WO2022253026A1 - 一种提高多能干细胞分化效能的方法 - Google Patents

一种提高多能干细胞分化效能的方法 Download PDF

Info

Publication number
WO2022253026A1
WO2022253026A1 PCT/CN2022/094433 CN2022094433W WO2022253026A1 WO 2022253026 A1 WO2022253026 A1 WO 2022253026A1 CN 2022094433 W CN2022094433 W CN 2022094433W WO 2022253026 A1 WO2022253026 A1 WO 2022253026A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
cells
stem cells
pluripotent stem
treatment
Prior art date
Application number
PCT/CN2022/094433
Other languages
English (en)
French (fr)
Inventor
吴理达
顾雨春
刘润琪
Original Assignee
呈诺再生医学科技(珠海横琴新区)有限公司
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 呈诺再生医学科技(珠海横琴新区)有限公司 filed Critical 呈诺再生医学科技(珠海横琴新区)有限公司
Publication of WO2022253026A1 publication Critical patent/WO2022253026A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C12N5/0657—Cardiomyocytes; Heart 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C12N2500/62—DMSO
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for improving the differentiation efficiency of pluripotent stem cells, a culture medium, a cell culture system and a kit.
  • Pluripotent Stem Cells are a type of pluripotent cells with self-renewal and self-replication capabilities, and are the hot spot and focus of current stem cell research. It holds great promise in cell therapy, tissue engineering and regenerative medicine, as well as pharmaceutical and biotechnological applications. It can differentiate into all cells in the body, and then form all tissues and organs of the body, but it loses the ability to develop into a complete individual, and its developmental potential is limited to a certain extent. Therefore, the research on pluripotent stem cells not only has important theoretical significance, but also has great application value in organ regeneration, repair and disease treatment. A hot spot in research fields such as medicine or tissue engineering, paving the way for regenerative medicine therapies.
  • the directional differentiation of stem cells is the basis of the development, growth and repair of organisms, and it is realized by the joint control of internal and external factors and regulation of the expression of specific genes.
  • stemness maintenance factors bFGF and TGF ⁇
  • stemness maintenance factors bFGF and TGF ⁇
  • small molecular compounds that inhibit signaling pathways will be added to maintain the stemness of ESC/iPSC.
  • ESC/iPSC can differentiate into any type of cell in our body, but different ESC/iPSC cell lines have inconsistent stemness maintenance ability, resulting in different differentiation ability, which in turn affects the differentiation efficiency. Since ESC/iPSC have been expanded and cultured in dry maintenance medium for a long time, sudden use of differentiation medium will cause a large number of death of ESC/iPSC and low differentiation efficiency.
  • This patent provides a method to improve the differentiation efficiency of ESC/iPSC.
  • a micro-differentiation treatment process is added.
  • pluripotent stem cells are cultivated with E6 medium or treated with DMSO.
  • the differentiation experiment of pluripotent stem cells after a certain period of time can improve the differentiation efficiency and activity rate of pluripotent stem cells.
  • the invention provides a method, system and kit for improving the differentiation efficiency of pluripotent stem cells.
  • the present invention provides a method for improving the differentiation efficiency of pluripotent stem cells, the method comprising performing microdifferentiation treatment on pluripotent stem cells.
  • the microdifferentiation treatment comprises using E6 medium to cultivate pluripotent stem cells and/or DMSO to treat pluripotent stem cells, and the microdifferentiation treatment is to use E6 medium to cultivate pluripotent stem cells and/or DMSO to treat pluripotent stem cells .
  • the components of the E6 medium include one or more of DMEM/F12, sodium bicarbonate, L-ascorbic acid, insulin, insulin transferrin and sodium selenite; preferably, the The components of the E6 medium are DMEM/F12, sodium bicarbonate, L-ascorbic acid, insulin, insulin transferrin and sodium selenite; preferably, the E6 medium is added to the DMEM/F12 medium with 15- 22mM sodium bicarbonate, 60-220 ⁇ M L-ascorbic acid, 20 ⁇ g/ml insulin, 10 ⁇ g/ml insulin transferrin and 20ng/ml sodium selenite.
  • the E6 medium can also be Essential 6 TM Medium; preferably, the E6 medium is a commercially available medium; more preferably, it is Thermo Fisher, the product number is A1516401 Medium.
  • the E6 medium can also be added with stemness maintenance factors or small molecular compounds; preferably, the E6 medium can also be added with TGF ⁇ and/or bFGF; preferably, the working concentration of TGF ⁇ is 1-10ng/ml, and the working concentration of bFGF is 50-100ng/ml.
  • the E6 medium can be a purchased commercial medium, or a culture medium prepared artificially.
  • culturing pluripotent stem cells in the E6 medium refers to cultivating pluripotent stem cells in E6 medium for 0-72 hours; preferably, cultivating pluripotent stem cells in E6 medium refers to cultivating pluripotent stem cells in E6 medium Pluripotent stem cells for 0-48 hours; preferably, the E6 medium for cultivating pluripotent stem cells refers to using E6 medium for cultivating pluripotent stem cells for 0-24 hours, specifically including: 1 hour, 2 hours, 3 hours, 4 hours , 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours hours, 22 hours, 23 hours, 24 hours; preferably, the E6 medium for culturing pluripotent stem cells refers to using E6 medium for culturing pluripotent stem cells for 24 hours.
  • the DMSO treatment of the pluripotent stem cells comprises adding DMSO to the microdifferentiation treatment medium to culture the cells; preferably, the DMSO treatment of the pluripotent stem cells is adding DMSO to the microdifferentiation treatment medium cells were cultured in.
  • the working concentration of DMSO is 1%-2%, specifically: 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%; preferably, the working concentration of DMSO is 1.5%-2%; more preferably, the working concentration of DMSO is 2%.
  • the microdifferentiation treatment medium includes Essential 8 TM Medium, Dulbecco's Modified Eagle's Medium (DMEM, Dulbecco's Modified Eagle's Medium), Minimal Essential Medium (MEM, Minimal Essential Medium), Eagle basic medium (BME, Basal Medium Eagle), F-10, F-12, ⁇ -minimal essential medium ( ⁇ -MEM, ⁇ -Minimal Essential Medium), G-minimal essential medium (G-MEM , Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Dulbecco's Medium), AmnioMax, new second-generation amniotic fluid medium (Amino MaxIIcomplete Medium, Gibco, Newyork, USA), Chang's medium, MesemCult-XF medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, HepatoZYME-SFM, William's EMedium, Waymouth'
  • the DMSO treatment of the pluripotent stem cells refers to the use of DMSO to treat the pluripotent stem cells for 0-72 hours; preferably, the DMSO treatment of the pluripotent stem cells refers to the use of DMSO to treat the pluripotent stem cells for 0-48 hours, 0-36 hours; preferably, the DMSO treatment of pluripotent stem cells refers to DMSO treatment of pluripotent stem cells for 0-24 hours, specifically including: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours , 24 hours; preferably, the DMSO treatment of the pluripotent stem cells means using DMSO to treat the pluripotent stem cells for 24 hours.
  • the pluripotent stem cells include one or more of ESCs, iPSCs, embryoid bodies, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, fat stem cells, and umbilical cord blood stem cells;
  • the pluripotent stem cells comprise ESCs and/or iPSCs;
  • said pluripotent stem cells comprise iPSCs
  • said pluripotent stem cells are iPSCs.
  • the iPSC can be a commercially available cell line, or can be induced from donor cells.
  • the source of the donor cells includes villi cells, skin (fibroblasts and keratinocytes) , amniotic fluid, extraembryonic tissue (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, amnion-derived mesenchymal stem cells, amnion-derived peripheral blood cells, breast epithelial cells, adipose stem cells, umbilical cord One or more of stroma and placenta.
  • the pluripotent stem cells can be human pluripotent stem cells or non-human pluripotent stem cells; the non-human pluripotent stem cells include mammals (such as mice, monkeys, cows, sheep, pigs, Pluripotent stem cells derived from horses and chickens.
  • mammals such as mice, monkeys, cows, sheep, pigs, Pluripotent stem cells derived from horses and chickens.
  • the embryonic stem cells include E14.1 mouse embryonic stem cell line, R1/E mouse embryonic stem cell line, Oct4-Neo mouse embryonic stem cell line, J1 mouse embryonic stem cell line, CE3 mouse embryonic stem cell line, Nanog -GFP mouse embryonic stem cell line, RW.4 mouse embryonic stem cell line, R1 mouse embryonic stem cell line, G-Olig2 mouse embryonic stem cell line, ESF158 mouse embryonic stem cell line, EDJ22 mouse embryonic stem cell line, C57BL/6 Mouse embryonic stem cell line, HN4 human embryonic stem cell line, H9 human embryonic stem cell line, H1 human embryonic stem cell line.
  • the human embryonic stem cells include one or more of the HN4 human embryonic stem cell line, the H9 human embryonic stem cell line and the H1 human embryonic stem cell line.
  • the human induced pluripotent stem cells include UiPSC-013 (UC-013) cell line and/or UiPS C-015 (UC-015) cell line.
  • the improvement of differentiation efficiency refers to the improvement of the ability of pluripotent stem cells to differentiate into terminal cells or their precursor cells; preferably, the improvement of differentiation efficiency refers to the differentiation of pluripotent stem cells into more terminal cells ; Preferably, the improvement of differentiation efficiency can be reflected by cell detection.
  • the cell detection can be a quantitative detection and/or a qualitative detection.
  • the cell detection can be cell viability detection, immune-based detection, flow cytometry detection, colorimetric detection, gold nanoparticle-based detection, fluorescence detection, ultraviolet detection, precursor of target terminal cells.
  • detection of somatic cell markers and target terminal cell markers preferably, the cell detection is fluorescence detection, detection of precursor cell markers of target terminal cells and target terminal cell markers one or more;
  • the precursor cell marker is one or more of MESP2, NKX2.5 and PDGFRA;
  • the target terminal cell marker is a cardiomyocyte marker; preferably, the target terminal cell marker is a cardiomyocyte marker TNNT and/or ⁇ -MHC.
  • the cell detection can be performed after 0-50 days of inducing differentiation treatment; preferably, the cell detection can be performed after 0-30 days of inducing differentiation treatment, and the cell detection It can be performed 3-15 days after the differentiation-inducing treatment.
  • the terminal cells include cardiomyocytes, progenitor cells, organoids, neural stem cells, dopaminergic neuron cells, oligodendrocytes, oligodendrocyte precursor cells, dopaminergic neurons, liver cells, kidney-related cells, islet body cells, endothelial progenitor cells, pigment epithelial cells, cone cells, skin cells, blood-related cells, mesenchymal hepatocytes;
  • said terminal cells are cardiomyocytes.
  • the method may further include one or more of basal culture before micro-differentiation treatment, differentiation-inducing treatment after micro-differentiation treatment, and expansion culture after differentiation-inducing treatment.
  • the stemness maintenance culture before the microdifferentiation treatment is to use a stemness maintenance medium to cultivate pluripotent stem cells.
  • the dry maintenance medium comprises one or more of the following combinations: Essential 8 TM Medium, Dulbecco's Modified Eagle's Medium (DMEM, Dulbecco's Modified Eagle's Medium), Minimal Essential Medium (MEM, Minimal Essential Medium), Eagle Basic Medium (BME, Basal Medium Eagle), F-10, F-12, ⁇ -Minimal Essential Medium ( ⁇ -MEM, ⁇ -Minimal Essential Medium), G-minimal essential medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Dulbecco's Medium), AmnioMax, new second-generation amniotic fluid medium (Amino MaxIIcomplete Medium, Gibco, New York, USA), Chang's culture Base, MesemCult-XF medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, HepatoZYME-SFM, William's EMedium
  • the dry maintenance medium can be a commercial medium or a self-prepared medium.
  • the stemness maintenance culture of the pluripotent stem cells is carried out before the microdifferentiation treatment; preferably, the stemness maintenance culture of the pluripotent stem cells is continued for 0-60 hours before the microdifferentiation treatment of the pluripotent stem cells ; Preferably, 0-48 hours, 0-36 hours, 0-24 hours, 12-24 hours, 24 hours.
  • the dry maintenance culture further includes detecting cell content; preferably, the step of detecting cells is performed 0-5 hours before the microdifferentiation treatment; preferably, 0-2 hours.
  • the inducing differentiation treatment after the microdifferentiation treatment includes using the first differentiation medium and/or the second differentiation medium to cultivate the cells after the microdifferentiation treatment, and the first differentiation medium can be used in the second differentiation medium. culturing the cells after the differentiation treatment before or after the differentiation medium;
  • the first differentiation medium cultures the microdifferentiated cells for 0-96 hours; preferably, 0-48 hours; preferably, 48 hours.
  • said second differentiation medium cultures cells for 0-96 hours; preferably, 0-48 hours; preferably, 48 hours.
  • the first differentiation medium and the second differentiation medium may contain one or more of substances that promote cardiomyocyte differentiation, substances that inhibit Wnt signaling pathway and substances that inhibit mTOR signaling pathway.
  • the substances that promote cardiomyocyte differentiation include BMP4, basic fibroblast growth factor (bFGF), activin A (Activin A), Noggin, Dorsomorphin, 6-bromoindirubin-3 One or more of '-oxime (6-bromoindirubin-3'-oxime, IO).
  • BMP4 basic fibroblast growth factor
  • Activin A activin A
  • Noggin Noggin
  • Dorsomorphin Dorsomorphin
  • 6-bromoindirubin-3 One or more of '-oxime (6-bromoindirubin-3'-oxime, IO).
  • the substance that inhibits the Wnt signaling pathway includes one or more of Wnt-C59, dickkopf homolog 1 (dickkopf homolog 1, KK1), IWP, and IWR (inhibitors of Wntresponse).
  • the substances that inhibit the mTOR signaling pathway include Rapamycin, Everolimus (RAD001), KU-0063794, AZD8055, Temsirolimus, INK128, Ridaforolimus.
  • the components of the first differentiation medium include at least one of DMEM/F12, 1 ⁇ B27 (minus vitaminA) supplement, L-glutamine, 1-thioglycerol, L-AA, BMP4, and CHIR99021
  • the The components of the first differentiation medium include DMEM/F12, 1 ⁇ B27 (minus vitaminA) supplement, 1% L-glutamine, 400 ⁇ M 1-thioglycerol, 50 ⁇ g/ml L-AA, 25ng/mL BMP4, 3 ⁇ M CHIR99021
  • the first The composition of a differentiation medium is DMEM/F12, 1 ⁇ B27(minus vitaminA)supplement, 1% L-glutamine, 400 ⁇ M 1-thioglycerol, 50 ⁇ g/ml L-AA, 25ng/mL BMP4, 3 ⁇ M CHIR99021.
  • the composition of the second differentiation medium includes one or more of RPMI 1640, 1 ⁇ B27 (minus insulin) supplement, L-glutamine, BMP4, Wnt-C59;
  • the second differentiation The components of the medium include RPMI 1640, 1 ⁇ B27 (minus insulin) supplement, 1% L-glutamine, 10ng/ml BMP4, 2 ⁇ M Wnt-C59;
  • the components of the second differentiation medium are RPMI 1640, 1 ⁇ B27(minus insulin)supplement, 1% L-glutamine, 10ng/ml BMP4, 2 ⁇ M Wnt-C59.
  • the first differentiation medium and the second differentiation medium can be purchased commercial medium or self-prepared medium.
  • the expansion culture after the differentiation-inducing treatment uses expansion medium to culture cells.
  • the expansion medium is a medium suitable for terminal cell expansion; preferably, the expansion medium is a medium suitable for cardiomyocyte expansion.
  • the components of the expansion medium include RPMI 1640, 1 ⁇ B27supplement, L-glutamine; Preferably, the components of the expansion medium include RPMI 1640, 1 ⁇ B27supplement, 1% L-glutamine.
  • the expansion medium can be a purchased commercial medium or self-prepared.
  • the present invention provides a cell subjected to microdifferentiation.
  • the cells are cardiomyocytes.
  • the present invention provides compositions and medicaments comprising cells that have been subjected to microdifferentiation.
  • the composition may also include other cell culture-related media, growth factors or additives.
  • the medicament may further include one or more of a pharmaceutically acceptable carrier, excipient and pharmaceutically active agent.
  • the present invention provides the application of the aforementioned cells, agents and compositions in the preparation of medicines for cell transplantation; preferably, the medicines can treat heart diseases.
  • the present invention provides a medium for improving the differentiation efficiency of pluripotent stem cells, said medium comprising E6 medium and/or a microdifferentiation treatment medium added with DMSO; preferably, said medium is E6 culture medium base and/or DMSO-supplemented microdifferentiation treatment medium.
  • the present invention provides a medium combination for improving the differentiation efficiency of pluripotent stem cells, the medium combination comprising the aforementioned E6 medium and/or a microdifferentiation treatment medium added with DMSO; preferably, the medium
  • the combination also includes one or more of the aforementioned dry maintenance medium, first differentiation medium, second differentiation medium, and expansion medium.
  • the present invention also provides a cell culture system for improving the differentiation efficiency of pluripotent stem cells, the cell culture system uses reagents required for microdifferentiation treatment, and the cell culture system includes a unit for microdifferentiation treatment of cells ;
  • the cell culture system also includes a dry maintenance culture unit and/or a differentiation induction unit; preferably, the system can also include a unit for monitoring cell status and/or a unit for separating and washing cells.
  • the cell culture system includes a unit for performing microdifferentiation on cells; preferably, the cell culture system further includes one of a stem maintenance culture unit, an induction differentiation unit and an expansion culture unit or more; preferably, the system may also include a unit for monitoring the state of the cells and/or a unit for separating and washing the cells.
  • the present invention also provides a kit for improving the differentiation efficiency of pluripotent stem cells, said kit including reagents required for microdifferentiation treatment; preferably, the reagents required for microdifferentiation treatment include E6 medium and/or Or a microdifferentiation treatment medium with DMSO added; more preferably, the reagent required for the microdifferentiation treatment is E6 medium and/or a microdifferentiation treatment medium with DMSO added.
  • the kit further includes instruments and/or devices required for the microdifferentiation of the cells.
  • the present invention also provides the application of DMSO, E6 medium or microdifferentiation treatment medium added with DMSO in the preparation of medium, medium combination, cell culture system or kit for improving the differentiation efficiency of pluripotent stem cells.
  • the present invention also provides the application of the above-mentioned DMSO, microdifferentiation treatment medium, E6 medium, medium, medium combination, cell culture system or kit in improving the differentiation efficiency of pluripotent stem cells.
  • the present invention also provides a culture medium for improving the differentiation efficiency of pluripotent stem cells, a medium combination for improving the differentiation efficiency of pluripotent stem cells, a cell culture system for improving the differentiation efficiency of pluripotent stem cells, or a kit for improving the differentiation efficiency of pluripotent stem cells Application in the preparation of cells.
  • the present invention also provides the application of the above-mentioned cells, medium, medium combination, cell culture system or kit in the preparation of cells, medicaments or compositions for cell transplantation therapy; preferably, in the preparation Application of cells, medicaments or compositions for cell transplantation to treat heart disease.
  • the present invention also provides the application of DMSO, DMSO-added micro-differentiation treatment medium or E6 medium, the above-mentioned medium combination, the above-mentioned cell culture system or the above-mentioned kit in preparing cells undergoing micro-differentiation treatment;
  • the cells are cells used for cell transplantation to treat diseases; more preferably, the cells are cells used for cell transplantation to treat heart diseases; more preferably, the cells are used for cell transplantation to treat heart diseases Cardiomyocytes.
  • the present invention also provides the application of the cells undergoing micro-differentiation treatment in cell transplantation to treat diseases; preferably, the application of said cells in cell transplantation to treat heart diseases.
  • the medium of the present invention includes, but is not limited to, Essential 8 TM Medium, Dulbecco's Modified Eagle's Medium (DMEM, Dulbecco's Modified Eagle's Medium), Minimal Essential Medium (MEM, Minimal Essential Medium), Eagle's Basic Medium (BME, Basal Medium Eagle), F-10, F-12, ⁇ -Minimal Essential Medium ( ⁇ -MEM, ⁇ -Minimal Essential Medium), G-Minimal Essential Medium (G-MEM, Glasgow's Minimal Essential Medium) , IMPM (IMDM, Iscove's Modified Dulbecco's Medium), AmnioMax, new second-generation amniotic fluid medium (Amino MaxIIcomplete Medium, Gibco, Newyork, USA), Chang's medium, MesemCult-XF medium (STEMCELL Technologies, Vancouver, Canada), RPMI1640 , Ham's F12, DMEM/F12, Ham's F-12K Medium, HepatoZYME-SFM, William'
  • the medium of the present invention may also contain cytokines, growth factors and small molecular compounds required for cell growth, and the small molecular compounds are selected from TGF- ⁇ signaling pathways, appearance modifiers, calcium ion channel agonists and metabolic pathway regulators and other functional groups, the cytokines and protein polypeptides include fibroblast growth factor 1, fibroblast growth factor 2, epidermal growth factor, platelet-derived growth factor, insulin, insulin-like growth factor 1, vascular endothelial cell growth factor, placenta Growth factors, leukemia inhibitory factor, stem cell factor, transferrin, and human serum albumin.
  • cytokines and protein polypeptides include fibroblast growth factor 1, fibroblast growth factor 2, epidermal growth factor, platelet-derived growth factor, insulin, insulin-like growth factor 1, vascular endothelial cell growth factor, placenta Growth factors, leukemia inhibitory factor, stem cell factor, transferrin, and human serum albumin.
  • the culture medium of the present invention may also include a nutrient mixture (Nutrient Mixture), which is a mixture containing various amino acids, vitamins, inorganic salts, etc. that are usually used in cell culture, and can be prepared by mixing the above amino acids, vitamins, inorganic salts, etc. Nutrient mixes are obtained; alternatively, commercially prepared nutrient mixes can be used.
  • Nutrient Mixture is a mixture containing various amino acids, vitamins, inorganic salts, etc. that are usually used in cell culture, and can be prepared by mixing the above amino acids, vitamins, inorganic salts, etc. Nutrient mixes are obtained; alternatively, commercially prepared nutrient mixes can be used.
  • the vitamins include biotin, choline chloride, D-calcium pantothenate, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, lime hydrochloride, coenzyme Q10, vitamin B12, putrescine hydrochloride, vitamin C and vitamin E;
  • Said salts include sodium bicarbonate, calcium chloride, potassium chloride, magnesium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate and sodium pyruvate;
  • Typical nutrient mixtures include one or more of M199, MCDB110, MCDB202, MCDB302.
  • Pluripotent stem cells are cells that have the potential to differentiate into a variety of cell tissues, but have lost the ability to develop into a complete individual, and their developmental potential is limited to a certain extent.
  • This stem cell has the potential to differentiate into a variety of cellular tissues, but loses the ability to develop a complete individual.
  • embryonic stem cells induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, etc.
  • pluripotent stem cells also include embryoid bodies formed from embryonic stem cells or induced pluripotent stem cells under certain culture conditions in vitro.
  • pluripotent stem cells also include ectoderm progenitor cells, mesoderm progenitor cells and endoderm progenitor cells formed from embryonic stem cells or induced pluripotent stem cells under certain culture conditions in vitro.
  • ESC embryonic stem cell, ES cells
  • Embryonic stem cells can participate in the developmental potential of various tissues including gonads under the condition of releasing differentiation inhibition, but cannot develop into an individual by itself. It can develop into the cell tissue of the three germ layers of ectoderm, mesoderm and endoderm.
  • iPSC induced pluripotent stem cells, iPS, iPSCs
  • Induced pluripotent stem cells are cells that are reprogrammed from somatic cells through the introduction of transcription factors (Oct4, Sox2, Klf4, and c-Myc), and have similar differentiation capabilities to embryonic stem cells. It is an important source of cells for treatment, and there are no ethical issues, and the application prospects in the medical field are very broad.
  • iPSCs as the source cells, they can be expanded in vitro and induced to differentiate into specific tissue cells.
  • the application of iPSCs has successfully cultured and differentiated a variety of somatic cells and different tissues such as cardiac muscle, nerve, pancreas, and bone.
  • Differentiation is the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (eg, cardiomyocytes).
  • a differentiated or differentiation-induced cell is one that has a more specialized ("committed") position within a cell lineage.
  • Maintenance generally refers to cells placed in growth medium under conditions that facilitate cell growth and/or division, which may or may not give rise to larger populations of cells.
  • Passaging refers to the process of removing cells from one culture vessel and placing them in a second culture vessel under conditions that facilitate cell growth and/or division.
  • Cell transplantation therapy is to transplant healthy cells into the patient's body to repair or replace damaged cells or tissues, so as to achieve the purpose of healing.
  • Myocardial precursor cells are cells with cardiomyocyte-specific differentiation ability and self-proliferation ability
  • Fig. 1 is a map of myocardial differentiation observed under a microscope on day 6-15 of cells subjected to microdifferentiation treatment using E6 medium.
  • Fig. 2 is a graph showing the results of immunofluorescence detection of cells subjected to differential differentiation using E6 medium.
  • Fig. 3 is a map of myocardial differentiation observed under a microscope on days 6-15 of cells treated with DMSO for differentiation.
  • Fig. 4 is a graph showing the results of immunofluorescence detection of cells differentiated using DMSO.
  • Fig. 5 is a graph showing the expression levels of myocardial precursor cell marker genes MESP2, NKX2.5 and PDGFRA in cells detected by qPCR method under different treatment methods.
  • Fig. 6 is a graph showing the results of expression levels of cardiomyocyte marker genes TNNT and ⁇ -MHC in cells detected by qPCR under different treatment methods.
  • Day -2 Sow iPSCs on a matrigel-coated 12-well culture plate at a density of 5w/cm 2 . Before placing in the incubator, shake up and down, left and right 5 times to make the cells evenly seeded.
  • Use Essential 8 TM Medium Medium (Thermo Fisher, A1517001), plus 10 ⁇ M Y-27632 2HCl.
  • Control group continue to use Essential 8 TM Medium for culture;
  • E6 experimental group replace the Essential 8 TM Medium medium with Essential 6 TM Medium (purchased from Thermo Fisher, Cat. No. A1516401) medium and culture for 24 hours;
  • DMSO experimental group the Essential 8 TM Medium medium was replaced with Essential 8 TM Medium containing 2% dimethyl sulfoxide (DMSO, purchased from Sigma, catalog number D2650), and cultured for 24 hours.
  • DMSO dimethyl sulfoxide
  • the main components include: DMEM/F12, 1 ⁇ B27(minus vitaminA)supplement, 1% L-glutamine, 400 ⁇ M 1-thioglycerol, 50 ⁇ g /ml L-AA, 25ng/mL BMP4, 3 ⁇ M CHIR99021;
  • the main components include: RPMI 1640, 1 ⁇ B27(minus insulin)supplement, 1% L-glutamine, 10ng/ml BMP4, 2 ⁇ M Wnt -C59.
  • the main components include: RPMI 1640, 1 ⁇ B27supplement, 1% L-glutamine.
  • cTNT is a marker gene of cardiomyocytes. Immunofluorescence detection of mature myocardium on day 15 proved that cTNT was expressed in cardiomyocytes derived from iPSCs, indicating that cardiomyocytes can be differentiated by the above method. The results are shown in Figure 2 As shown, the light gray in the figure is the part emitting fluorescence.
  • cTNT is a marker gene of cardiomyocytes. Immunofluorescence detection of mature myocardium on day 15 proved that cTNT was expressed in cardiomyocytes derived from iPSCs, indicating that cardiomyocytes could be differentiated by the above method. The results are shown in Figure 4 As shown, the light gray in the figure is the part emitting fluorescence.
  • qPCR detection of myocardial precursor cell marker genes on the third day of myocardial differentiation, the marker genes MESP2, NKX2.
  • the expression of marker genes of cardiac precursor cells in cells cultured in E6 medium or treated with DMSO was significantly increased, indicating that microdifferentiation treatment can improve the differentiation efficiency of iPSCs.
  • the results of qPCR detection are shown in Figure 5.
  • qPCR detection of cardiomyocyte marker genes On the 15th day of the myocardial differentiation process, the cardiomyocyte marker genes TNNT and ⁇ -MHC were detected by qPCR, and it was found that compared with iPSC cells and cells that had not been differentiated , the expression of cardiomyocyte marker genes such as TNNT and ⁇ -MHC in cells cultured in E6 medium or treated with DMSO was significantly increased, indicating that microdifferentiation treatment can improve the differentiation efficiency of iPSCs. The results of qPCR detection are shown in Figure 6.
  • microdifferentiation treatment can effectively improve the differentiation efficiency of pluripotent stem cells, especially the differentiation efficiency of iPSCs into myocardial precursor cells and cardiomyocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cardiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Rheumatology (AREA)
  • Transplantation (AREA)
  • Vascular Medicine (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种提高多能干细胞分化效能的方法，所述提高多能干细胞分化效能的方法包括对多能干细胞进行微分化处理，具体的，所述微分化处理包括使用E6培养基培养多能干细胞和/或DMSO处理多能干细胞。

Description

一种提高多能干细胞分化效能的方法 技术领域
本发明涉及生物技术领域，尤其涉及提高多能干细胞分化效能的方法、培养基、细胞培养系统和试剂盒。
背景技术
多能干细胞(Pluripotent Stem Cells)是一类具有自我更新、自我复制能力的多潜能细胞，是当前干细胞研究的热点和焦点。在细胞治疗、组织工程学和再生医学以及药物和生物技术应用上有着巨大的前景。它可以分化成体内所有的细胞，进而形成身体的所有组织和器官，但失去了发育成完整个体的能力，发育潜能受到一定的限制。因此，多能干细胞的研究不仅具有重要的理论意义，而且在器官再生、修复和疾病治疗方面极具应用价值，基于其具有的强大的自我更新能力和多向分化潜能使之成为创伤修复、再生医学或组织工程学等研究领域的热点，为再生医学疗法铺平了道路。
干细胞的定向分化是生物体发育、生长和修复的基础，是由细胞内外因素共同控制、调节特定基因的表达来实现的。在干性维持培养基中，ESC/iPSC会逐步的自动分化成不同的胚层细胞，这也是ESC/iPSC干性的体现。因此，在ESC/iPSC培养时，会在干性维持培养基中添加干性维持因子(bFGF和TGFβ)，或者添加抑制信号通路的小分子化合物，从而维持ESC/iPSC的干性。在我们进行定向分化时，会通过添加特定的小分子化合物到分化培养基中，引导ESC/iPSC分化。
ESC/iPSC可以分化为我们体内任何类型的细胞，但是不同的ESC/iPSC细胞系干性维持能力不一致，导致分化能力不一，进而影响分化效率。由于ESC/iPSC长期在干性维持培养基中扩增培养，突然地使用分化培养基，会引起ESC/iPSC的大量死亡，分化效率低下等状况。
本专利提供了一种提高ESC/iPSC的分化效能的方法，在干性维持和分化中间，添加了一个微分化处理的过程，在分化实验之前用E6培养基培养多能干细胞或用DMSO处理多能干细胞一定时间之后再进行分化实验，可以提高多能干细胞的分化效能及活率。
发明内容
本发明提供了一种提高多能干细胞分化效能的方法、系统和试剂盒。
方法
一方面，本发明提供了一种提高多能干细胞分化效能的方法，所述方法包括对多能干细胞进行微分化处理。
在一种实施方式中，所述微分化处理包括使用E6培养基培养多能干细胞和/或DMSO处理多能干细胞，微分化处理是使用E6培养基培养多能干细胞和/或DMSO处理多能干细胞。
在一种实施方式中，所述E6培养基的成分包括DMEM/F12，碳酸氢钠，L-抗坏血酸，胰岛素，胰岛素转铁蛋白和亚硒酸钠中的一种或多种；优选地，所述E6培养基的成分是 DMEM/F12，碳酸氢钠，L-抗坏血酸，胰岛素，胰岛素转铁蛋白和亚硒酸钠；优选地，所述E6培养基是向DMEM/F12培养基中添加15–22mM的碳酸氢钠，60-220μM的L-抗坏血酸，20μg/ml胰岛素，10μg/ml胰岛素转铁蛋白和20ng/ml亚硒酸钠配置而成。
在一种实施方式中，所述E6培养基还可以是Essential 6 TMMedium；优选地，所述E6培养基是商品化的培养基；更优选地，是赛默飞世尔，货号为A1516401的培养基。
在一种实施方式中，所述E6培养基还可以添加干性维持因子或小分子化合物；优选地，所述E6培养基还可以添加TGFβ和/或bFGF；优选地，所述TGFβ的工作浓度是1-10ng/ml，所述bFGF的工作浓度是50-100ng/ml。
在一种实施方式中，所述E6培养基可以是购买的商品化培养基，也可以是人为配置的培养基。
在一种实施方式中，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞0-72小时；优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞0-48小时；优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞0-24小时，具体的包括：1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、13小时、14小时、15小时、16小时、17小时、18小时、19小时、20小时、21小时、22小时、23小时、24小时；优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞24小时。
在一种实施方式中，所述DMSO处理多能干细胞包括将DMSO添加到微分化处理培养基中对细胞进行培养；优选地，所述DMSO处理多能干细胞是将DMSO添加到微分化处理培养基中对细胞进行培养。
在一种实施方式中，所述DMSO的工作浓度是1％-2％，具体的：1％，1.1％，1.2％，1.3％，1.4％，1.5％，1.6％，1.7％，1.8％，1.9％，2％；优选地，所述DMSO的工作浓度是1.5％-2％；更优选地，所述DMSO的工作浓度是2％。
在一种实施方式中，所述微分化处理培养基包括Essential 8 TMMedium、达尔伯克改良伊格尔培养基(DMEM，Dulbecco’s Modified Eagle’s Medium)、最小必需培养基(MEM，Minimal Essential Medium)、伊格尔基本培养基(BME，Basal Medium Eagle)、F-10，F-12、α-最小必需培养基(α-MEM，α-Minimal Essential Medium)、G-最小必需培养基(G-MEM，Glasgow’s Minimal Essential Medium)、IMPM(IMDM，Iscove’s Modified Dulbecco’s Medium)、AmnioMax、新型二代羊水培养基(Amino MaxⅡcomplete Medium，Gibco，Newyork，USA)、Chang’s培养基、MesemCult-XF培养基(STEMCELL Technologies，Vancouver，Canada)、RPMI 1640、Ham’s F12、DMEM/F12、Ham's F-12K Medium，HepatoZYME-SFM、William’s EMedium、Waymouth’s Medium或Hepatocyte Culture Medium中的任意一种或多种的组合；优选地，微分化处理培养基是Essential 8 TMMedium培养基。
在一种实施方式中，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞0-72小时；优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞0-48小时，0-36小时；优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞0-24小时，具体的包括：1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、13小时、14小时、15小时、16小时、17小时、18小时、19小时、20小时、21小时、22小时、23小时、24小时；优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞24小时。
在一种实施方式中，所述多能干细胞包括ESC、iPSC、拟胚体、细胞造血干细胞、神经干细胞、间充质干细胞、皮肤干细胞、脂肪干细胞、脐血干细胞中的一种或多种；
优选地，所述多能干细胞包括ESC和/或iPSC；
优选地，所述多能干细胞包括iPSC；
更优选地，所述多能干细胞是iPSC。
在一种实施方式中，所述iPSC可以是商品化的细胞系，也可以是由供体细胞诱导而来，所述供体细胞的来源包括绒毛细胞，皮肤(成纤维细胞和角质形成细胞)，羊水，胚外组织(胎盘和脐带)，脐带血，骨膜，牙组织，脂肪组织，神经干细胞，肝细胞，羊膜来源的间质干细胞，羊膜来源的外周血细胞，乳腺上皮细胞，脂肪干细胞，脐带基质和胎盘中的一种或多种。
在一种实施方式中，所述多能干细胞可以是人源多能干细胞或非人源多能干细胞；所述非人源多能干细胞包括哺乳动物(如鼠、猴、牛、羊、猪、马、鸡)来源的多能干细胞。
优选地，所述胚胎干细胞包括E14.1小鼠胚胎干细胞系、R1/E小鼠胚胎干细胞系、Oct4-Neo小鼠胚胎干细胞系、J1小鼠胚胎干细胞系、CE3小鼠胚胎干细胞系、Nanog-GFP小鼠胚胎干细胞系、RW.4小鼠胚胎干细胞系、R1小鼠胚胎干细胞系、G-Olig2小鼠胚胎干细胞系、ESF158小鼠胚胎干细胞系、EDJ22小鼠胚胎干细胞系、C57BL/6小鼠胚胎干细胞系、HN4人类胚胎干细胞系、H9人类胚胎干细胞系、H1人类胚胎干细胞系。
优选地，所述人胚胎干细胞包括HN4人类胚胎干细胞系、H9人类胚胎干细胞系和H1人类胚胎干细胞系中的一种或多种。
优选地，所述人诱导多能干细胞包括UiPSC-013(UC-013)细胞系和/或UiPS C-015(UC-015)细胞系。
在一种实施方式中，所述提高分化效能是指提高多能干细胞分化为终端细胞或其前体细胞的能力；优选地，所述提高分化效能是指多能干细胞分化出更多的终端细胞；优选地，所述提高分化效能可以通过细胞检测体现。
在一种实施方式中，所述细胞检测可以是定量检测和/或定性检测。
在一种实施方式中，所述细胞检测可以是细胞活性检测、基于免疫的检测、流式细胞仪检测、比色检测、基于金纳米颗粒的检测、荧光检测、紫外检测、目标终端细胞的前体细胞标志物和目标终端细胞标志物的检测中的一种或多种；优选地，所述细胞检测是荧光检测、目标终端细胞的前体细胞标志物和目标终端细胞标志物的检测中的一种或多种；
优选地，所述前体细胞标志物是MESP2，NKX2.5和PDGFRA中的一种或多种；
优选地，所述目标终端细胞标志物是心肌细胞标志物；优选地，所述目标终端细胞标志物是心肌细胞标志物TNNT和/或β-MHC。
在一种实施方式中，所述细胞检测可以在诱导分化处理进行了0-50天之后进行；优选地，所述细胞检测可以在诱导分化处理进行了0-30天之后进行，所述细胞检测可以在诱导分化处理进行了3-15天之后进行。
在一种实施方式中，所述终端细胞包括心肌细胞，祖细胞，类器官，神经干细胞，多巴胺能神经元细胞，少突胶质细胞，少突胶质前体细胞，多巴胺能神经元，肝细胞，肾相关细胞，胰岛小体细胞，内皮祖细胞，色素上皮细胞，视锥细胞，皮肤细胞，血液相关细胞，间充质肝细胞；
优选地，所述终端细胞是心肌细胞。
在一种实施方式中，所述方法还可以包括微分化处理前的基础培养、微分化处理后的诱导分化处理和诱导分化处理后的扩增培养中的一种或多种。
在一种实施方式中，所述微分化处理前的干性维持培养是使用干性维持培养基培养多能干细胞。
在一种实施方式中，所述干性维持培养基包括以下一种或多种的组合：Esse ntial 8 TMMedium、达尔伯克改良伊格尔培养基(DMEM，Dulbecco’s Modified Eagle’s Medium)、最小必需培养基(MEM，Minimal Essential Medium)、伊格尔基本培养基(BME，Basal Medium Eagle)、F-10，F-12、α-最小必需培养基(α-MEM，α-Minimal Essential Medium)、G-最小必需培养基(G-MEM，Glasgow’s Minimal Essential Medium)、IMPM(IMDM，Iscove’s Modified Dulbecco’s Medium)、AmnioMax、新型二代羊水培养基(Amino MaxⅡcomplete Medium，Gibco，New york，USA)、Chang’s培养基、MesemCult-XF培养基(STEMCELL Technologies，Vancouver，Canada)、RPMI 1640、Ham’s F12、DMEM/F12、Ham's F-12K Medium，HepatoZYME-SFM、William’s EMedium、Waymouth’s Medium或Hepatocyte Culture Medium；优选地，所述干性维持培养基包括Essential 8 TMMedium；优选地，所述干性维持培养基是Essential 8 TMMedium。
在一种实施方式中，所述干性维持培养基可以是商品化的培养基或自行配制的培养基。
优选地，所述对多能干细胞进行干性维持培养在微分化处理之前进行；优选地，所述对多能干细胞进行干性维持培养在对多能干细胞进行微分化处理之前持续0-60小时；优选地，0-48小时，0-36小时，0-24小时，12-24小时，24小时。
优选地，所述干性维持培养还包括检测细胞含量；优选地，所述检测细胞步骤在微分化处理前0-5小时进行；优选地，0-2小时。
在一种实施方式中，所述微分化处理后的诱导分化处理包括使用第一分化培养基和/或第二分化培养基培养微分化处理后的细胞，所述第一分化培养基可以在第二分化培养基之前或之后培养微分化处理后的细胞；
优选地，所述第一分化培养基培养微分化处理后的细胞持续0-96小时；优选的，0-48小时；优选地，48小时。
优选地，所述第二分化培养基培养细胞持续0-96小时；优选的，0-48小时；优选地，48小时。
优选地，所述第一分化培养基和第二分化培养基可以包含促进心肌细胞分化的物质、抑制Wnt信号通路的物质和抑制mTOR信号通路的物质中的一种或多种。
在一种实施方式中，所述具有促进心肌细胞分化的物质包括BMP4、碱性成纤维细胞生长因子(bFGF)、激活素A(Activin A)、Noggin、Dorsomorphin、6-溴靛玉红-3'-肟(6-bromoindirubin-3'-oxime，IO)中的一种或多种。
在一种实施方式中，所述具有抑制Wnt信号通路的物质包括Wnt-C59，dickkopf同源物1(dickkopf homolog 1，KKl)、IWP、IWR(inhibitors of Wntresponse)中的一种或多种。
在一种实施方式中，所述具有抑制mTOR信号通路的物质包括雷帕霉素 (Rapamycin)、依维莫司(RAD001)、KU-0063794、AZD8055、Temsirolimus、INK128、Ridaforolimus。
优选地，所述第一分化培养基的成分中包括DMEM/F12，1×B27(minus vitaminA)supplement，L-glutamine，1-thioglycerol，L-AA，BMP4，CHIR99021中的至少一种，所述第一分化培养基的成分中包括DMEM/F12，1×B27(minus vitaminA)supplement，1％L-glutamine，400μM1-thioglycerol，50μg/ml L-AA，25ng/mL BMP4，3μM CHIR99021，所述第一分化培养基的成分是DMEM/F12，1×B27(minus vitaminA)supplement，1％L-glutamine，400μM 1-thioglycerol，50μg/ml L-AA，25ng/mL BMP4，3μM CHIR99021。
优选地，所述第二分化培养基的成分包括RPMI 1640，1×B27(minus insulin)supplement，L-glutamine，BMP4，Wnt-C59中的一种或多种；优选地，所述第二分化培养基的成分包括RPMI 1640，1×B27(minus insulin)supplement，1％L-glutamine，10ng/ml BMP4，2μM Wnt-C59；优选地，所述第二分化培养基的成分是RPMI 1640，1×B27(minus insulin)supplement，1％L-glutamine，10ng/ml BMP4，2μM Wnt-C59。
在一种实施方式中，所述第一分化培养基和第二分化培养基可以是购买的商品化培养基或者自行配制的培养基。
在一种实施方式中，所述诱导分化处理后的扩增培养是使用扩增培养基培养细胞。
在一种实施方式中，所述扩增培养基是适用于终端细胞扩增的培养基；优选地，所述扩增培养基是适用于心肌细胞扩增的培养基。
优选地，所述扩增培养基的成分包括RPMI 1640，1×B27supplement，L-glutamine；优选地，所述扩增培养基的成分是包括RPMI 1640，1×B27supplement，1％L-glutamine。
在一种实施方式中，所述扩增培养基可以是购买的商品化培养基或者自行配制。
细胞
另一方面，本发明提供了一种经过微分化处理的细胞。
在一种实施方式中，所述细胞是心肌细胞。
另一方面，本发明提供了由经过微分化处理的细胞组成的组合物和药剂。
在一种实施方式中，所述组合物还可以包括其他细胞培养相关的培养基、生长因子或添加剂。
在一种实施方式中，所述药剂还可以包括药学上可接受的载体、赋形剂和药学活性剂中的一种或多种。
另一方面，本发明提供了前述细胞、药剂和组合物在制备细胞移植治疗所用药物中的应用；优选地，所述药物可以治疗心脏疾病。
培养基
另一方面，本发明提供了一种提高多能干细胞分化效能的培养基，所述培养基包括E6培养基和/或添加DMSO的微分化处理培养基；优选地，所述培养基是E6培养基和/或添加DMSO的微分化处理培养基。
培养基组合
另一方面，本发明提供了一种提高多能干细胞分化效能的培养基组合，所述培养 基组合包含前述E6培养基和/或添加DMSO的微分化处理培养基；优选地，所述培养基组合还包括前述干性维持培养基、第一分化培养基、第二分化培养基和扩增培养基中的一种或多种。
细胞培养系统
另一方面，本发明还提供了一种提高多能干细胞分化效能的细胞培养系统，所述细胞培养系统使用微分化处理所需的试剂，所述细胞培养系统包括对细胞进行微分化处理的单元；优选地，所述细胞培养系统还包括干性维持培养单元和/或诱导分化单元；优选地，所述系统还可以包括监测细胞状态的单元和/或对细胞进行分离清洗的单元。
在一种实施方式中，所述细胞培养系统包括对细胞进行微分化处理的单元；优选地，所述细胞培养系统还包括干性维持培养单元、诱导分化单元和扩增培养单元中的一种或多种；优选地，所述系统还可以包括监测细胞状态的单元和/或对细胞进行分离清洗的单元。
试剂盒
另一方面，本发明还提供了一种提高多能干细胞分化效能的试剂盒，所述试剂盒包括微分化处理所需的试剂；优选地，微分化处理所需的试剂包括E6培养基和/或添加DMSO的微分化处理培养基；更优选地，微分化处理所需的试剂是E6培养基和/或添加DMSO的微分化处理培养基。
在一种实施方式中，所述试剂盒还包括对细胞进行微分化处理所需要的仪器和/或装置。
应用
另一方面，本发明还提供了DMSO、E6培养基或添加了DMSO的微分化处理培养基在制备提高多能干细胞分化效能的培养基、培养基组合、细胞培养系统或试剂盒中的应用。
另一方面，本发明还提供了上述DMSO、微分化处理培养基、E6培养基、培养基、培养基组合、细胞培养系统或试剂盒在提高多能干细胞分化效能中的应用。
另一方面，本发明还提供了提高多能干细胞分化效能的培养基、提高多能干细胞分化效能的培养基组合、提高多能干细胞分化效能的细胞培养系统或提高多能干细胞分化效能的试剂盒在制备细胞中的应用。
另一方面，本发明还提供了上述细胞、培养基、培养基组合、细胞培养系统或试剂盒，在制备用于细胞移植治疗的细胞、药剂或组合物中的应用；优选地，在制备用于细胞移植治疗心脏疾病的细胞、药剂或组合物中的应用。
另一方面，本发明还提供了DMSO、添加了DMSO的微分化处理培养基或E6培养基、前述培养基组合、前述细胞培养系统或前述试剂盒在制备经过微分化处理的细胞中的应用；
优选地，所述细胞是用于细胞移植治疗疾病的细胞；更优选的，所述细胞是用于细胞移植治疗心脏疾病的细胞；更优选地，所述细胞是用于细胞移植治疗心脏疾病的心肌细胞。
另一方面，本发明还提供了一种经过微分化处理的细胞在细胞移植治疗疾病中的应用；优选地，所述的细胞在细胞移植治疗心脏疾病中的应用。
一般定义：
除非另有定义，否则本文所用的技术和科学术语具有与所属领域的普通技术人员 之一通常理解的相同的含义。
培养基
本发明的培养基包括但不限于Essential 8 TMMedium、达尔伯克改良伊格尔培养基(DMEM，Dulbecco’s Modified Eagle’s Medium)、最小必需培养基(MEM，Minimal Essential Medium)、伊格尔基本培养基(BME，Basal Medium Eagle)、F-10，F-12、α-最小必需培养基(α-MEM，α-Minimal Essential Medium)、G-最小必需培养基(G-MEM，Glasgow’s Minimal Essential Medium)、IMPM(IMDM，Iscove’s Modified Dulbecco’s Medium)、AmnioMax、新型二代羊水培养基(Amino MaxⅡcomplete Medium，Gibco，Newyork，USA)、Chang’s培养基、MesemCult-XF培养基(STEMCELL Technologies，Vancouver，Canada)、RPMI1640、Ham’s F12、DMEM/F12、Ham's F-12K Medium，HepatoZYME-SFM、William’s EMedium、Waymouth’s Medium或Hepatocyte Culture Medium。
本发明的培养基还可包含细胞生长所需细胞因子、生长因子和小分子化合物，所述小分子化合物选自TGF-β信号通路、表观修饰剂、钙离子通道激动剂和代谢通路调节剂等功能群，所述细胞因子和蛋白多肽包括成纤维细胞生长因子1、成纤维细胞生长因子2、表皮生长因子、血小板衍生生长因子、胰岛素、胰岛素样生长因子1、血管内皮细胞生长因子、胎盘生长因子、白血病抑制因子、干细胞因子、转铁蛋白和人血清白蛋白。
本发明的培养基还可包含营养混合物(Nutrient Mixture)，所述营养混合物为包含通常用于细胞培养的各种氨基酸、维生素、无机盐等的混合物，混合上述氨基酸、维生素、无机盐等可以制备获得营养混合物；或者可使用商业性制备的营养混合物。
所述维生素包括生物素、氯化胆碱、D-泛酸钙、叶酸、肌醇、烟酰胺、吡哆醇盐酸盐、核黄素、盐酸石克氨、辅酶Q10、维生素B12、腐胺二盐酸盐、维生素C和维生素E；
所述盐类包括碳酸氢钠、氯化钙、氯化钾、氯化镁、硫酸镁、氯化钠、一水磷酸二氢钠、磷酸氢二钠和丙酮酸钠；
商业性制备的营养混合物包括M199、MCDB110、MCDB202、MCDB302中的一种或多种。
多能干细胞
多能干细胞(pluripotent stem cell，psc)是具有分化出多种细胞组织的潜能，但失去了发育成完整个体的能力，发育潜能受到一定的限制的细胞。这种干细胞具有分化出多种细胞组织的潜能，但失去了发育完整个体的能力。例如：胚胎干细胞、诱导多能干细胞、造血干细胞、神经干细胞、间充质干细胞、皮肤干细胞等。
在本发明中多能干细胞还包括胚胎干细胞或诱导多能干细胞在体外一定的培养条件下形成的拟胚体。
在本发明中多能干细胞还包括胚胎干细胞或诱导多能干细胞在体外一定的培养条件下形成的外胚层祖细胞、中胚层祖细胞和内胚层祖细胞。
ESC(embryonic stem cell，ES cells)胚胎干细胞
当胚胎发育到囊胚时，其干细胞具有分化为各种细胞类型的能力，是多能干细胞。胚胎干细胞在解除分化抑制的条件下能参与包括生殖腺在内的各种组织的发育潜力，但无法独自发育成一个个体。它可以发育成为外胚层、中胚层及内胚层三种胚层的细胞组织。
iPSC(induced pluripotent stem cells，iPS，iPSCs)诱导多能干细胞
诱导多能干细胞是由体细胞经转录因子(Oct4、Sox2、Klf4和c-Myc)导入重编程而 来的，并且具有与胚胎干细胞相似分化能力的细胞，成为研究人类疾病发病机制、组织细胞替代治疗的重要细胞来源，并且不存在伦理问题，医疗领域的应用前景非常广阔。以iPSCs作为来源细胞，可以将其在体外扩增并诱导分化为特定的组织细胞，应用iPSCs已经成功培养和分化出心肌、神经、胰腺、骨等多种体细胞和不同的组织。
分化
分化是非特化的(“未定型的”)或较少特化的细胞获得特化细胞(例如心肌细胞)特征的过程。已分化的或分化诱导的细胞是在细胞谱系中具有更加特化的(“定型的”)位置的细胞。
维持
维持通常指的是在易于细胞生长和/或分裂的条件下放入生长培养基中的细胞，其可能或可能不产生更大群的细胞。
传代
传代指的是把细胞从一个培养容器移去，并在易于细胞生长和/或分裂的条件下把它们置于第二个培养容器的过程。
细胞移植治疗
细胞移植治疗是把健康的细胞移植到患者体内，以达到修复或替换受损细胞或组织，从而达到治愈的目的。
前体细胞
一类只能向特定终端分化的，较祖细胞更有限的增殖能力的成体细胞。心肌前体细胞即为具有心肌特异性分化能力且具有自我增殖能力的细胞
附图说明
图1是使用E6培养基进行微分化处理的细胞在第6-15天显微镜下观察到的心肌分化形态图。
图2是使用E6培养基进行微分化处理的细胞进行免疫荧光检测的结果图。
图3是使用DMSO进行微分化处理的细胞在第6-15天显微镜下观察到的心肌分化形态图。
图4是使用DMSO进行微分化处理的细胞进行免疫荧光检测的结果图。
图5是通过qPCR方法检测，不同处理方式下，细胞中心肌前体细胞标志性基因MESP2，NKX2.5以及PDGFRA表达量的结果图。
图6是通过qPCR方法检测，不同处理方式下，细胞中心肌细胞标志性基因TNNT以及β-MHC表达量的结果图。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
实施例1、心肌细胞的诱导分化
本实施例中使用到的试剂详细信息如下：
Figure PCTCN2022094433-appb-000001
1、前期准备-干性维持培养
第-2天：铺种iPSC到matrigel包被的12孔培养板，密度为5w/cm 2，放入培养箱前，上下左右各5下摇匀，使细胞均匀铺种，使用Essential 8 TMMedium培养基(赛默飞世尔，A1517001)，加10μM Y-27632 2HCl。
2、微分化处理
第-1天：
对照组：继续使用Essential 8 TMMedium培养；
E6实验组：将Essential 8 TMMedium培养基换为Essential 6 TMMedium(购买于赛默飞世尔，货号A1516401)培养基，培养24小时；
DMSO实验组：将Essential 8 TMMedium培养基换为含有2％二甲基亚砜(DMSO，购买于西格玛，货号D2650)的Essential 8 TMMedium，培养24小时。
3、分化实验-诱导分化
第0-2天：更换为中胚层分化培养基(第一分化培养基)，主要成分包括：DMEM/F12，1×B27(minus vitaminA)supplement，1％L-glutamine，400μM 1-thioglycerol，50μg/ml L-AA，25ng/mL BMP4，3μM CHIR99021；
第3-5天，更换为心肌细胞分化培养基(第二分化培养基)，主要成分包括：RPMI 1640，1×B27(minus insulin)supplement，1％L-glutamine，10ng/ml BMP4，2μM Wnt-C59。
4、扩增培养
第6-14天，更换为心肌细胞扩增培养基，主要成分包括：RPMI 1640，1×B27supplement，1％L-glutamine。
5、检测
E6实验组的细胞：
显微镜下观察：在分化实验的过程中，在第6-15天观察，记录第6、8、10、12、14、15天时分化的细胞形态，细胞形态如图1所示：心肌分化过程中，在第6-8天开始形成细胞网状结构，并在第8天细胞开始跳动，第10天形成非常明显的心肌纤维网，说明通过上述方法可以分化得到心肌细胞。
荧光检测：cTNT是心肌细胞的标记性基因，通过对第15天成熟的心肌进行免疫荧光检测，证明cTNT在iPSC来源的心肌细胞中表达，说明通过上述方法可以分化得到心肌细胞，结果如图2所示，如图浅灰色为散发荧光的部位。
DMSO实验组：
显微镜下观察：在分化实验的过程中，在第6-15天观察细胞，记录第6、8、10、12、14、15天时分化的细胞形态，细胞形态如图3所示：心肌分化过程中，在第6-8天开始形成细胞网状结构，并在第8天细胞开始跳动，第10天形成非常明显的心肌纤维网，说明通过上述方法可以分化得到心肌细胞。
荧光检测：cTNT是心肌细胞的标记性基因，通过对第15天成熟的心肌进行免疫荧光检测，证明cTNT在iPSC来源的心肌细胞中表达，说明通过上述方法可以分化得到心肌细胞，结果如图4所示，如图浅灰色为散发荧光的部位。
qPCR检测心肌前体细胞标志基因：在心肌分化过程中的第三天，通过qPCR方法，对心肌前体细胞标志性基因MESP2，NKX2.5以及PDGFRA进行检测，发现相较于iPSC细胞和未经过微分化处理的细胞，经过E6培养基培养或DMSO处理的细胞中心肌前体细胞标志基因的表达显著提高，说明微分化处理能够提高iPSC分化效能，qPCR检测结果如图5所示。
qPCR检测心肌细胞标志性基因：在心肌分化过程中的第15天，通过qPCR方法，对心肌细胞标志性基因TNNT以及β-MHC进行检测，发现相较于iPSC细胞和未经过微分化处理的细胞，经过E6培养基培养或DMSO处理的细胞中TNNT以及β-MHC等心肌细胞标志基因表达显著提高，说明微分化处理能够提高iPSC分化效能，qPCR检测结果如图6所示。
以上检测结果表明微分化处理可以有效提高多能干细胞的分化效能，尤其是iPSC分化为心肌前体细胞和心肌细胞的效能。

Claims (10)

  1. 一种提高多能干细胞分化效能的方法，其特征在于，所述方法包括对多能干细胞进行微分化处理；优选地，所述微分化处理包括使用E6培养基培养多能干细胞和/或DMSO处理多能干细胞；优选地，微分化处理是使用E6培养基培养多能干细胞和/或DMSO处理多能干细胞；
    优选地，所述E6培养基的成分包括DMEM/F12，碳酸氢钠，L-抗坏血酸，胰岛素，胰岛素转铁蛋白和亚硒酸钠中的一种或多种；优选地，所述E6培养基的成分是DMEM/F12，碳酸氢钠，L-抗坏血酸，胰岛素，胰岛素转铁蛋白和亚硒酸钠；优选地，所述E6培养基是向DMEM/F12培养基中添加15–22mM的碳酸氢钠，60-220μM的L-抗坏血酸，20μg/ml胰岛素，10μg/ml胰岛素转铁蛋白和20ng/ml亚硒酸钠配置而成；
    优选地，所述E6培养基还可以是Essential 6 TMMedium；优选地，所述E6培养基是商品化的培养基；更优选地，是赛默飞世尔的货号为A1516401的培养基；
    优选地，所述E6培养基还可以添加干性维持因子或小分子化合物；优选地，所述E6培养基还可以添加TGFβ和/或bFGF；优选地，所述TGFβ的工作浓度是1-10ng/ml，所述bFGF的工作浓度是50-100ng/ml；
    优选地，所述DMSO处理多能干细胞包括将DMSO添加到微分化处理培养基中对细胞进行培养；优选地，所述DMSO处理多能干细胞是将DMSO添加到微分化处理培养基中对细胞进行培养；
    优选地，所述微分化处理培养基包括Essential 8 TMMedium、达尔伯克改良伊格尔培养基、最小必需培养基、伊格尔基本培养基、RPMI1640、F-10、F-12、α-最小必需培养基、G-最小必需培养基、IMPM、AmnioMax、新型二代羊水培养基、Chang’s培养基、MesemCult-XF培养基、Ham’s F12、DMEM/F12、Ham’s F-12K Medium、HepatoZYME-SFM、William’s EMediu m、Waymouth’s Medium和Hepatocyte Culture Medium中的任意一种或多种的组合；优选地，微分化处理培养基是Essential 8 TMMedium培养基；
    优选地，所述DMSO的工作浓度是1％-2％；优选地，所述DMSO的工作浓度是1.5％-2％；更优选地，所述DMSO的工作浓度是2％；
    优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞0-72小时；优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞0-48小时；优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞0-24小时；优选地，所述E6培养基培养多能干细胞是指使用E6培养基培养多能干细胞24小时；
    优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞0-72小时；优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞0-48小时；优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞0-24小时；优选地，所述DMSO处理多能干细胞是指使用DMSO处理多能干细胞24小时。
  2. 根据权利要求1所述的方法，其特征在于，所述多能干细胞包括ESC、iPSC、拟胚体、细胞造血干细胞、神经干细胞、间充质干细胞、皮肤干细胞、脂肪干细胞、脐血干细胞中的一种或多种；优选地，所述多能干细胞包括ESC和/或iPSC；优选地，所述多能干细胞包括iPSC；更优选地，所述多能干细胞是iPSC；
    优选地，所述iPSC可以是商品化的细胞系，也可以是由供体细胞诱导而来；
    优选地，所述多能干细胞可以是人源多能干细胞或非人源多能干细胞。
  3. 根据权利要求1所述的方法，其特征在于，所述提高分化效能是指提高多能干细胞分化为终端细胞的能力；优选地，所述提高分化效能是指多能干细胞分化出更多的终端细胞；所述终端细胞包括心肌细胞，祖细胞，类器官，神经干细胞，多巴胺能神经元细胞，少突胶质细胞，少突胶质前体细胞，多巴胺能神经元，肝细胞，肾相关细胞，胰岛小体细胞，内皮祖细胞，色素上皮细胞，视锥细胞，皮肤细胞，血液相关细胞和间充质肝细胞中的一种或多种；优选地，所述终端细胞是心肌细胞；
    优选地，所述提高分化效能可以通过细胞检测体现，所述细胞检测可以是定量检测和/或定性检测；
    优选地，所述细胞检测可以是细胞活性检测、基于免疫的检测、流式细胞仪检测、比色检测、基于金纳米颗粒的检测、荧光检测、紫外检测、目标终端细胞的前体细胞标志物和目标终端细胞标志物的检测中的一种或多种；优选地，所述细胞检测是荧光检测、目标终端细胞的前体细胞标志物和目标终端细胞标志物的检测中的一种或多种；
    优选地，所述前体细胞标志物是MESP2，NKX2.5和PDGFRA中的一种或多种；
    优选地，所述目标终端细胞标志物是心肌细胞标志物；优选地，所述目标终端细胞标志物是心肌细胞标志物TNNT和/或β-MHC。
  4. 根据权利要求1所述的方法，其特征在于，所述方法还可以包括微分化处理前的干性维持培养、微分化处理后的诱导分化处理和诱导分化处理后的扩增培养中的一种或多种；
    优选地，所述微分化处理前的干性维持培养使用的培养基是干性维持培养基；优选地，所述干性维持培养基是Essential 8 TMMedium；
    优选地，所述微分化处理后的诱导分化处理使用的培养基是第一分化培养基和/或第二分化培养基；优选地，所述第一分化培养基的成分是DMEM/F12，1×B27(minus vitaminA)supplement，1％L-glutamine，400μM 1-thioglycerol，50ug/ml L-AA，25ng/mL BMP4，3μM CHIR99021；优选地，所述第二分化培养基的成分是RPMI 1640，1×B27(minus insulin)supplement，1％L-glutamine，10ng/ml BMP4，2μM Wnt-C59；
    优选地，所述诱导分化处理后的扩增培养使用的培养基是扩增培养基；优选地，所述扩增培养基的成分是RPMI 1640，1×B27 supplement，1％L-glutamine；
    优选地，所述微分化处理前的干性维持培养基、第一分化培养基、第二分化培养基和扩增培养基可以是购买的商品化培养基或者自行配制的。
  5. 一种细胞或其构成的组合物或药剂，其特征在于，所述细胞经过了微分化处理；优选地，所述细胞是心肌细胞；优选地，所述细胞是经过权利要求1-4任一方法制得的细胞；
    优选地，所述组合物还可以包括其他细胞培养相关的培养基、生长因子或添加剂；
    优选地，所述药剂还可以包括药学上可接受的载体、赋形剂和药学活性剂中的一种或多种。
  6. 一种提高多能干细胞分化效能的培养基，所述培养基包括E6培养基和/或权利要求1-4任一方法中使用的添加DMSO的微分化处理培养基；优选地，所述培养基是E6培养基和/或权利要求1-4任一方法中使用的添加DMSO的微分化处理培养基。
  7. 一种提高多能干细胞分化效能的培养基组合，所述培养基组合包含E6培养基和/或权利要求1-4任一方法中使用的添加DMSO的微分化处理培养基；优选地，所述培养基组合还包括权利要求1-4任一方法中使用的干性维持培养基、权利要求1-4任一方法中使用的第一 分化培养基、权利要求1-4任一方法中使用的第二分化培养基和权利要求1-4任一方法中使用的扩增培养基中的一种或多种。
  8. 一种提高多能干细胞分化效能的细胞培养系统，所述细胞培养系统包括对细胞进行微分化处理的单元；优选地，所述微分化处理的单元是指对细胞进行权利要求1-4任一方法中的微分化处理；
    优选地，所述细胞培养系统还包括干性维持培养单元、诱导分化单元和扩增培养单元中的一种或多种；优选地，所述干性维持培养单元是指对细胞进行权利要求1-4任一方法中的干性维持培养，所述诱导分化单元是指对细胞进行权利要求1-4任一方法中的诱导分化处理，所述干扩增培养单元是指对细胞进行权利要求1-4任一方法中的扩增培养；
    优选地，所述系统还可以包括监测细胞状态的单元和/或对细胞进行分离清洗的单元。
  9. 一种提高多能干细胞分化效能的试剂盒，所述试剂盒包括微分化处理所需的试剂；优选地，所述微分化处理是指权利要求1-4任一方法中的微分化处理；优选地，所述试剂盒还包括对细胞进行微分化处理所需要的仪器和/或装置。
  10. 一种应用，其特征在于，所述应用包括以下任一一种：
    1)DMSO、权利要求1-4任一方法中使用的添加DMSO的微分化处理培养基或权利要求1-4任一方法中使用的E6培养基在制备提高多能干细胞分化效能的培养基、提高多能干细胞分化效能的培养基组合、提高多能干细胞分化效能的细胞培养系统或提高多能干细胞分化效能的试剂盒的应用；
    优选地，所述提高多能干细胞分化效能的培养基包括权利要求6所述的培养基；优选地，所述提高多能干细胞分化效能的培养基是权利要求6所述的培养基；
    优选地，所述提高多能干细胞分化效能的培养基组合包括权利要求7所述的培养基组合；优选地，所述提高多能干细胞分化效能的培养基组合是权利要求7所述的培养基组合；
    优选地，所述提高多能干细胞分化效能的试剂盒包括权利要求8所述的试剂盒；优选地，所述提高多能干细胞分化效能的试剂盒是权利要求8所述的试剂盒；
    优选地，所述提高多能干细胞分化效能的细胞培养系统包括权利要求9所述的细胞培养系统；优选地，所述提高多能干细胞分化效能的细胞培养系统是权利要求9所述的细胞培养系统；
    2)DMSO、权利要求1-4任一方法中使用的添加DMSO的微分化处理培养基或权利要求1-4任一方法中使用的E6培养基、权利要求6所述的培养基、权利要求7所述的培养基组合、权利要求8所述的细胞培养系统或权利要求9所述的试剂盒在提高多能干细胞分化效能中的应用；
    3)DMSO、权利要求1-4任一方法中使用的添加DMSO的微分化处理培养基或权利要求1-4任一方法中使用的E6培养基、权利要求6所述的培养基、权利要求7所述的培养基组合、权利要求8所述的细胞培养系统或权利要求9所述的试剂盒在制备经过微分化处理的细胞中的应用；
    优选地，所述细胞是经过权利要求1-4任一方法中微分化处理的细胞；
    优选地，所述细胞是权利要求5中所述的细胞；
    优选地，所述细胞是用于细胞移植治疗疾病的细胞；优选的，所述细胞是用于细胞移植治疗心脏疾病的细胞；优选地，所述细胞是心肌细胞；
    4)权利要求5所述的细胞、组合物或药剂在制备细胞移植治疗药物中的应用；优选地，所述细胞移植治疗的是心脏疾病；
    5)权利要求5所述的细胞在制备权利要求5所述的组合物和药剂中的应用。
PCT/CN2022/094433 2021-06-02 2022-05-23 一种提高多能干细胞分化效能的方法 WO2022253026A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110612291.X 2021-06-02
CN202110612291.XA CN113337459B (zh) 2021-06-02 2021-06-02 一种提高多能干细胞分化效能的方法

Publications (1)

Publication Number Publication Date
WO2022253026A1 true WO2022253026A1 (zh) 2022-12-08

Family

ID=77474648

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/094433 WO2022253026A1 (zh) 2021-06-02 2022-05-23 一种提高多能干细胞分化效能的方法

Country Status (2)

Country Link
CN (1) CN113337459B (zh)
WO (1) WO2022253026A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116445408A (zh) * 2023-05-22 2023-07-18 呈诺再生医学科技(北京)有限公司 LSD1抑制剂在促进iPSC向HSC分化和HSC干性维持中的应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113337459B (zh) * 2021-06-02 2022-09-06 呈诺再生医学科技(珠海横琴新区)有限公司 一种提高多能干细胞分化效能的方法
CN114561356B (zh) * 2022-03-09 2022-09-13 北京呈诺医学科技有限公司 影响细胞因子分泌的方法及应用
CN116590219A (zh) * 2022-09-19 2023-08-15 苏州南医大创新中心 一种干细胞来源的类原肠胚模型及其构建方法和应用
CN116042516A (zh) * 2023-02-22 2023-05-02 深圳市寰宇生物科技有限公司 一种从人诱导性多能干细胞分化的心肌细胞球及其制备方法和应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101037669A (zh) * 2006-03-13 2007-09-19 上海交通大学医学院附属新华医院 一种从人胚胎干细胞诱导肝细胞的方法
CN105849253A (zh) * 2013-09-20 2016-08-10 修复股份有限公司 一种多能干细胞定向分化为功能性心肌的方法
CN107460164A (zh) * 2012-07-23 2017-12-12 中国科学院生物物理研究所 体外诱导多能干细胞分化为心室肌细胞的方法
CN108384755A (zh) * 2018-02-08 2018-08-10 北京呈诺医学科技有限公司 一种高效、快捷的诱导性多能干细胞向神经干细胞分化的方法
CN109563485A (zh) * 2016-08-10 2019-04-02 加图立大学校产学协力团 通过诱导诱导多能干细胞的分化来培养角膜上皮细胞的方法及系统
CN112105718A (zh) * 2018-03-22 2020-12-18 东方酵母工业株式会社 多能干细胞分化促进剂
CN112359012A (zh) * 2020-10-22 2021-02-12 中国科学院水生生物研究所 诱导多能干细胞分化制备心脏瓣膜内皮细胞的方法及其应用
CN112831461A (zh) * 2021-02-26 2021-05-25 澳门大学 一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物
CN113337459A (zh) * 2021-06-02 2021-09-03 呈诺再生医学科技(珠海横琴新区)有限公司 一种提高多能干细胞分化效能的方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101037669A (zh) * 2006-03-13 2007-09-19 上海交通大学医学院附属新华医院 一种从人胚胎干细胞诱导肝细胞的方法
CN107460164A (zh) * 2012-07-23 2017-12-12 中国科学院生物物理研究所 体外诱导多能干细胞分化为心室肌细胞的方法
CN105849253A (zh) * 2013-09-20 2016-08-10 修复股份有限公司 一种多能干细胞定向分化为功能性心肌的方法
CN109563485A (zh) * 2016-08-10 2019-04-02 加图立大学校产学协力团 通过诱导诱导多能干细胞的分化来培养角膜上皮细胞的方法及系统
CN108384755A (zh) * 2018-02-08 2018-08-10 北京呈诺医学科技有限公司 一种高效、快捷的诱导性多能干细胞向神经干细胞分化的方法
CN112105718A (zh) * 2018-03-22 2020-12-18 东方酵母工业株式会社 多能干细胞分化促进剂
CN112359012A (zh) * 2020-10-22 2021-02-12 中国科学院水生生物研究所 诱导多能干细胞分化制备心脏瓣膜内皮细胞的方法及其应用
CN112831461A (zh) * 2021-02-26 2021-05-25 澳门大学 一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物
CN113337459A (zh) * 2021-06-02 2021-09-03 呈诺再生医学科技(珠海横琴新区)有限公司 一种提高多能干细胞分化效能的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MUMMERY, C.L. ; FEIJEN, A. ; MOOLENAAR, W.H. ; VAN DEN BRINK, C.E. ; DE LAAT, S.W.: "Establishment of a differentiated mesodermal line from P19 EC cells expressing functional PDGF and EGF receptors", EXPERIMENTAL CELL RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 165, no. 1, 1 July 1986 (1986-07-01), AMSTERDAM, NL , pages 229 - 242, XP024858450, ISSN: 0014-4827, DOI: 10.1016/0014-4827(86)90547-1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116445408A (zh) * 2023-05-22 2023-07-18 呈诺再生医学科技(北京)有限公司 LSD1抑制剂在促进iPSC向HSC分化和HSC干性维持中的应用
CN116445408B (zh) * 2023-05-22 2024-02-02 呈诺再生医学科技(北京)有限公司 LSD1抑制剂在促进iPSC向HSC分化和HSC干性维持中的应用

Also Published As

Publication number Publication date
CN113337459A (zh) 2021-09-03
CN113337459B (zh) 2022-09-06

Similar Documents

Publication Publication Date Title
WO2022253026A1 (zh) 一种提高多能干细胞分化效能的方法
JP5227318B2 (ja) 細胞増殖培地
EP4001403A1 (en) Methods for differentiating pluripotent cells
JP2012523240A (ja) 幹細胞培養のための方法および組成物
US10724000B2 (en) Small molecule based conversion of somatic cells into neural crest cells
WO2023221438A1 (zh) 一种通过调节NGN3表达促进iPSC向胰岛细胞分化的方法
WO2023011251A1 (zh) 快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用
US9290740B2 (en) Use of basic fibroblast growth factor in the de-differentiation of animal connective tissue cells
JP2023531975A (ja) 間葉系幹細胞を産生するための培地及び方法
Sauerzweig et al. A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells
KR102141641B1 (ko) 인간지방 유래 중간엽 줄기세포로부터 모유두세포로의 분화방법
H Parsons et al. Patents on technologies of human tissue and organ regeneration from pluripotent human embryonic stem cells
CN115975914A (zh) 利用化学小分子药物重编程诱导多能干细胞的方法
EP4257678A1 (en) Production method for producing pluripotent stem cell population
WO2010108008A2 (en) Bsc cell differentiation and use in therapy
EP3950933A1 (en) Cell population including pluripotent stem cells and production method thereof
JP2016026490A (ja) 多能性幹細胞から肝芽細胞を分化誘導する培養培地および方法
WO2019199230A1 (en) A method of transporting mesenchymal stem cells by means of a cell culture medium and a method of administering stem cells to wounds
CN116769695A (zh) 从畸胎瘤、类器官和拟胚体中产生人细胞和组织的培养基和方法
Poudel et al. Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea
CN110564673A (zh) 一种干细胞培养液及其应用
Katolikova et al. Cell replacement therapy in Parkinson’s disease—history of development and prospects for use in clinical practice
US20230235279A1 (en) Amniotic-like epithelial cell generation
EP3875581A1 (en) Automated method for preparing keratinocytes
JP2024024550A (ja) 未分化幹細胞の細胞死誘導剤、及び分化細胞の純化方法

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: 22815073

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE