CN117327643A - Method for obtaining definitive endoderm cells from human pluripotent stem cells by using YAP inhibitor - Google Patents
Method for obtaining definitive endoderm cells from human pluripotent stem cells by using YAP inhibitor Download PDFInfo
- Publication number
- CN117327643A CN117327643A CN202311127162.7A CN202311127162A CN117327643A CN 117327643 A CN117327643 A CN 117327643A CN 202311127162 A CN202311127162 A CN 202311127162A CN 117327643 A CN117327643 A CN 117327643A
- Authority
- CN
- China
- Prior art keywords
- stem cells
- cells
- pluripotent stem
- definitive endoderm
- medium
- 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.)
- Pending
Links
- 210000004039 endoderm cell Anatomy 0.000 title claims abstract description 54
- 210000001778 pluripotent stem cell Anatomy 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000003112 inhibitor Substances 0.000 title claims abstract description 26
- 102100023267 YY1-associated protein 1 Human genes 0.000 title claims abstract description 23
- 230000004069 differentiation Effects 0.000 claims abstract description 59
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- 238000012258 culturing Methods 0.000 claims abstract description 17
- 239000007640 basal medium Substances 0.000 claims abstract description 6
- 241000124008 Mammalia Species 0.000 claims abstract description 4
- ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N verteporfin Chemical compound C=1C([C@@]2([C@H](C(=O)OC)C(=CC=C22)C(=O)OC)C)=NC2=CC(C(=C2C=C)C)=NC2=CC(C(=C2CCC(O)=O)C)=NC2=CC2=NC=1C(C)=C2CCC(=O)OC ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N 0.000 claims description 48
- 229960003895 verteporfin Drugs 0.000 claims description 48
- 239000002609 medium Substances 0.000 claims description 21
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 19
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 16
- 229930006000 Sucrose Natural products 0.000 claims description 16
- 239000005720 sucrose Substances 0.000 claims description 16
- 108010082117 matrigel Proteins 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 108010023082 activin A Proteins 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 230000029087 digestion Effects 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 4
- 102000001267 GSK3 Human genes 0.000 claims description 3
- 108060006662 GSK3 Proteins 0.000 claims description 3
- NFVJNJQRWPQVOA-UHFFFAOYSA-N n-[2-chloro-5-(trifluoromethyl)phenyl]-2-[3-(4-ethyl-5-ethylsulfanyl-1,2,4-triazol-3-yl)piperidin-1-yl]acetamide Chemical compound CCN1C(SCC)=NN=C1C1CN(CC(=O)NC=2C(=CC=C(C=2)C(F)(F)F)Cl)CCC1 NFVJNJQRWPQVOA-UHFFFAOYSA-N 0.000 claims description 3
- -1 B27 Proteins 0.000 claims description 2
- 210000001900 endoderm Anatomy 0.000 abstract description 13
- 238000011161 development Methods 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 5
- 238000000338 in vitro Methods 0.000 abstract description 4
- 230000001172 regenerating effect Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 58
- 230000000052 comparative effect Effects 0.000 description 29
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 9
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000003550 marker Substances 0.000 description 9
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 8
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 101001062347 Homo sapiens Hepatocyte nuclear factor 3-beta Proteins 0.000 description 7
- 238000010166 immunofluorescence Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 102100029284 Hepatocyte nuclear factor 3-beta Human genes 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 230000024245 cell differentiation Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 101000652324 Homo sapiens Transcription factor SOX-17 Proteins 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 102100030243 Transcription factor SOX-17 Human genes 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 239000013630 prepared media Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- OBKXEAXTFZPCHS-UHFFFAOYSA-N 4-phenylbutyric acid Chemical compound OC(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-N 0.000 description 2
- RTRQQBHATOEIAF-UHFFFAOYSA-N AICA riboside Natural products NC1=C(C(=O)N)N=CN1C1C(O)C(O)C(CO)O1 RTRQQBHATOEIAF-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091092584 GDNA Proteins 0.000 description 2
- 102000003964 Histone deacetylase Human genes 0.000 description 2
- 108090000353 Histone deacetylase Proteins 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 108700038175 YAP-Signaling Proteins Proteins 0.000 description 2
- RTRQQBHATOEIAF-UUOKFMHZSA-N acadesine Chemical compound NC1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RTRQQBHATOEIAF-UUOKFMHZSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- INVTYAOGFAGBOE-UHFFFAOYSA-N entinostat Chemical compound NC1=CC=CC=C1NC(=O)C(C=C1)=CC=C1CNC(=O)OCC1=CC=CN=C1 INVTYAOGFAGBOE-UHFFFAOYSA-N 0.000 description 2
- 229950005837 entinostat Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000002220 organoid Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229950009215 phenylbutanoic acid Drugs 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- AEQFSUDEHCCHBT-UHFFFAOYSA-M sodium valproate Chemical compound [Na+].CCCC(C([O-])=O)CCC AEQFSUDEHCCHBT-UHFFFAOYSA-M 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229940102566 valproate Drugs 0.000 description 2
- 108010059616 Activins Proteins 0.000 description 1
- 102000005606 Activins Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 208000012239 Developmental disease Diseases 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 101001060274 Homo sapiens Fibroblast growth factor 4 Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- RTKIYFITIVXBLE-UHFFFAOYSA-N Trichostatin A Natural products ONC(=O)C=CC(C)=CC(C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102000013814 Wnt Human genes 0.000 description 1
- 108050003627 Wnt Proteins 0.000 description 1
- 102000044880 Wnt3A Human genes 0.000 description 1
- 108700013515 Wnt3A Proteins 0.000 description 1
- 229940126697 YAP-TEAD PPI inhibitor Drugs 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002247 constant time method Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- RTKIYFITIVXBLE-QEQCGCAPSA-N trichostatin A Chemical compound ONC(=O)/C=C/C(/C)=C/[C@@H](C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-QEQCGCAPSA-N 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Reproductive Health (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for obtaining definitive endoderm cells by differentiating human pluripotent stem cells by using YAP inhibitor, wherein the culture medium comprises the following components: YAP inhibitor is added on the basal medium. The method comprises the following steps: the culture medium is used for culturing the mammalian pluripotent stem cells so as to differentiate into definitive endoderm cells. According to the invention, YAP inhibitor is added into the basic culture medium to improve the differentiation efficiency of the pluripotent stem cells of the mammal into definitive endoderm cells; the culture medium provided by the invention has the advantages of low cost and stable performance, and can efficiently differentiate human pluripotent stem cells into definitive endoderm cells. This provides a low cost and high efficiency research system for in vitro research of endoderm related development problems. And simultaneously, the application cost of the regenerative medicine is reduced.
Description
Technical Field
The invention relates to the field of stem cells and regenerative medicine, in particular to a method for obtaining definitive endoderm cells by differentiating human pluripotent stem cells by using YAP inhibitors.
Background
Embryonic stem cells (embryonic stem cell, ESC) and induced pluripotent stem cells (induced pluripotent stem cell, iPSC) with multipotency are a group of specialized cells that are capable of developing into all types of cells in the fetus and self-renew almost indefinitely. The research on the pluripotent stem cells not only can reveal the process of human development, fills the current knowledge blank of early development of human embryo, and searches for signal molecules or signal stimuli playing an important role in the development process. Meanwhile, the multipotent stem cells can also form similar tissues and organoids to in-vivo organs in an in-vitro differentiation mode, and the process can be used as a model of developmental diseases, and a mechanism in the occurrence and development processes of the diseases is revealed so as to realize the effective and disciplined treatment; can also be used as a donor source of regenerative medicine to alleviate the dilemma of donor shortage in organ transplantation at present.
Pluripotent stem cells can differentiate towards the endo-, meso-, ectotricodeermal lineages, where the endodermal layers can subsequently develop into the digestive system of the liver, stomach, pancreas, intestine, etc. In vitro, liver organoids obtained by utilizing definitive endoderm cell differentiation can effectively simulate the disease development process of fatty liver and drug liver injury, and provide a new treatment idea for liver diseases; the islet-like organ obtained by utilizing the definitive endoderm cell differentiation can effectively control the blood sugar level of diabetics after transplantation, and is hopeful to solve the problem of serious shortage of donors in islet transplantation.
In view of the above, research on definitive endoderm cells has extremely high scientific and commercial values, and therefore, it is necessary to develop a culture medium and a culture method capable of obtaining definitive endoderm cells more rapidly and efficiently.
Disclosure of Invention
The invention aims to provide a culture medium and a method for obtaining definitive endoderm cells by differentiating human pluripotent stem cells by using YAP inhibitors, and the differentiation efficiency of differentiating and culturing the mammalian pluripotent stem cells into the definitive endoderm cells is improved by adding Verteporfin into a basic culture medium.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention there is provided the use of the YAP inhibitor Verteporfin, said use comprising: the YAP inhibitor is added to improve the differentiation efficiency of the mammalian pluripotent stem cells into definitive endoderm cells.
In a second aspect of the present invention, there is provided a method for increasing the differentiation efficiency of mammalian pluripotent stem cells into definitive endoderm cells by differentiation culture using YAP inhibitors, the method comprising: mammalian pluripotent stem cells were cultured using Verteporfin as YAP inhibitor.
In a third aspect of the present invention, there is provided a medium for increasing differentiation efficiency of a mammalian pluripotent stem cell differentiated into definitive endoderm cells using YAP inhibition, the medium comprising the components of: YAP inhibitor is added on the basal medium.
Further, the components of the culture medium include: 1-100 ng/ml of Activin A, B27, bovine serum albumin, diabody and Verteporfin are added on a basic culture medium.
Further, the components of the culture medium include: adding 1-100 ng/ml of Activin A, 0.5-1.5% of B27, 0.1-0.3% of bovine serum albumin, 0.5-1.5% of diabody and one of the following compounds to a basal medium:
compound a: 0.1-0.5. Mu.M Verteporfin;
compound B: 0.5. Mu.M Verteporfin+2% sucrose.
Further, the components of the culture medium further comprise: 2-3 mu M GSK3 inhibitor. In particular, the GSK3 inhibitor may be CHIR99021 or a Wnt signaling activator, such as Wnt3A, for example.
In other embodiments, the medium may further comprise a Histone Deacetylase (HDAC) inhibitor, e.g., trichostatin a, sodium butyrate (NaB), entinostat (Entinostat), phenyl Butyrate (PB), panobinstat (Panobinstat), or valproate (valproate).
In other embodiments, the differentiation medium may further comprise one or more growth factors, such as FGF1, FGF2, and FGF4, and/or serum, such as FBS or FCS.
In other embodiments, the differentiation medium may further comprise a PI3K (phosphoinositide 3-kinase) inhibitor, such as LY294002.
Further, the types of mammalian pluripotent stem cells include: mammalian embryonic stem cells or mammalian induced pluripotent stem cells.
Specifically, the mammalian pluripotent stem cells comprise: human pluripotent stem cells, primate pluripotent stem cells, mouse pluripotent stem cells, rat pluripotent stem cells, canine pluripotent stem cells, feline pluripotent stem cells, porcine pluripotent stem cells, bovine pluripotent stem cells, and equine pluripotent stem cells.
In a fourth aspect of the present invention, there is provided a method of culturing a mammalian pluripotent stem cell to a definitive endoderm cell, the method comprising: the culture medium is used for culturing the mammalian pluripotent stem cells so as to differentiate into definitive endoderm cells.
Further, the method comprises:
culturing the pluripotent stem cells of the mammal by using a growth medium for 3-6 days, then performing digestion counting, inoculating the pluripotent stem cells into an orifice plate coated with matrigel, and culturing by using a normal growth medium;
and culturing the cells after the density of 75-85% is reached by changing the culture medium to obtain the definitive endoderm cells.
In the above technical scheme, the growth medium may be mtesr+1% diabody, and the matrigel is 1: the 100 dilution ratio is coated in the plate holes, and the addition ratio can be properly adjusted in other embodiments;
in the above technical scheme, for a 24-well plate, the digestion count is followed by 0.8X10 5 Density per well cells were seeded into a 24-well plate coated with matrigel; in other embodiments, the number of cells seeded is adjusted accordingly for different dishes.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
according to the culture medium and the culture method for improving the differentiation efficiency of the pluripotent mammalian stem cells into the definitive endoderm cells by utilizing the YAP inhibitor, the Verteporfin is added into the basic culture medium to improve the differentiation efficiency of the pluripotent mammalian stem cells into the definitive endoderm cells; specifically:
(1) At least one of Verteporfin is added into the culture medium, so that the differentiation efficiency of the pluripotent stem cells can be higher at day 3, and the differentiation time can be shortened.
(2) The culture medium provided by the invention has the advantages of low cost and stable performance, and can efficiently differentiate human pluripotent stem cells into definitive endoderm cells. This provides a low cost and high efficiency research system for in vitro research of endoderm related development problems. Meanwhile, the application cost of regenerative medicine is reduced, so that the research and application of cell therapy are greatly promoted, and the method has a good application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart showing the effect of Verteporfin on the positive rate of differentiation of human embryonic stem cells toward endoderm for comparison of example 1 and comparative example 1;
FIG. 2 shows immunofluorescence and statistical results of the effect of Verteporfin and Verteporfin+2% sucrose combination on the expression of human embryonic stem cells toward endodermal differentiation marker genes in comparison of example 2, example 3 and comparative example 2;
FIG. 3 is a flow chart showing the effect of Verteporfin and Verteporfin+2% sucrose in combination on expression of human embryonic stem cells toward endodermal differentiation marker genes, as shown in comparison of example 2, example 3 and comparative example 2;
FIG. 4 is a quantitative PCR result showing the effect of Verteporfin and Verteporfin+2% sucrose in combination on the expression of human embryonic stem cells toward endodermal differentiation marker genes, as shown in comparison of example 2, example 3 and comparative example 2;
FIG. 5 is a flow chart of a method for culturing definitive endoderm cells from human pluripotent stem cells by YAP inhibitor differentiation according to an embodiment of the invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.
The terms in the present invention are explained as follows:
YAP is known as Yes-associated protein (Yes-associated protein, YAP);
verteporfin (CL 318952, BPD-MA, CL 318952), also known as Verteporfin, is an inhibitor of YAP-TEAD interactions and is also a potent photosensitizing agent. The CAS number (CAS No.) is 129497-78-5.
AICAR, known as 5-Aminoidazole-4-carboxamide 1-. Beta. -D-ribofuranoside, also known as AICARiboside or Acadsin.
K-975, CAS number: 2563855-03-6, molecular formula C 16 H 14 ClNO 2 。
Activin a, CAS number: 104625-48-1, molecular formula: c (C) 27 H 34 O 3 。
B-27, purchased from Shanghai BasalMedia Technologies, cat# S441J7.
The double antibody, namely P/S double antibody (penicillin-streptomycin) is purchased from Thermo Fisher Scientific with the product number of 15140122;
a culture medium and a culture method for differentiating pluripotent mammalian stem cells into definitive endoderm cells according to the present application will be described in detail with reference to examples, comparative examples and experimental data.
Example 1
1. A medium for differentiating human pluripotent stem cells into qualitative endodermal cells, the medium based on IMDM/F12, further comprising the following components: 100ng/ml Activin A,1% B27,0.2% bovine serum albumin, 1% diabody, 0.1-0.5. Mu.M Verteporfin (0.1. Mu.M and 0.5. Mu.M in two experimental groups respectively).
2. A method for culturing human pluripotent stem cells into definitive endoderm cells by using the culture medium comprises the following steps:
(1) Mtesr+1% diabodies were used as growth medium, at 1: human embryonic stem cells were cultured on matrigel at a dilution ratio of 100.
(2) The human embryonic stem cells in the step (1) are cloned and grown after 4-5 days of culture, the cell density is about 80% -90% of the bottom area of the culture plate hole, the cells are kept stand for 3 minutes in a cell culture box at 37 ℃ by using Ackutase, the cells are observed to be round balls under a microscope, DMEM/F12 culture medium is immediately added to stop digestion, and the cells are counted after being lightly blown and resuspended according to 0.8X10 5 Density per well cells were seeded into matrigel coated 24 well plates and cultured in normal growth medium.
(3) After the cells in the step (2) were cultured for one day, the density reached about 80%, and at this time, definitive endoderm differentiation was started by changing the freshly prepared medium, which was designated as differentiation D0 day.
(4) The culture medium was replaced every 24 hours after the initiation of differentiation, and definitive endoderm cells were obtained and examined at day D3.
Example 2
1. A medium for differentiating human pluripotent stem cells into qualitative endodermal cells, the medium based on IMDM/F12, further comprising the following components: 100ng/ml Activin A,1% B27,0.2% bovine serum albumin, 1% diabody, 0.5. Mu.M Verteporfin.
2. A method for culturing human pluripotent stem cells into definitive endoderm cells by using the culture medium comprises the following steps:
(1) Mtesr+1% diabodies were used as growth medium, at 1: human embryonic stem cells were cultured on matrigel at a dilution ratio of 100.
(2) The human embryonic stem cells in the step (1) are cloned and grown after 4-5 days of culture, the cell density is about 80% -90% of the bottom area of the culture plate hole, the cells are kept stand for 3 minutes in a cell culture box at 37 ℃ by using Ackutase, the cells are observed to be round balls under a microscope, DMEM/F12 culture medium is immediately added to stop digestion, and the cells are counted after being lightly blown and resuspended according to 0.8X10 5 Density per well cells were seeded into matrigel coated 24 well plates and cultured in normal growth medium.
(3) After the cells in the step (2) were cultured for one day, the density reached about 80%, and at this time, definitive endoderm differentiation was started by changing the freshly prepared medium, which was designated as differentiation D0 day.
(4) The culture medium was replaced every 24 hours after the initiation of differentiation, and definitive endoderm cells were obtained and examined at day D3.
Example 3
1. A medium for differentiating human pluripotent stem cells into qualitative endodermal cells, the medium based on IMDM/F12, further comprising the following components: 100ng/ml Activin A,1% B27,0.2% bovine serum albumin, 1% diabody, 0.5. Mu.M Verteporfin+2% sucrose.
2. A method for culturing human pluripotent stem cells into definitive endoderm cells by using the culture medium comprises the following steps:
(1) Mtesr+1% diabodies were used as growth medium, at 1: human embryonic stem cells were cultured on matrigel at a dilution ratio of 100.
(2) The human embryonic stem cells in the step (1) are cloned and grown after 4-5 days of culture, the cell density is about 80% -90% of the bottom area of the culture plate hole, the cells are kept stand for 3 minutes in a cell culture box at 37 ℃ by using Ackutase, the cells are observed to be round balls under a microscope, DMEM/F12 culture medium is immediately added to stop digestion, and the cells are counted after being lightly blown and resuspended according to 0.8X10 5 Density per well cells were seeded into matrigel coated 24 well plates, normalAnd (5) culturing in a growth medium.
(3) After the cells in the step (2) were cultured for one day, the density reached about 80%, and at this time, definitive endoderm differentiation was started by changing the freshly prepared medium, which was designated as differentiation D0 day.
(4) The culture medium was replaced every 24 hours after the initiation of differentiation, and definitive endoderm cells were obtained and examined at day D3-4.
Comparative example 1
In comparative example 1, the procedure of example 1 was followed except that Verteporfin was not added to the differentiation medium. Comparative example 1 is a negative control.
Comparative example 2
In comparative example 2, the procedure of example 2 was followed except that 0.5. Mu.M Verteporfin was not added to the differentiation medium. Comparative example 2 is a blank negative control.
Experimental example 1
The positive rate of definitive endoderm cells of each example and each comparative example was examined for mRNA levels of definitive endoderm marker genes or immunofluorescence of the positive rate of definitive endoderm cells.
1. The detection method comprises the following steps:
1. positive rate detection of definitive endoderm cells derived from human embryonic stem cells:
the cells obtained by the differentiation are treated with TrypLE at 37℃CO 2 The incubator was allowed to stand for 1 minute before terminating digestion with 2% FBS and gently pipetting the collected cell fluids into a 1.5ml EP tube. Then, the mixture was centrifuged at 3000rpm for 3 minutes in a centrifuge at 4℃and the supernatant was discarded to obtain a cell pellet. 1 with 2% FBS: 200 proportion of CXCR4-APC antibody dilutions were prepared, 200. Mu.l of antibody dilutions were gently blown against the cell pellet for each sample and placed on ice for incubation for 30 minutes in the dark. A sample was also prepared and an APC-Isotype diluent (1:200) was added as a negative control. After that, the mixture was centrifuged at 3000rpm at 4℃for 3 minutes, and the supernatant was discarded and washed twice with 2% FBS. Finally, cell suspension is obtained by re-suspending cells by precooled PBS, the cell suspension is transferred into a flow tube, and the CXCR4 positive rate is detected by an up-flow instrument. The antibody used in this study was Anti-CXCR4 (555976,1:200,BD Biosciences).
2. Real-time fluorescent quantitative PCR (polymerase chain reaction) detection of mRNA (messenger ribonucleic acid) level change of definitive endoderm cell marker genes:
(1) Total RNA extraction from cells
The experiment adopts a small total RNA extraction kit (double column type) of the Meiya to extract RNA of cells, and the specific operation steps are as follows:
digesting differentiated definitive endoderm cells with TrypLE, centrifuging, collecting cell precipitate, adding appropriate amount of Buffer RL into cell sample, and beating with a pipette gun to break up cells (generally cell amount is not more than 5X10 6 350 μl Buffer RL is added and stored at-80deg.C; preparing a proper amount of ice, taking out a sample to be extracted from a refrigerator, transferring the sample to a gDNA filtering column (the column is placed in a collecting pipe) after melting and mixing uniformly, and centrifuging for 2 minutes at 14000 g; discarding the gDNA filter column, adding 70% ethanol with the volume equal to that of the sample, and blowing for 3-5 times by using a pipetting gun; the RNA purification column was mounted on a collection tube, and the sample solution was transferred to the RNA column and centrifuged at 12000g for 1 minute; the filtrate was decanted, the column was packed into a recovery header and 500. Mu.l Buffer RW1 was added and 12000g was centrifuged for 1 minute; the filtrate was discarded, the column was packed into a recovery header and 500. Mu.l Buffer RW2 was added; centrifuging 12000g for 1 min, and repeating the step once; the filtrate was decanted and the column was packed into a recovery header and 12000g was air-separated for 2 minutes; the RNA column was transferred to a fresh 1.5ml EP tube, 35. Mu.l of RNase Free water was added to the center of the column membrane and allowed to stand at room temperature for 2 minutes; 12000g was centrifuged for 1 min, the column was discarded, and the concentration and quality of RNA were measured and stored at-80 ℃.
(2) Reverse transcription to obtain cDNA
1ug of the above-extracted RNA was used to prepare cDNA using an Abclonal reverse transcription kit. Taking out the extracted RNA from the refrigerator, placing the RNA on ice, melting and mixing uniformly, taking 1 mug of the RNA into a PCR eight-joint tube, adding 4 mu l of 5 xqRT Supermix of Abclonal, and finally supplementing the total volume to 20ul with RNase Free water; the reaction mixture was gently stirred with a finger, collected at the bottom of the tube by a palm centrifuge, and subjected to reverse transcription in a Bio-rad PCR apparatus. Reverse transcription conditions were 25℃for 5 minutes; 42 ℃ for 20 minutes; 5 seconds at 85 ℃, and preserving at-20 ℃ after finishing.
(3) Real-time fluorescent quantitative PCR
Taking a proper amount of cDNA obtained by reverse transcription (the corresponding RNA amount is 10-20ng per well), adding 5ul of Bimake 2X SYBR Green qPCR Master Mix, 5 mu M of upstream primer and 5 mu M of downstream primer (1 mu l of upstream and downstream primer mixture), and finally supplementing the total volume to 10 mu l by RNase Free water; amplification was performed using a CFX384 quantitative PCR instrument of Bio-rad under the following reaction conditions: the 39 cycles were repeated for 5min at 95 ℃, 15s at 95 ℃ and 30s at 60 ℃. The experimental results were analyzed against housekeeping gene GAPDH using the delta CT method. The primers used are shown in Table 1.
TABLE 1
3. Immunofluorescence detection of positive rate of definitive endoderm cells derived from human embryonic stem cells:
discarding the culture medium in the hole after the cell differentiation is finished, and lightly cleaning the cells twice by using DPBS; 200ul of pre-chilled 4% paraformaldehyde was then carefully added to each well and allowed to stand at room temperature for 15 minutes; after fixation, the cells were gently washed with DPBS 3 times for 5 minutes each; 200ul of blocking solution (8.7 ml DPBS+1ml donkey serum+0.3 ml 10% Triton-X100) was carefully added to each well to cover the wells evenly and incubated for 1-2 hours at room temperature; then adding 150-200ul of blocking solution containing primary antibody to cover the plate hole, and incubating for 1-2 hours at room temperature; then the primary antibody solution is recovered and stored in a refrigerator at 4 ℃, and DPBS is used for gently cleaning the cells for 3 times, each time for 5 minutes; adding 150-200ul of blocking solution containing secondary antibody into each hole to cover the hole of the plate, and incubating for 1-2 hours at room temperature in a dark place; DPBS gently washes the cells 3 times for 5 minutes each; 150-200ul DAPI solution (1:5000 diluted in DPBS) was added to each well and incubated at room temperature for 5-10 min in the absence of light; DPBS gently washes the cells 3 times for 5 minutes each, finally, the cells are soaked with DPBS, placed under an inverted fluorescence microscope for fluorescent observation and pictures are taken. The primary antibody used in this study was FOXA2 (R & D, # AF2400, 1:200). The second fluorescent antibody used in this study was DoαGt-488 (1:200,Jackson ImmunoResearch); nuclear dye: DAPI (roche, USA).
2. Detection results and analysis
1. Example 1 and comparative example 1
Specific results of measuring the positive rate of CXCR4 in the cells of example 1 and comparative example 1 are shown in FIG. 1.
FIG. 1 is a flow chart showing positive rates of definitive endoderm cells of example 1 and comparative example 1, wherein "0" is comparative example 1. The results show that the use of Verteporfin in example 1 significantly increases the proportion of CXCR4 positive cells compared to the 56% or so positive rate of comparative example 1. At this time, the cell state is still normal, and the proportion of CXCR4 positive cells can be as high as about 93-96% under the control of 0.1 and 0.5 mu M Verteporfin. For comparative example 1, differentiation efficiency of about 90% was usually achieved by 4-5 days, whereas the addition of Verteporfin in example 1 reduced the differentiation time to 3 days to achieve comparable differentiation levels, reducing the time required for definitive endoderm cell differentiation. The addition of Verteporfin can effectively improve the differentiation efficiency of definitive endoderm and reduce the time required for differentiation.
2. Example 2, example 3 and comparative example 2
The mRNA expression levels of the flow type, definitive endoderm marker genes FOXA2, SOX17 of the cell CXCR4 positive rate in example 2, example 3 and comparative example 2 were examined, and immunofluorescence results were shown in fig. 2 to 4.
FIG. 2 shows immunofluorescence and statistical results of the effect of Verteporfin and Verteporfin+2% sucrose combination on the expression of human embryonic stem cells toward endodermal differentiation marker genes in comparison of example 2, example 3 and comparative example 2. In the "blank" group of comparative example 2, the proportion of cells positive for the definitive endoderm marker gene FOXA2 was detected by immunofluorescence, and the statistical result was about 50%, and the same result was seen from the immunofluorescence photograph. Example 2 is a "0.5 μ MVerteporfin" treated cell in which the FOXA2 positive cell fraction was statistically about 70%, demonstrating that the Verteporfin treatment was effective in increasing the differentiation efficiency of definitive endoderm cells. Example 3 is a "0.5. Mu.M Verteporfin+2% sucrose" treated cell in which the ratio of FOXA2 positive cells was statistically about 75%, demonstrating that the combined use of Verteporfin and sucrose was effective in promoting differentiation of definitive endoderm cells.
FIG. 3 is a flow chart showing the effect of Verteporfin and Verteporfin+2% sucrose in combination on expression of human embryonic stem cells toward endodermal differentiation marker genes, as shown in comparison of example 2, example 3 and comparative example 2. By flow cytometric detection of the proportion of CXCR4 positive cells, we can see that the proportion of CXCR4 positive cells in the "control group of comparative example 2 is approximately 50%. The "0.5 μm Verteporfin treated" group of example 2 had a CXCR4 positive cell fraction of approximately 85% with a very significant improvement in definitive endoderm differentiation efficiency compared to comparative example 2. Example 3 is a "0.5 μm verteporfin+2% sucrose" treated cell, wherein the proportion of CXCR4 positive cells was statistically about 85%, demonstrating that the combined use of Verteporfin and sucrose effectively increases the differentiation efficiency of definitive endoderm cells.
FIG. 4 shows the quantitative PCR results of the effects of Verteporfin and Verteporfin+2% sucrose combination on the expression of human embryonic stem cells toward endodermal differentiation marker genes in comparison of example 2, example 3 and comparative example 2. The 2.5-fold increase in FOXA2 expression and 5-fold increase in SOX17 expression levels in example 2 compared to comparative example 2, indicates that 0.5 μ MVerteporfin in example 2 can effectively increase the differentiation efficiency of definitive endoderm cells. The 2.7-fold increase in FOXA2 expression and 4.5-fold increase in SOX17 expression levels in example 3 compared to comparative example 2, indicates that 0.5 μm verteporfin+2% sucrose in example 3 can effectively increase the differentiation efficiency of definitive endoderm cells.
The results in FIGS. 2, 3 and 4 are consistent, and all show that the addition of Verteporfin can significantly promote the differentiation of definitive endoderm, and that the combination of Verteporfin and sucrose can also promote the differentiation of definitive endoderm. Meanwhile, for comparative example 2, differentiation efficiency of about 90% is often required to be achieved by 4-5 days, while the use of Verteporfin or the combined use of Verteporfin and sucrose in examples 2 and 3 can achieve a comparable differentiation level by shortening the differentiation time to 3 days, thereby reducing the time required for differentiation of definitive endoderm cells.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. Use of YAP inhibitor Verteporfin, comprising: the YAP inhibitor Verteporfin is added to improve the differentiation efficiency of the pluripotent stem cells of the mammal into definitive endoderm cells by differentiation culture.
2. A method for increasing the differentiation efficiency of mammalian pluripotent stem cells into definitive endoderm cells by differentiation culture using YAP inhibitors, the method comprising: mammalian pluripotent stem cells were cultured using YAP inhibitors.
3. A culture medium for increasing the differentiation efficiency of mammalian pluripotent stem cells into definitive endoderm cells by means of YAP inhibitors, the culture medium comprising the following components: YAP inhibitor is added on the basal medium.
4. A medium for the differentiation of mammalian pluripotent stem cells into definitive endoderm cells according to claim 3, wherein the medium comprises the components: 100ng/ml of Activin A, B27, bovine serum albumin, diabodies, and Verteporfin were added to the basal medium.
5. The medium of claim 4 for differentiation of mammalian pluripotent stem cells into definitive endoderm cells, wherein the medium comprises the components of: adding 1-100 ng/ml of Activin A, 0.5-1.5% of B27, 0.1-0.3% of bovine serum albumin, 0.5-1.5% of diabody and one of the following compounds to a basal medium:
compound a: 0.1-0.5. Mu.M Verteporfin;
compound B: 0.5. Mu.M Verteporfin+2% sucrose.
6. The medium for differentiation of mammalian pluripotent stem cells into definitive endoderm cells of claim 5, wherein the medium further comprises the components of: 2-3 mu M GSK3 inhibitor.
7. The medium of claim 5 for differentiation of mammalian pluripotent stem cells into definitive endoderm cells, wherein the types of mammalian pluripotent stem cells comprise: mammalian embryonic stem cells or mammalian induced pluripotent stem cells.
8. A method of culturing a mammalian pluripotent stem cell to differentiate into definitive endoderm cells, the method comprising: culturing mammalian pluripotent stem cells with the medium of any one of claims 3 to 7 to differentiate into definitive endoderm cells.
9. The method of claim 8, wherein the method comprises the steps of:
culturing the pluripotent stem cells of the mammal by using a growth medium for 3-6 days, then performing digestion counting, inoculating the pluripotent stem cells into an orifice plate coated with matrigel, and culturing by using a normal growth medium;
when the density of 75-85% is reached, culturing by replacing the culture medium according to any one of claims 4-8 to obtain the definitive endoderm cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311127162.7A CN117327643A (en) | 2023-09-01 | 2023-09-01 | Method for obtaining definitive endoderm cells from human pluripotent stem cells by using YAP inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311127162.7A CN117327643A (en) | 2023-09-01 | 2023-09-01 | Method for obtaining definitive endoderm cells from human pluripotent stem cells by using YAP inhibitor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117327643A true CN117327643A (en) | 2024-01-02 |
Family
ID=89294145
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311127162.7A Pending CN117327643A (en) | 2023-09-01 | 2023-09-01 | Method for obtaining definitive endoderm cells from human pluripotent stem cells by using YAP inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117327643A (en) |
-
2023
- 2023-09-01 CN CN202311127162.7A patent/CN117327643A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11155782B2 (en) | Method for preparing pluripotent stem cells | |
| CN109749986B (en) | Method for obtaining pancreatic precursor cells and pancreatic beta cells by differentiation of human pluripotent stem cells | |
| CN104894060B (en) | Inducing somatic transdifferentiation is the method and its application of neural stem cell | |
| US20100151574A1 (en) | Multipotent progenitor cell derived from adipose tissue | |
| JP2023512453A (en) | Methods of Differentiating Neuronal Cells and Related Compositions and Methods of Use | |
| CN103834613A (en) | Methods for preparing pleuripotent cardiovascular progenitor cells and maintaining cardiovascular differentiation capacity | |
| CN111269875A (en) | Method for directionally differentiating into islet cells by using autoimmune cells | |
| CN111269878B (en) | Special culture medium for converting human pluripotent stem cells into expanded pluripotent stem cells and application of special culture medium | |
| Goodrich et al. | In vivo generation of β-cell–like cells from CD34+ cells differentiated from human embryonic stem cells | |
| CN115011553A (en) | Preparation method and application of stem neural crest-derived bone marrow mesenchymal stem cells | |
| US20100190251A1 (en) | Method for the differentiation of human adult stem cells into insulin-secreting cells | |
| JP5636174B2 (en) | Method for producing mesenchymal cells or chondrocytes and method for suppressing carcinogenicity | |
| CN117327643A (en) | Method for obtaining definitive endoderm cells from human pluripotent stem cells by using YAP inhibitor | |
| CN117305224A (en) | Method for obtaining definitive endoderm cells from human pluripotent stem cells by differentiation using myosin activator or Piezo1 activator | |
| CN117165513A (en) | Culture medium and culture method for differentiation of mammalian pluripotent stem cells into definitive endoderm cells | |
| CN113583937B (en) | Culture medium and culture method for differentiating pluripotent mammalian stem cells into definitive endoderm cells | |
| RU2430158C1 (en) | Method for differentiation of adult human stem cells in insulin-secreting cells | |
| CN110592007B (en) | Mesenchymal stem cell and preparation method and application thereof | |
| CN114891737B (en) | Preparation method and application of multipotent stem cell-derived kidney mesenchymal stem cells | |
| CN116144584B (en) | Pancreatic progenitor cells differentiated from human induced pluripotent stem cells, and preparation method and application thereof | |
| CN109486770A (en) | A kind of microRNA-181c-5p promotion source of people iPS directed differentiation is the method for beta Cell of islet | |
| CN116064376A (en) | Application of sodium acetate in improving differentiation efficiency of mammal pluripotent stem cells to definitive endoderm cells | |
| CN115011544B (en) | Method for obtaining islet delta cells by in vitro induction and application thereof | |
| US20230313145A1 (en) | Method for screening in vitro population of stem cell derived beta like cells and novel markers thereof | |
| CN115975911A (en) | Use of AMPK activators to increase the efficiency of differentiation of mammalian pluripotent stem cells into definitive endoderm cells |
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 |