CN113337459B - Method for improving differentiation efficiency of pluripotent stem cells - Google Patents
Method for improving differentiation efficiency of pluripotent stem cells Download PDFInfo
- Publication number
- CN113337459B CN113337459B CN202110612291.XA CN202110612291A CN113337459B CN 113337459 B CN113337459 B CN 113337459B CN 202110612291 A CN202110612291 A CN 202110612291A CN 113337459 B CN113337459 B CN 113337459B
- Authority
- CN
- China
- Prior art keywords
- medium
- differentiation
- cells
- stem cells
- cell
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
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
Abstract
The invention provides a method for improving differentiation efficiency of pluripotent stem cells, which comprises the step of differentiating the pluripotent stem cells, specifically, culturing the pluripotent stem cells by using an E6 culture medium and/or treating the pluripotent stem cells by DMSO.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method, a culture medium, a cell culture system and a kit for improving differentiation efficiency of pluripotent stem cells.
Background
Pluripotent Stem Cells (Pluripotent Stem Cells) are a type of Pluripotent Cells with self-renewal and self-replication capacity, and are the hot spot and focus of current Stem cell research. Has great prospect in cell therapy, tissue engineering and regenerative medicine as well as drug and biotechnology application. It can differentiate into all cells in the body, thus forming all tissues and organs of the body, but loses the ability to develop into a complete individual, and the development potential is limited to a certain extent. Therefore, the research of the pluripotent stem cells not only has important theoretical significance, but also has great application value in the aspects of organ regeneration, repair and disease treatment, and the research becomes a hotspot in the research fields of wound repair, regenerative medicine or tissue engineering and the like based on the strong self-renewal capacity and the multidirectional differentiation potential of the pluripotent stem cells, thereby paving a way for regenerative medicine therapy.
The directional differentiation of stem cells is the basis of the development, growth and repair of organisms and is realized by controlling and regulating the expression of specific genes together by factors inside and outside cells. In the dry maintenance medium, ESC/iPSC will gradually and automatically differentiate into different germ layer cells, which is also the manifestation of ESC/iPSC dryness. Therefore, in the ESC/iPSC culture, dryness of ESC/iPSC is maintained by adding dryness-maintaining factors (bFGF and TGF. beta.) or small molecule compounds inhibiting the signal pathway to the dryness-maintaining medium. When we performed directed differentiation, ESC/iPSC differentiation was directed by adding specific small molecule compounds to the differentiation medium.
ESC/iPSC can differentiate into any type of cells in our body, but different ESC/iPSC cell lines have inconsistent stem maintenance capability, so that the differentiation capability is inconsistent, and the differentiation efficiency is further influenced. When ESC/iPSC is expanded and cultured in a dry maintenance medium for a long period of time, a differentiation medium is suddenly used, which causes a large amount of death of ESC/iPSC and a low differentiation efficiency.
The patent provides a method for improving differentiation efficiency of ESC/iPSC, wherein a differentiation process is added in the process of dryness maintenance and differentiation, and before a differentiation experiment, a differentiation experiment is carried out after pluripotent stem cells are cultured by an E6 culture medium or treated by DMSO for a certain time, so that the differentiation efficiency and the survival rate of the pluripotent stem cells can be improved.
Disclosure of Invention
The invention provides a method, a system and a kit for improving differentiation efficiency of pluripotent stem cells.
Method
In one aspect, the present invention provides a method for increasing differentiation efficiency of pluripotent stem cells, the method comprising differentiating pluripotent stem cells.
In one embodiment, the differentiating treatment comprises culturing the pluripotent stem cells with an E6 medium and/or treating the pluripotent stem cells with DMSO, and the differentiating treatment is culturing the pluripotent stem cells with an E6 medium and/or treating the pluripotent stem cells with DMSO.
In one embodiment, the components of the E6 medium include one or more of DMEM/F12, sodium bicarbonate, L-ascorbic acid, insulin transferrin, and sodium selenite; preferably, the composition of the E6 medium is DMEM/F12, sodium bicarbonate, L-ascorbic acid, insulin transferrin and sodium selenite; preferably, the E6 medium is prepared by adding 15-22 mM sodium bicarbonate, 60-220. mu.M L-ascorbic acid, 20. mu.g/ml insulin, 10. mu.g/ml insulin transferrin and 20ng/ml sodium selenite into DMEM/F12 medium.
In one embodiment, the E6 medium can also be Essential 6 TM Medium; preferably, the E6 medium is a commercial available medium; more preferably, it is a medium of Saimer Feishale, cat # A1516401.
In one embodiment, the E6 medium may also be supplemented with a sternness maintenance factor or a small molecule compound; preferably, the E6 medium can also be added with TGF beta and/or bFGF; preferably, the working concentration of TGF beta is 1-10ng/ml, and the working concentration of bFGF is 50-100 ng/ml.
In one embodiment, the E6 medium may be a commercially available medium or a medium prepared by human.
In one embodiment, the E6 medium culturing the pluripotent stem cells refers to culturing the pluripotent stem cells using E6 medium for 0-72 hours; preferably, the culture of the pluripotent stem cells by the E6 culture medium refers to the culture of the pluripotent stem cells for 0-48 hours by using the E6 culture medium; preferably, the culture of the pluripotent stem cells by the E6 culture medium refers to the culture of the pluripotent stem cells for 0-24 hours by using the E6 culture medium, and specifically comprises the following steps: 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 culture of the pluripotent stem cells in the E6 medium refers to the culture of the pluripotent stem cells for 24 hours by using the E6 medium.
In one embodiment, the DMSO-treated pluripotent stem cells comprise cells cultured by adding DMSO to a differential processing medium; preferably, the DMSO-treated pluripotent stem cells are cultured by adding DMSO to a differentiation treatment medium.
In one embodiment, the working concentration of DMSO is 1% to 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% to 2%; more preferably, the working concentration of DMSO is 2%.
In one embodiment, the differential processing medium comprises Essential 8 TM Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Medium (BME, basic Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel Secondary amniotic fluid Medium (Aminomax II complex, Gibco, Newrk, USA), Chang's Medium, Meseseult-XF Medium (MCELL Technologies, MCMI, Hancadk's Medium, Hadam's Medium, Hadame's Medium, and Hadame's Medium (DMEM, alpha-MEM, alpha-minimum Essential Medium), G-minimum Essential Medium (G-MEM, alpha-minimum Essential Medium), G-M, G-minimum Essential Medium (G-MEM, G-M, Iscoyo's Modified Eagle's Medium, M, Iscoz's Medium, Iscoz, Ishoech, Ishogel, Isho, any one or more of the group consisting of Hepatozyme-SFM, William's Electron, Waymouth's Medium, and Heapatcyte Culture Medium; preferably, the differentiation treatment medium is Essential 8 TM Medium.
In one embodiment, the DMSO treatment of the pluripotent stem cells refers to treatment of the pluripotent stem cells with DMSO for 0-72 hours; preferably, the DMSO-treated pluripotent stem cells are treated with DMSO for 0-48 hours, 0-36 hours; preferably, the DMSO-treated pluripotent stem cells refer to the DMSO-treated pluripotent stem cells for 0 to 24 hours, and specifically include: 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 treatment of the pluripotent stem cells with DMSO for 24 hours.
In one embodiment, the pluripotent stem cells comprise one or more of ESCs, ipscs, embryoid bodies, cellular hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, cord blood stem cells;
preferably, the pluripotent stem cells comprise ESCs and/or ipscs;
preferably, the pluripotent stem cells comprise ipscs;
more preferably, the pluripotent stem cells are ipscs.
In one embodiment, the iPSC may be a commercial cell line or may be induced from donor cells derived from one or more of villous cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic tissue (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, hepatocytes, amnion-derived mesenchymal stem cells, amnion-derived peripheral blood cells, mammary epithelial cells, adipose stem cells, umbilical cord stroma, and placenta.
In one embodiment, the pluripotent stem cells may be human-derived pluripotent stem cells or non-human-derived pluripotent stem cells; the non-human pluripotent stem cells include pluripotent stem cells derived from mammals (e.g., mouse, monkey, cow, sheep, pig, horse, chicken).
Preferably, the embryonic stem cells comprise an E14.1 mouse embryonic stem cell line, an R1/E mouse embryonic stem cell line, an Oct4-Neo mouse embryonic stem cell line, a J1 mouse embryonic stem cell line, a CE3 mouse embryonic stem cell line, a Nanog-GFP mouse embryonic stem cell line, a RW.4 mouse embryonic stem cell line, a R1 mouse embryonic stem cell line, a G-Olig2 mouse embryonic stem cell line, an ESF158 mouse embryonic stem cell line, a EDJ22 mouse embryonic stem cell line, a C57BL/6 mouse embryonic stem cell line, an HN4 human embryonic stem cell line, an H9 human embryonic stem cell line, and an H1 human embryonic stem cell line.
Preferably, the human embryonic stem cells comprise one or more of the HN4, H9 and H1 human embryonic stem cell lines.
Preferably, the human induced pluripotent stem cells comprise a UiPSC-013(UC-013) cell line and/or a UiPS C-015(UC-015) cell line.
In one embodiment, the improvement in differentiation potency refers to an improvement in the ability of pluripotent stem cells to differentiate into terminal cells or their precursor cells; preferably, the improvement of differentiation potency refers to differentiation of more terminal cells from the pluripotent stem cells; preferably, the increased differentiation potency is manifested by cellular detection.
In one embodiment, the cellular detection may be a quantitative detection and/or a qualitative detection.
In one embodiment, the cellular assay may be one or more of a cellular activity assay, an immune-based assay, a flow cytometry assay, a colorimetric assay, a gold nanoparticle-based assay, a fluorescence assay, an ultraviolet assay, a detection of a precursor cell marker of a target terminal cell, and a detection of a target terminal cell marker; preferably, the cellular detection is one or more of fluorescence detection, detection of a precursor cell marker of the terminal cell of interest and detection of a terminal cell marker of interest;
preferably, the precursor cell marker is one or more of MESP2, NKX2.5 and PDGFRA;
preferably, the target terminal cell marker is a cardiomyocyte marker; preferably, the target terminal cellular marker is a cardiomyocyte marker TNNT and/or β -MHC.
In one embodiment, the cell assay may be performed 0 to 50 days after the differentiation-inducing treatment; preferably, the cell assay may be performed after the differentiation-inducing treatment is performed for 0 to 30 days, and the cell assay may be performed after the differentiation-inducing treatment is performed for 3 to 15 days.
In one embodiment, the terminal cell comprises a cardiomyocyte, progenitor cell, organoid, neural stem cell, dopaminergic neuron cell, oligodendrocyte precursor cell, dopaminergic neuron, hepatocyte, kidney-related cell, islet corpus cell, endothelial progenitor cell, pigment epithelial cell, cone cell, skin cell, blood-related cell, mesenchymal hepatocyte;
preferably, the terminal cell is a cardiomyocyte.
In one embodiment, the method may further comprise one or more of a basal culture before differentiation treatment, an induced differentiation treatment after differentiation treatment, and an expansion culture after induced differentiation treatment.
In one embodiment, the dry maintenance culture before the differential differentiation treatment is a culture of pluripotent stem cells using a dry maintenance medium.
In one embodiment, the dry maintenance medium comprises a combination of one or more of: essential 8 TM Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Medium (BME, basic Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM, alpha-minimum Essential Medium), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel Secondary aqueous Medium (Amino Max II complex, Gibco, New erk, USA), Chang's Medium, Meseseult-XF Medium (MCL Technologies, MCMI, Hancadm's Medium), Hadame's Medium, and Hadame's Medium (DMEM, alpha-MEM, alpha-minimum Essential Medium), hepatoZYME-SFM, William's EMedium, Waymouth's Medium or Hepatocyte Culture Medium; preferably, the dry maintenance medium comprises Essential 8 TM Medium; preferably, the dry maintenance medium is Essential 8 TM Medium。
In one embodiment, the dry maintenance medium may be a commercial or self-prepared medium.
Preferably, the dry-maintenance culture of pluripotent stem cells is performed before the differentiation treatment; preferably, the dry maintenance culture of the pluripotent stem cells lasts for 0 to 60 hours before the differentiation treatment of the pluripotent stem cells; preferably 0-48 hours, 0-36 hours, 0-24 hours, 12-24 hours, 24 hours.
Preferably, the dry maintenance culture further comprises detecting cell content; preferably, the step of detecting cells is performed 0 to 5 hours before the differential differentiation treatment; preferably, 0-2 hours.
In one embodiment, the differentiation-induced process after the differentiation process comprises culturing the differentiation-treated cells using a first differentiation medium and/or a second differentiation medium, the first differentiation medium may culture the differentiation-treated cells before or after the second differentiation medium;
preferably, the first differentiation medium cultures the cells after differentiation treatment for 0 to 96 hours; preferably, 0 to 48 hours; preferably, 48 hours.
Preferably, the second differentiation medium cultures the cells for 0-96 hours; preferably, 0 to 48 hours; preferably, 48 hours.
Preferably, the first differentiation medium and the second differentiation medium may contain one or more of an agent promoting differentiation of cardiomyocytes, an agent inhibiting the Wnt signaling pathway, and an agent inhibiting the mTOR signaling pathway.
In one embodiment, the substance that promotes cardiomyocyte differentiation includes one or more of BMP4, basic fibroblast growth factor (bFGF), activin a (activin a), Noggin, Dorsomorphin, 6-bromoindirubin-3'-oxime (6-bromoindirubin-3' -oxime, IO).
In one embodiment, the agent that inhibits the Wnt signaling pathway comprises one or more of Wnt-C59, dickkopf homolog 1(dickkopf homolog 1, KKl), IWP, IWR (inhibitors of Wntresponse).
In one embodiment, the agent that inhibits the mTOR signaling pathway comprises Rapamycin (Rapamycin), everolimus (RAD001), KU-0063794, AZD8055, Temsiriolimus, INK128, Ridaforolimus.
Preferably, the composition of the first differentiation medium comprises at least one of DMEM/F12, 1 XB 27(minus vitamin A) supplement, L-glutamine, 1-thioglycol, L-AA, BMP4 and CHIR99021, the composition of the first differentiation medium comprises DMEM/F12, 1 XB 27(minus vitamin A) supplement, 1% L-glutamine, 400 μ M1-thioglycol, 50 μ g/mL L-AA, 25ng/mL BMP4 and 3 μ M CHIR99021, and the composition of the first differentiation medium is DMEM/F12, 1 XB 27(minus vitamin A) supplement, 1% L-glutamine, 400 μ M1-thioglycol, 50 μ g/mL BMP-AA, 25ng/mL BMP 4/mL BMP 863 μ M.
Preferably, the composition of the second differentiation medium comprises one or more of RPMI 1640, 1 XB 27(minus insulin) supplement, L-glutamine, BMP4, Wnt-C59; preferably, the composition of the second differentiation medium comprises RPMI 1640, 1 XB 27(minus insulin) supplement, 1% L-glutamine, 10ng/ml BMP4, 2. mu.M Wnt-C59; preferably, the composition of the second differentiation medium is RPMI 1640, 1 XB 27(minus insulin) supplement, 1% L-glutamine, 10ng/ml BMP4, 2. mu.M Wnt-C59.
In one embodiment, the first and second differentiation media may be purchased commercial media or self-prepared media.
In one embodiment, the expansion culture after the differentiation-inducing treatment is a culture of cells using an expansion medium.
In one embodiment, the expansion medium is a medium suitable for terminal cell expansion; preferably, the expansion medium is a medium suitable for expansion of cardiomyocytes.
Preferably, the components of the amplification medium include RPMI 1640, 1 XB 27supplement, L-glutamine; preferably, the components of the amplification medium include RPMI 1640, 1 XB 27supplement, 1% L-glutamine.
In one embodiment, the amplification medium may be a commercial medium purchased or self-prepared.
Cells
In another aspect, the invention provides a cell that has been subjected to differential differentiation.
In one embodiment, the cell is a cardiomyocyte.
In another aspect, the present invention provides compositions and agents consisting of cells subjected to differential differentiation.
In one embodiment, the composition may also include other cell culture related media, growth factors, or additives.
In one embodiment, the medicament may further comprise one or more of a pharmaceutically acceptable carrier, excipient, and pharmaceutically active agent.
In another aspect, the invention provides the use of the aforementioned cells, agents and compositions in the preparation of a medicament for cell transplantation therapy; preferably, the medicament can treat heart diseases.
Culture medium
In another aspect, the present invention provides a medium for improving differentiation potency of pluripotent stem cells, the medium comprising E6 medium and/or differentiation medium supplemented with DMSO; preferably, the medium is E6 medium and/or a differential processing medium supplemented with DMSO.
Culture medium combination
In another aspect, the present invention provides a culture medium composition for improving differentiation potency of pluripotent stem cells, the culture medium composition comprising the aforementioned E6 culture medium and/or a differentiation-treated culture medium supplemented with DMSO; preferably, the culture medium combination further comprises one or more of the aforementioned dry maintenance medium, first differentiation medium, second differentiation medium and amplification medium.
Cell culture system
In another aspect, the present invention provides a cell culture system for improving differentiation efficiency of pluripotent stem cells, the cell culture system using reagents required for differentiation processing, the cell culture system comprising a unit for performing differentiation processing on cells; preferably, the cell culture system further comprises a dry maintenance culture unit and/or a differentiation-inducing unit; preferably, the system may further comprise a unit for monitoring the state of the cells and/or a unit for performing separate washing of the cells.
In one embodiment, the cell culture system comprises a unit for subjecting cells to differential processing; preferably, the cell culture system further comprises one or more of a dry maintenance culture unit, a differentiation induction unit, and an expansion culture unit; preferably, the system may further comprise a unit for monitoring the state of the cells and/or a unit for performing separate washing of the cells.
Reagent kit
In another aspect, the present invention also provides a kit for improving differentiation potency of pluripotent stem cells, the kit comprising reagents required for differential differentiation treatment; preferably, the reagents required for the differential differentiation treatment include an E6 medium and/or a differential differentiation treatment medium to which DMSO is added; more preferably, the reagent required for the differentiation treatment is an E6 medium and/or a differentiation treatment medium to which DMSO is added.
In one embodiment, the kit further comprises the instruments and/or devices required for the differential differentiation process of the cells.
Applications of
On the other hand, the invention also provides application of the DMSO, the E6 culture medium or the differentiation treatment culture medium added with the DMSO in preparation of a culture medium, a culture medium combination, a cell culture system or a kit for improving the differentiation efficiency of the pluripotent stem cells.
On the other hand, the invention also provides application of the DMSO, the differential differentiation treatment culture medium, the E6 culture medium, the culture medium combination, the cell culture system or the kit in improving the differentiation efficiency of the pluripotent stem cells.
On the other hand, the invention also provides a culture medium for improving the differentiation efficiency of the pluripotent stem cells, a culture medium combination for improving the differentiation efficiency of the pluripotent stem cells, a cell culture system for improving the differentiation efficiency of the pluripotent stem cells or an application of a kit for improving the differentiation efficiency of the pluripotent stem cells in preparing the cells.
In another aspect, the invention also provides the use of a cell, culture medium combination, cell culture system or kit as described above, in the preparation of a cell, medicament or composition for cell transplantation therapy; preferably, for the preparation of cells, medicaments or compositions for cell transplantation in the treatment of heart diseases.
On the other hand, the present invention also provides the use of DMSO, a differentiation-treated medium or E6 medium to which DMSO is added, the combination of the above-mentioned media, the above-mentioned cell culture system, or the above-mentioned kit for preparing cells subjected to differentiation treatment;
preferably, the cells are cells for use in cell transplantation therapy for diseases; more preferably, the cells are cells for use in cell transplantation for the treatment of a cardiac disorder; more preferably, the cells are cardiomyocytes for cell transplantation in the treatment of cardiac disorders.
In another aspect, the invention also provides the use of cells subjected to differential differentiation in the treatment of diseases by cell transplantation; preferably, the use of said cells in cell transplantation for the treatment of heart diseases.
General definition:
unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Culture medium
The culture medium of the present invention includes, but is not limited to, Essential 8 TM Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Medium (BME, basic Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel Secondary amniotic fluid Medium (Aminomax II complex, Gibco, Newrk, USA), Chang's Medium, Meseseult-XF Medium (MCELL Technologies, MCMI, Hancadk's Medium, Hadam's Medium, Hadame's Medium, and Hadame's Medium (DMEM, alpha-MEM, alpha-minimum Essential Medium), G-minimum Essential Medium (G-MEM, alpha-minimum Essential Medium), G-M, G-minimum Essential Medium (G-MEM, G-M, Iscoyo's Modified Eagle's Medium, M, Iscoz's Medium, Iscoz, Ishoech, Ishogel, Isho, Hepatozyme-SFM, William's EMedium, Waymouth's Medium, or Heapatocyte Culture Medium.
The culture medium of the present invention may further comprise a cytokine required for cell growth, a growth factor, and a small molecule compound selected from the group consisting of TGF- β signaling pathway, an appearance modifier, a calcium channel agonist, and a metabolic pathway regulator, wherein the cytokine and protein polypeptides include fibroblast growth factor 1, fibroblast growth factor 2, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor 1, vascular endothelial growth factor, placental growth factor, leukemia inhibitory factor, stem cell factor, transferrin, and human serum albumin.
The medium of the present invention may further comprise a Nutrient Mixture (Nutrient mix) which is a Mixture comprising various amino acids, vitamins, inorganic salts, etc. generally used for cell culture, and the Nutrient Mixture can be prepared by mixing the above amino acids, vitamins, inorganic salts, etc.; alternatively, commercially prepared nutrient mixtures may be used.
The vitamins include biotin, choline chloride, calcium D-pantothenate, folic acid, inositol, niacinamide, pyridoxine hydrochloride, riboflavin, ammonium rockii hydrochloride, coenzyme Q10, vitamin B12, putrescine dihydrochloride, vitamin C, and vitamin E;
the salts comprise sodium bicarbonate, calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate and sodium pyruvate;
commercially prepared nutrient mixtures include one or more of M199, MCDB110, MCDB202, MCDB 302.
Pluripotent stem cells
Pluripotent stem cells (psc) are cells that have the potential to differentiate into multiple cell tissues, but lose the ability to develop into an intact individual, with some limitations on developmental potential. Such stem cells have the potential to differentiate into multiple cell tissues, but lose the ability to develop an intact individual. For example: embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, and the like.
The pluripotent stem cells also include embryonic stem cells or embryoid bodies formed by inducing the pluripotent stem cells under certain culture conditions in vitro.
Pluripotent stem cells in the present invention also include embryonic stem cells or ectodermal, mesodermal and endodermal progenitor cells derived from pluripotent stem cells under certain in vitro culture conditions.
ESC (embryonic stem cells, ES cells) embryonic stem cells
When the embryo develops into a blastocyst, its stem cells have the ability to differentiate into various cell types, and are pluripotent stem cells. Embryonic stem cells can participate in the development potential of various tissues including gonads under the condition of releasing differentiation inhibition, but cannot develop into an individual independently. It can develop into cell tissues of ectoderm, mesoderm and endoderm.
iPSC (induced pluripotent stem cells, iPS, iPSCs) induces pluripotent stem cells
The induced pluripotent stem cells are obtained by reprogramming somatic cells through the introduction of transcription factors (Oct4, Sox2, Klf4 and c-Myc) and have the differentiation capacity similar to that of embryonic stem cells, become important cell sources for researching human disease pathogenesis and tissue cell replacement therapy, do not have ethical problems, and have wide application prospects in the field of medical treatment. iPSCs are used as source cells, can be amplified in vitro and induced to differentiate into specific tissue cells, and various somatic cells and different tissues such as cardiac muscle, nerve, pancreas, bone and the like are successfully cultured and differentiated by applying the iPSCs.
Differentiation
Differentiation is a process by which non-specialized ("uncommitted") or less specialized cells acquire the characteristics of a specialized cell (e.g., cardiomyocyte). Differentiated or differentiation-induced cells are cells that have a more specialized ("committed") location in the cell lineage.
Maintenance of
Maintenance generally refers to cells placed in a growth medium under conditions conducive to cell growth and/or division, which may or may not result in a larger population of cells.
Passage of culture
Passaging refers to the process of removing cells from one culture vessel and placing them in a second culture vessel under conditions conducive to cell growth and/or division.
Cell transplantation therapy
Cell transplantation therapy is the transplantation of healthy cells into a patient's body to repair or replace damaged cells or tissues, thereby achieving the goal of healing.
Precursor cell
A class of adult cells that can only differentiate towards a specific terminal, with a more limited proliferative capacity than progenitor cells. The cardiac muscle precursor cell is a cell with cardiac muscle specific differentiation ability and self proliferation ability
Drawings
FIG. 1 is a morphological diagram of myocardial differentiation observed under a microscope on days 6 to 15 in cells differentiated by E6 medium.
FIG. 2 is a graph showing the results of immunofluorescence assay of cells differentiated by E6 medium.
FIG. 3 is a diagram showing the morphology of myocardial differentiation observed in the microscope on days 6 to 15 in the cells subjected to differential differentiation treatment with DMSO.
FIG. 4 is a graph showing the results of immunofluorescence assay of cells differentiated using DMSO.
FIG. 5 is a graph showing the results of the expression levels of cardiomyocyte precursor marker genes MESP2, NKX2.5 and PDGFRA in cells by qPCR method under different treatment modes.
FIG. 6 is a graph showing the results of the expression levels of the cardiomyocyte marker genes TNNT and β -MHC in the cells measured by qPCR.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 Induction of differentiation of cardiomyocytes
The detailed information of the reagents used in this example is as follows:
1. prophase preparation-dry maintenance culture
Day-2: spreading iPSC to matrigel-coated 12-well culture plate with density of 5w/cm 2 Before placing in an incubator, shaking up and down and left and right respectively for 5 times to uniformly lay the cells, and using Essential 8 TM Medium Medium (Saimenfeshiel, A1517001) with 10. mu. M Y-276322 HCl.
2. Differentiation process
Day-1:
control group: continued use of Essential 8 TM Culturing Medium;
e6 experimental group: mixing the Essential 8 TM Medium Medium was changed to Essential 6 TM Medium (purchased from Saimei Feishale, cat # A1516401) Medium, cultured for 24 hours;
DMSO experimental group: mixing the Essential 8 TM Medium Medium was changed to Essential 8 containing 2% dimethyl sulfoxide (DMSO, from Sigma, cat # D2650) TM Medium, cultured for 24 hours.
3. Differentiation experiment-induced differentiation
Day 0-2: replaced with mesoderm differentiation medium (first differentiation medium), the main components included: DMEM/F12, 1 XB 27(minus vitamine A) supplement, 1% L-glutamine, 400 μ M1-thioglycol, 50 μ g/mL L-AA, 25ng/mL BMP4, 3 μ M CHIR 99021;
on days 3-5, the medium was changed to cardiomyocyte differentiation medium (second differentiation medium) and the main ingredients included: RPMI 1640, 1 XB 27(minus insulin) supplement, 1% L-glutamine, 10ng/ml BMP4, 2. mu.M Wnt-C59.
4. Amplification culture
On days 6-14, the culture medium was changed to cardiomyocyte expansion medium, and the main ingredients included: RPMI 1640, 1 XB 27supplement, 1% L-glutamine.
5. Detection of
Cells of the E6 experimental group:
and (3) observing under a microscope: during the differentiation experiment, cell morphology differentiated at days 6, 8, 10, 12, 14, 15 was recorded, observed at days 6-15, and shown in figure 1: during the process of myocardial differentiation, cell network formation began on days 6-8, cells began to beat on day 8, and a very distinct myocardial fiber network was formed on day 10, indicating that cardiomyocytes could be differentiated by the above method.
Fluorescence detection: cTNT is a marker gene of cardiomyocytes, and immunofluorescence detection of myocardium matured on day 15 demonstrated that cTNT is expressed in cardiomyocytes derived from iPSC, indicating that cardiomyocytes can be differentiated by the above method, and the results are shown in fig. 2, where light gray is the site emitting fluorescence.
DMSO experimental group:
and (3) observing under a microscope: during the differentiation experiment, cells were observed on days 6-15, and cell morphology was recorded on days 6, 8, 10, 12, 14, 15 as shown in FIG. 3: during the process of myocardial differentiation, cell networks began to form on days 6-8, cells began to jump on day 8, and a very distinct myocardial fiber network was formed on day 10, indicating that cardiomyocytes could be differentiated by the above method.
Fluorescence detection: cTNT is a marker gene of cardiomyocytes, and immunofluorescence detection of myocardium matured on day 15 demonstrated that cTNT is expressed in cardiomyocytes derived from iPSC, indicating that cardiomyocytes can be differentiated by the above method, and the results are shown in fig. 4, where light gray is the site emitting fluorescence.
qPCR detection of cardiac muscle precursor cell marker genes: on the third day in the cardiac muscle differentiation process, the cardiac muscle precursor cell marker genes MESP2, NKX2.5 and PDGFRA were detected by qPCR method, and the expression of the cardiac muscle precursor cell marker genes in the cells cultured in E6 medium or treated with DMSO was significantly improved compared with iPSC cells and cells not subjected to differentiation treatment, which indicates that the differentiation treatment can improve the iPSC differentiation efficiency, and the qPCR detection result is shown in FIG. 5.
qPCR detection of cardiac myocyte marker genes: on the 15 th day in the myocardial differentiation process, myocardial cell marker genes TNNT and beta-MHC are detected by a qPCR method, and the fact that the expression of the myocardial cell marker genes such as TNNT and beta-MHC is remarkably improved in cells cultured by an E6 culture medium or treated by DMSO compared with iPSC cells and cells not subjected to differential treatment is found, and the differential treatment can improve the iPSC differentiation efficiency is shown, and the qPCR detection result is shown in FIG. 6.
The detection results show that differentiation treatment can effectively improve the differentiation efficiency of the pluripotent stem cells, particularly the efficiency of differentiation of iPSC into cardiac muscle precursor cells and cardiac muscle cells.
Claims (11)
1. A method for improving the differentiation potency of iPSC to cardiac muscle precursor cells, which comprises differentiating iPSC before inducing differentiation, and culturing the differentiated iPSC cells by using a first differentiation medium;
the differential treatment included treatment of the iPSC with E6 medium or use of Essential 8 with 2% DMSO TM Medium treatment iPSC, the differential differentiation treatment being performed for 24 hours;
the first differentiation medium was composed of DMEM/F12, 1 XB 27 supplement-minus nitamin A, 1% L-glutamine, 400. mu.M 1-thioglycol, 50. mu.g/mL L-AA, 25ng/mL BMP4, and 3. mu.M CHIR99021, and the culture time using the first differentiation medium lasted for 2 days.
2. The method of claim 1, wherein the E6 medium is a commercial medium sold under the trade designation a1516401 by seimer feishel.
3. The method of claim 1, wherein the ipscs are commercial cell lines or are induced from donor cells.
4. The method of claim 1, further comprising a dry maintenance culture before differential differentiation, wherein the dry maintenance culture uses a dry maintenance medium, and wherein the dry maintenance medium is Essential 8 TM Medium。
5. The method of claim 1, wherein the effectiveness of differentiation is demonstrated by cellular assays comprising cellular activity assays, immuno-based assays, flow cytometry assays, colorimetric assays, gold nanoparticle-based assays, fluorescence assays, ultraviolet assays for cardiac precursor cell marker genes.
6. The method of claim 5, wherein the cellular detection comprises quantitative detection and/or qualitative detection.
7. The method of claim 5, wherein the cellular assay is a fluorescent assay for a cardiac muscle precursor cell marker gene that is one or more of MESP2, NKX2.5, and PDGFRA.
8. A cell culture system for improving the differentiation efficiency of iPSC into myocardial precursor cells, which comprises a differentiation processing unit and a differentiation induction processing unit, wherein the differentiation processing unit comprises an E6 culture medium and/or Essential 8 containing 2% DMSO TM Medium processing of iPSC, the induction scoreThe differentiation processing unit comprises culturing the cells using a first differentiation medium whose composition is DMEM/F12, 1 XB 27 supplement-minus nitamin A, 1% L-glutamine, 400. mu.M 1-thioglycol, 50. mu.g/mL L-AA, 25ng/mL BMP4, 3. mu.M CHIR 99021.
9. The cell culture system of claim 8, further comprising a dry maintenance culture unit comprising culturing cells using a dry maintenance medium that is Essential 8 TM Medium。
10. Use of the cell culture system of claim 8 or 9 to increase the potency of ipscs to differentiate into cardiomyocyte precursor cells.
11. Use of the cell culture system of claim 8 or 9 for high potency induction of iPSC differentiation into cardiomyocyte precursor cells.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110612291.XA CN113337459B (en) | 2021-06-02 | 2021-06-02 | Method for improving differentiation efficiency of pluripotent stem cells |
| PCT/CN2022/094433 WO2022253026A1 (en) | 2021-06-02 | 2022-05-23 | Method for improving differentiation efficacy of pluripotent stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110612291.XA CN113337459B (en) | 2021-06-02 | 2021-06-02 | Method for improving differentiation efficiency of pluripotent stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113337459A CN113337459A (en) | 2021-09-03 |
| CN113337459B true CN113337459B (en) | 2022-09-06 |
Family
ID=77474648
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110612291.XA Active CN113337459B (en) | 2021-06-02 | 2021-06-02 | Method for improving differentiation efficiency of pluripotent stem cells |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113337459B (en) |
| WO (1) | WO2022253026A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337459B (en) * | 2021-06-02 | 2022-09-06 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Method for improving differentiation efficiency of pluripotent stem cells |
| CN114561356B (en) * | 2022-03-09 | 2022-09-13 | 北京呈诺医学科技有限公司 | Method for influencing cytokine secretion and application |
| CN116590219A (en) * | 2022-09-19 | 2023-08-15 | 苏州南医大创新中心 | Stem cell-derived gastrulation-like model, construction method and application thereof |
| CN116042516A (en) * | 2023-02-22 | 2023-05-02 | 深圳市寰宇生物科技有限公司 | Myocardial cell pellet differentiated from human induced pluripotent stem cells, and preparation method and application thereof |
| CN116445408B (en) * | 2023-05-22 | 2024-02-02 | 呈诺再生医学科技(北京)有限公司 | Use of LSD1 inhibitors to promote iPSC differentiation to HSCs and maintenance of HSC dryness |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037669A (en) * | 2006-03-13 | 2007-09-19 | 上海交通大学医学院附属新华医院 | Method for inducing liver cell from human embryonic stem cells |
| CN105849253A (en) * | 2013-09-20 | 2016-08-10 | 修复股份有限公司 | A method to direct differentiation of pluripotent stem cells into functional heart muscle |
| CN108384755A (en) * | 2018-02-08 | 2018-08-10 | 北京呈诺医学科技有限公司 | A method of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013295940B2 (en) * | 2012-07-23 | 2019-01-31 | Institute Of Biophysics, Chinese Academy Of Sciences | Method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro |
| KR102015815B1 (en) * | 2016-08-10 | 2019-08-29 | 가톨릭대학교 산학협력단 | Method for Culturing Cornea Epithealial Cell by Inducing Differentiation of Induced Pluripotent Stem Cell and System for the Same |
| EP3770250A4 (en) * | 2018-03-22 | 2021-11-24 | Oriental Yeast Co., Ltd. | Differentiation promoter for pluripotent stem cells |
| CN112359012B (en) * | 2020-10-22 | 2022-11-25 | 中国科学院水生生物研究所 | Method for preparing heart valve endothelial cells by inducing differentiation of pluripotent stem cells and application thereof |
| CN112831461B (en) * | 2021-02-26 | 2023-08-08 | 澳门大学 | Method and medicine for inducing stem cells to differentiate into mesoderm lineage or trophoblast lineage |
| CN113337459B (en) * | 2021-06-02 | 2022-09-06 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Method for improving differentiation efficiency of pluripotent stem cells |
-
2021
- 2021-06-02 CN CN202110612291.XA patent/CN113337459B/en active Active
-
2022
- 2022-05-23 WO PCT/CN2022/094433 patent/WO2022253026A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037669A (en) * | 2006-03-13 | 2007-09-19 | 上海交通大学医学院附属新华医院 | Method for inducing liver cell from human embryonic stem cells |
| CN105849253A (en) * | 2013-09-20 | 2016-08-10 | 修复股份有限公司 | A method to direct differentiation of pluripotent stem cells into functional heart muscle |
| CN108384755A (en) * | 2018-02-08 | 2018-08-10 | 北京呈诺医学科技有限公司 | A method of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113337459A (en) | 2021-09-03 |
| WO2022253026A1 (en) | 2022-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113337459B (en) | Method for improving differentiation efficiency of pluripotent stem cells | |
| JP5227318B2 (en) | Cell growth medium | |
| KR100973453B1 (en) | Chondrocyte precursors derived from human embryonic stem cells | |
| JP2012523240A (en) | Methods and compositions for stem cell culture | |
| Bartakova et al. | Regenerative cell therapy for corneal endothelium | |
| JP2016514481A (en) | Methods and compositions for culturing endoderm progenitor cells in suspension | |
| US10724000B2 (en) | Small molecule based conversion of somatic cells into neural crest cells | |
| US20230125741A1 (en) | Media and methods for producing mesenchymal stem cells | |
| JP2023078353A (en) | Medium for direct differentiation of mesenchymal stem cells derived from embryonic stem cells, method for producing mesenchymal stem cells using the same, and mesenchymal stem cells produced thereby | |
| CN113337458A (en) | Method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells | |
| KR102141641B1 (en) | Method for differentiation of dermal papilla cells from human adipose mesenchymal stem cells | |
| EP3950933A1 (en) | Cell population including pluripotent stem cells and production method thereof | |
| WO2023221438A1 (en) | Method for promoting differentiation of ipsc into islet cell by means of regulating ngn3 expression | |
| WO2023011251A1 (en) | Rapid and efficient clinical grade pigment epithelium induction method, kit, and application | |
| CN115975914A (en) | Method for inducing pluripotent stem cells by reprogramming of chemical small molecule drugs | |
| EP4257678A1 (en) | Production method for producing pluripotent stem cell population | |
| US11788062B2 (en) | Functional feline pancreatic cells from adipose tissue | |
| JP2011152111A (en) | Culture medium for culturing pluripotent stem cell | |
| US20230235279A1 (en) | Amniotic-like epithelial cell generation | |
| WO2005066330A1 (en) | A system for improving acceptance of tissue grafts made from embryonic stem cells | |
| US20220340871A1 (en) | Method for obtaining or maintaining abcg2-positive corneal limbal stem cells | |
| JPWO2017188082A1 (en) | Medium additive | |
| JP2024024550A (en) | Cell death inducer for undifferentiated stem cells and method for purifying differentiated cells | |
| CA2555021A1 (en) | Medium for preparing feeder cells for embryonic stem cells and feeder cells | |
| CN116769695A (en) | Culture medium and method for producing human cells and tissues from teratomas, organoids and embryoid bodies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |