微类器官以及制造和使用它们的方法
CN107427537A (zh) * 2015-03-03 2017-12-01 哈佛学院院长及董事 产生功能性人体组织的方法
CN109072179A (zh) * 2016-02-18 2018-12-21 学校法人庆应义塾 细胞培养基、培养方法以及类器官

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, FEI ET AL: "Organoid and Its Application in Cancer Research", CHINESE JOURNAL OF CELL BIOLOGY, vol. 39, no. 4, 30 April 2017 (2017-04-30), CN, pages 394 - 400, XP009522412, ISSN: 1674-7666, DOI: 10.11844/cjcb.2017.04.9002 *

Also Published As

Publication number Publication date
CN111500533B (zh) 2024-07-16
CN111500533A (zh) 2020-08-07
US20220106568A1 (en) 2022-04-07

Similar Documents

Publication Publication Date Title
Shang et al. Advancing application of mesenchymal stem cell-based bone tissue regeneration
Oryan et al. Role of mesenchymal stem cells in bone regenerative medicine: what is the evidence?
Vériter et al. Human adipose-derived mesenchymal stem cells in cell therapy: safety and feasibility in different" hospital exemption" clinical applications
Monaco et al. Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells
Bruder et al. Tissue engineering of bone: cell based strategies.
Dupont et al. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair
Chung et al. Human perivascular stem cell-based bone graft substitute induces rat spinal fusion
Zhao et al. Osteogenic media and rhBMP-2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate-chitosan fibrous scaffold
Chen et al. Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects
He et al. Remote control of the recruitment and capture of endogenous stem cells by ultrasound for in situ repair of bone defects
AU2018335854B2 (en) Biomaterial comprising adipose-derived stem cells and method for producing the same
Vertenten et al. Enhancing bone healing and regeneration: present and future perspectives in veterinary orthopaedics
Haghighat et al. Adipose derived stem cells for treatment of mandibular bone defects: An autologous study in dogs
WO2020156388A1 (zh) 干细胞发生器制备骨缺损修复材料的新用途
Peng et al. Endogenous repair theory enriches construction strategies for orthopaedic biomaterials: a narrative review
Zhu et al. Insights into the use of genetically modified decellularized biomaterials for tissue engineering and regenerative medicine
Wen et al. Allogeneic adipose-derived stem cells regenerate bone in a critical-sized ulna segmental defect
WO2020156387A1 (zh) 干细胞发生器及其构建方法
JP2020534135A (ja) 脂肪由来幹細胞を含む生体材料およびその作製方法
Ben-David et al. Autologous cell-coated particles for the treatment of segmental bone defects—a new cell therapy approach
Murakami et al. Autologous bone grafts with MSCs or FGF-2 accelerate bone union in large bone defects
WO2020156390A1 (zh) 干细胞发生器产生的干细胞用于治疗造血损伤
Chailakhyan et al. Reconstruction of ligament and tendon defects using cell technologies
Zhang et al. Intraoperative construct preparation: a practical route for cell-based bone regeneration
Yi et al. Irradiated human chondrocytes expressing bone morphogenetic protein 2 promote healing of osteoporotic bone fracture in rats

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: 20747591

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20747591

Country of ref document: EP

Kind code of ref document: A1