CN116574672B - 一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 - Google Patents
一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 Download PDFInfo
- Publication number
- CN116574672B CN116574672B CN202310845386.5A CN202310845386A CN116574672B CN 116574672 B CN116574672 B CN 116574672B CN 202310845386 A CN202310845386 A CN 202310845386A CN 116574672 B CN116574672 B CN 116574672B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- cells
- stage
- differentiation
- pluripotent stem
- 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
- 230000004069 differentiation Effects 0.000 title claims abstract description 43
- 239000001963 growth medium Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 29
- 210000002889 endothelial cell Anatomy 0.000 title claims abstract description 21
- 230000002324 hematogenic effect Effects 0.000 title claims abstract description 16
- 210000004263 induced pluripotent stem cell Anatomy 0.000 title claims abstract description 13
- 230000001939 inductive effect Effects 0.000 title claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 26
- 239000000203 mixture Substances 0.000 claims abstract description 18
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims abstract description 14
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 14
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000002609 medium Substances 0.000 claims abstract description 14
- 239000003112 inhibitor Substances 0.000 claims abstract description 11
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims abstract description 7
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 7
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 7
- 102000004877 Insulin Human genes 0.000 claims abstract description 7
- 108090001061 Insulin Proteins 0.000 claims abstract description 7
- 102000004338 Transferrin Human genes 0.000 claims abstract description 7
- 108090000901 Transferrin Proteins 0.000 claims abstract description 7
- 229930003268 Vitamin C Natural products 0.000 claims abstract description 7
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229940125396 insulin Drugs 0.000 claims abstract description 7
- 150000002632 lipids Chemical class 0.000 claims abstract description 7
- 230000037361 pathway Effects 0.000 claims abstract description 7
- 239000011435 rock Substances 0.000 claims abstract description 7
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 7
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 7
- 239000011781 sodium selenite Substances 0.000 claims abstract description 7
- 239000012581 transferrin Substances 0.000 claims abstract description 7
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 7
- 239000011718 vitamin C Substances 0.000 claims abstract description 7
- 229940121396 wnt pathway inhibitor Drugs 0.000 claims abstract description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 6
- 229940024606 amino acid Drugs 0.000 claims abstract description 6
- 150000001413 amino acids Chemical class 0.000 claims abstract description 6
- 229940031098 ethanolamine Drugs 0.000 claims abstract description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960002743 glutamine Drugs 0.000 claims abstract description 6
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229940035024 thioglycerol Drugs 0.000 claims abstract description 6
- 239000007640 basal medium Substances 0.000 claims abstract description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims abstract description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims abstract description 3
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims abstract description 3
- 239000000835 fiber Substances 0.000 claims abstract description 3
- 239000003102 growth factor Substances 0.000 claims abstract description 3
- 210000003038 endothelium Anatomy 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 230000003394 haemopoietic effect Effects 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 9
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 9
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical group 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 claims description 7
- 239000000243 solution Substances 0.000 claims description 5
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 3
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims description 3
- 102100035140 Vitronectin Human genes 0.000 claims description 3
- 108010031318 Vitronectin Proteins 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000001079 digestive effect Effects 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000006911 enzymatic reaction Methods 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 239000002356 single layer Substances 0.000 claims description 3
- 239000012879 subculture medium Substances 0.000 claims description 3
- 210000000988 bone and bone Anatomy 0.000 claims 1
- 230000000921 morphogenic effect Effects 0.000 claims 1
- 210000000130 stem cell Anatomy 0.000 abstract description 7
- 230000006698 induction Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 abstract description 2
- 102000046148 human BMP4 Human genes 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract 1
- 230000001172 regenerating effect Effects 0.000 abstract 1
- 210000004369 blood Anatomy 0.000 description 12
- 239000008280 blood Substances 0.000 description 12
- 230000006872 improvement Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 210000002242 embryoid body Anatomy 0.000 description 6
- 210000000601 blood cell Anatomy 0.000 description 5
- 230000003511 endothelial effect Effects 0.000 description 4
- 230000009762 endothelial cell differentiation Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 3
- 230000011132 hemopoiesis Effects 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 210000002536 stromal cell Anatomy 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001325 yolk sac Anatomy 0.000 description 2
- 239000012583 B-27 Supplement Substances 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000004156 Wnt signaling pathway Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000003566 hemangioblast Anatomy 0.000 description 1
- 208000013210 hematogenous Diseases 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 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
- C12N5/069—Vascular Endothelial 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
- 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
- C12N2500/32—Amino acids
-
- 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
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
-
- 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
- C12N2500/35—Polyols, e.g. glycerin, inositol
-
- 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
- C12N2500/30—Organic components
- C12N2500/46—Amines, e.g. putrescine
-
- 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
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- 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
- C12N2501/998—Proteins 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Vascular Medicine (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提出了一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法，属于干细胞与再生医学领域。包括第一阶段培养基和第二阶段培养基，第一阶段培养基包括：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、亚硒酸钠、乙醇胺、维生素C、脂质混合物、人白蛋白、WNT通路抑制剂、ROCK通路抑制剂，第二阶段培养基包括：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、亚硒酸钠、乙醇胺、维生素C、脂质混合物、人白蛋白、重组人骨形态发生蛋白‑4、碱性纤维生长因子、血管内皮生长因子。本发明诱导培养体系方便进行调控和标准化，促进了其分化下游细胞的应用。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法。
背景技术
化学诱导重编程是指利用化学小分子将人体细胞转变为多能干细胞，称为化学诱导多能干细胞（hCiPSCs）。与胚胎干细胞和诱导多能干细胞相同，hCiPSCs具备自我更新、无限增殖和分化成所有功能细胞类型的能力。利用多能干细胞的分化特性可以用来研究体内功能细胞和疾病的发育。
造血干细胞移植和免疫细胞疗法等目前已经在临床上显示出广泛的治疗前景和疗效，用于治疗多种血液疾病和肿瘤。然而其来源受限，批量之间差异大，成本高，难以满足治疗的需要量；多能干细胞可以作为无限来源用于制备造血干细胞和各种类型的免疫细胞。等经过多年研究，利用多能干细胞分化能力已经能够制备各种血液谱系，在这个过程中对于血液和内皮谱系的发育关系也有了更深入的理解。在胚胎发育过程中，血液细胞与内皮细胞发育相关联。在卵黄囊血岛（yolk sac）、胚内腹侧中胚层-主动脉性腺-中肾（AGM区）和背侧主动脉的腹侧壁均发现血细胞团与内皮细胞一起形成分化。进而产生了一种假设，即血细胞与内皮细胞有共同的祖细胞-成血管血液干细胞（hemangioblast）。尽管成血管血液干细胞的性质仍有争议，但越来越多的证据表明生血内皮细胞（hemogenicendothelial）是一种短暂的中间产物，但对于胚胎发生过程中多能造血干细胞的从头产生是非常重要的。如何在体外高效制备生血内皮细胞对于进一步获得功能更好的造血干祖细胞以及下游免疫细胞至关重要。
已有的制备技术包内含2D和3D的分化方法，两种方法均有采用，但这些方法均有一些缺点。
已有的制备技术包括3D体系如EB（embryoid body类胚体）、2D体系如与饲养层基质细胞共培养分化或直接定向分化，通过添加诱导因子到特定的分化培养基，但是因为分化方式不同，分化效率存在明显差异。一方面分化体系中含有血清、异源成分、基质细胞，会导致微生物污染的风险；另一方面，成分复杂且不明确、周期长、不易调控和标准化，严重地限制了其分化下游细胞的临床研究和应用。
发明内容
本发明的目的在于提供一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法。该培养基成分明确、简单、无动物源、成本低，采用定向2D分化方法，操作简单、效率高，有利于未来规模化和产业化。
本发明的技术方案是这样实现的：
本发明提供一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，包括第一阶段培养基和第二阶段培养基，所述第一阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、亚硒酸钠、乙醇胺、维生素C、脂质混合物、人白蛋白、WNT通路抑制剂、ROCK通路抑制剂，所述第二阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、亚硒酸钠、乙醇胺、维生素C、脂质混合物、人白蛋白、重组人骨形态发生蛋白-4、碱性纤维生长因子、血管内皮生长因子。
作为本发明的进一步改进，所述WNT通路抑制剂为CHIR99021；所述ROCK通路抑制剂为Y27632。
作为本发明的进一步改进，所述第一阶段培养基的配方如下：
作为本发明的进一步改进，所述第二阶段培养基的配方如下：
本发明进一步保护一种诱导化学诱导多能干细胞向生血内皮细胞分化的方法，包括以下步骤：
S1.准备分化用单层化学诱导多能干细胞，培养；
S2.细胞完全换液，换成上述第一阶段培养基，培养，得到中胚层样细胞；
S3.细胞完全换液，将所有培养基替换成上述第二阶段培养基，培养，得到CD31+CD34+生血内皮细胞。
作为本发明的进一步改进，步骤S1的具体方法为：使用消化液Accutase消化对数期生长的hCiPSCs，37℃，5min，使用培养液3倍稀释中止酶反应，收集细胞离心300g，3min；用传代培养基重悬细胞，计数，以30万每板的密度接种到包被好玻连蛋白的培养板。
作为本发明的进一步改进，步骤S2的具体方法为：按照第一阶段培养基组成配制培养基，分别在0天和2天换液，连续培养3天。
作为本发明的进一步改进，步骤S3的具体方法为：按照第二阶段培养基组成配制培养基，分别在3天和5天换液，连续培养3天，第6天检测生血内皮的分化效率。
本发明具有如下有益效果：本发明诱导培养体系中不含有血清、异源成分和基质细胞且成分明确、诱导周期短、方便进行调控和标准化，促进了其分化下游细胞的临床研究和应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为多潜能干细胞向生血内皮分化流程图；
图2为hCiPSCs向生血内皮分化6天的效率图；
图3为EB和spin EB法分化体系对比图；
图4为小分子CHIR99021的使用浓度对分化效率的影响对比图；
图5为不同接种密度对分化效率的影响对比图；
图6为流式分析分化最终分化产物的纯度图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
如图1，方法包括以下步骤：
S1.准备分化用单层化学诱导多能干细胞，培养两天；具体方法为：使用消化液Accutase消化对数期生长的hCiPSCs，37℃，5min，使用培养液3倍稀释中止酶反应，收集细胞离心300g，3min；用传代培养基重悬细胞，计数，以30万每板的密度接种到包被好Vitronectin（玻连蛋白）的培养板。
S2.将细胞完全换液，换成第一阶段培养基（配方见表1），得到中胚层样细胞,培养3天；具体方法为，按照培养基组成配制培养基，分别在0天和2天换液。
表1. 第一阶段培养基配方
S3.将细胞完全换液，将所有培养基替换成第二阶段培养基（配方见表2），得到CD31+ CD34+生血内皮细胞，培养3天。具体为，按照培养基组成配制培养基，分别在3天和5天换液，第6天检测生血内皮的分化效率。
表2. 第二阶段培养基配方
测试例1
1、WNT通路抑制剂与ROCK通路抑制剂协同促进生血内皮分化：
对于成分确定的培养体系的研究结果以及我们之前的专利，表明胰岛素、转铁蛋白、亚硒酸钠、乙醇胺的组合是细胞存活和增殖的最基本的成分，通过在分化培养基中进一步添加维生素C，能够促进细胞的存活和增殖，而脂质混合物和人白蛋白的添加能够进一步促进分化体系的稳定，此7种成分构成了分化体系中的添加物组合。在2D无饲养层分化体系中，WNT通路抑制剂CHIR99021与ROCK通路抑制剂Y27632协同，6天即能有效诱导hCiPSCs向CD34+CD31+生血内皮的分化（图2）。
相同条件下，3D的EB培养体系和spin EB法培养体系，只能诱导低比例的生血内皮细胞；而商售的常用于分化的B27添加物在相同条件的生血内皮的比例也低于7种添加物组合添加CHIR99021和Y27632的条件（图3）。上述实验结果表明，在生血内皮的分化过程中，通过简单的7中添加物与两个小分子抑制剂的组合即可诱导高效、短时诱导生血内皮。
2、小分子抑制剂的浓度对生血内皮分化的影响
WNT信号通路抑制剂CHIR99021的剂量对CD31+CD34+生血内皮细胞效率的影响。流式细胞检测结果表明，相比于DMSO组，分化前3天添加5-10 μM CHIR99021与2 μMY27632组合均可高效促进生血内皮产生，呈一定范围的浓度依赖性，大于10 μM 会影响细胞的存活（图4）。
3、hCiPSCs细胞起始密度对生血内皮分化的影响
为了探究起始细胞密度对于生血内皮的影响，在铺板的时候采用不同接种密度的方式来摸索。实验结果显示，细胞密度影响CD31+CD34+生血内皮的数量，30-40万每细胞培养板的密度是适宜的（图5）。
4、分化的生血内皮具有向造血干祖细胞的分化能力
通过基础培养基中添加各种成分和信号通路抑制剂的组合，我们确定了成分明确的、无异源的、高效、6天即可诱导生血内皮的培养基和方法。还需确认生血内皮通过EHT(内皮-造血转换)过程，能够有效分化为造血干祖细胞。实验结果显示，在分化D8天即可观察到，非贴壁的、圆形的血细胞，随着分化进程，数量也越来越多。流式结果表明，上层的血细胞90%以上均是CD34+CD45+（图6）。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，其特征在于，包括第一阶段培养基和第二阶段培养基，所述第一阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、亚硒酸钠、乙醇胺、维生素C、脂质混合物、人白蛋白、WNT通路抑制剂和ROCK通路抑制剂，所述第二阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、亚硒酸钠、乙醇胺、维生素C、脂质混合物、人白蛋白、重组人骨形态发生蛋白-4、碱性纤维生长因子和血管内皮生长因子；所述WNT通路抑制剂为CHIR99021；所述ROCK通路抑制剂为Y27632；
所述第一阶段培养基的配方如下：
；
所述第二阶段培养基的配方如下：
。
2.一种诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，包括以下步骤：
S1.准备分化用单层化学诱导多能干细胞，培养；
S2.细胞完全换液，换成权利要求1中所述第一阶段培养基，培养，得到中胚层样细胞；
S3.细胞完全换液，将所有培养基替换成权利要求1中所述第二阶段培养基，培养，得到CD31+ CD34+生血内皮细胞。
3.根据权利要求2所述诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，步骤S1的具体方法为：使用消化液Accutase消化对数期生长的hCiPSCs，37℃，5min，使用培养液3倍稀释中止酶反应，收集细胞离心300g，3min；用传代培养基重悬细胞，计数，以30万每板的密度接种到包被好玻连蛋白的培养板。
4.根据权利要求2所述诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，步骤S2的具体方法为：按照第一阶段培养基组成配制培养基，分别在0天和2天换液，连续培养3天。
5.根据权利要求2所述诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，步骤S3的具体方法为：按照第二阶段培养基组成配制培养基，分别在3天和5天换液，连续培养3天，第6天检测生血内皮的分化效率。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310845386.5A CN116574672B (zh) | 2023-07-11 | 2023-07-11 | 一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310845386.5A CN116574672B (zh) | 2023-07-11 | 2023-07-11 | 一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116574672A CN116574672A (zh) | 2023-08-11 |
| CN116574672B true CN116574672B (zh) | 2023-10-20 |
Family
ID=87539984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310845386.5A Active CN116574672B (zh) | 2023-07-11 | 2023-07-11 | 一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116574672B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117965430A (zh) * | 2024-01-16 | 2024-05-03 | 深圳市济因生物科技有限公司 | 一种促进多能干细胞存活的方法及其应用 |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107496457A (zh) * | 2017-08-03 | 2017-12-22 | 北京再生生物科技研究院有限公司 | 一种MSCs来源的OPCs滋养感觉神经元轴突生长的方法 |
| CN109797132A (zh) * | 2019-03-15 | 2019-05-24 | 苏州大学 | 一种促进人多能干细胞定向分化为内皮细胞的方法 |
| CN112725261A (zh) * | 2021-01-19 | 2021-04-30 | 上海爱萨尔生物科技有限公司 | 多能干细胞分化培养内皮细胞的培养液及分化方法 |
| CN113265377A (zh) * | 2021-06-11 | 2021-08-17 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | 一种动脉内皮促进功能性t细胞再生的方法 |
| CN114181968A (zh) * | 2022-02-11 | 2022-03-15 | 北京干细胞与再生医学研究院 | 一种具备b谱系分化潜能的人造血祖细胞的制备方法及其应用 |
| CN115029314A (zh) * | 2022-06-06 | 2022-09-09 | 北京呈诺医学科技有限公司 | Cd34+细胞分化培养基、方法及应用 |
| WO2022212514A1 (en) * | 2021-03-31 | 2022-10-06 | Garuda Therapeutics, Inc. | Pluripotent stem cell-derived hematopoietic lineages |
| WO2022217955A1 (zh) * | 2021-04-13 | 2022-10-20 | 中国科学院深圳先进技术研究院 | 一种诱导多能干细胞向造血前体细胞分化的培养基以及方法 |
| CN115247151A (zh) * | 2022-09-21 | 2022-10-28 | 呈诺再生医学科技(北京)有限公司 | 制备造血内皮细胞的方法及制备造血干细胞或造血干祖细胞的方法 |
| CN115772496A (zh) * | 2023-02-10 | 2023-03-10 | 山东兴瑞生物科技有限公司 | 一种诱导性多能干细胞制备通用血小板的方法 |
| CN115927199A (zh) * | 2015-11-04 | 2023-04-07 | 菲特治疗公司 | 用于诱导造血细胞分化的方法和组合物 |
| CN116396940A (zh) * | 2022-01-06 | 2023-07-07 | 北京干细胞与再生医学研究院 | 造血干祖细胞及其制备方法和用途 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2015353853B2 (en) * | 2014-11-25 | 2020-10-15 | President And Fellows Of Harvard College | Methods for generation of podocytes from pluripotent stem cells and cells produced by the same |
| CN110218696A (zh) * | 2018-03-01 | 2019-09-10 | 中国科学院广州生物医药与健康研究院 | 一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法 |
-
2023
- 2023-07-11 CN CN202310845386.5A patent/CN116574672B/zh active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927199A (zh) * | 2015-11-04 | 2023-04-07 | 菲特治疗公司 | 用于诱导造血细胞分化的方法和组合物 |
| CN107496457A (zh) * | 2017-08-03 | 2017-12-22 | 北京再生生物科技研究院有限公司 | 一种MSCs来源的OPCs滋养感觉神经元轴突生长的方法 |
| CN109797132A (zh) * | 2019-03-15 | 2019-05-24 | 苏州大学 | 一种促进人多能干细胞定向分化为内皮细胞的方法 |
| CN112725261A (zh) * | 2021-01-19 | 2021-04-30 | 上海爱萨尔生物科技有限公司 | 多能干细胞分化培养内皮细胞的培养液及分化方法 |
| WO2022212514A1 (en) * | 2021-03-31 | 2022-10-06 | Garuda Therapeutics, Inc. | Pluripotent stem cell-derived hematopoietic lineages |
| WO2022217955A1 (zh) * | 2021-04-13 | 2022-10-20 | 中国科学院深圳先进技术研究院 | 一种诱导多能干细胞向造血前体细胞分化的培养基以及方法 |
| CN113265377A (zh) * | 2021-06-11 | 2021-08-17 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | 一种动脉内皮促进功能性t细胞再生的方法 |
| CN116396940A (zh) * | 2022-01-06 | 2023-07-07 | 北京干细胞与再生医学研究院 | 造血干祖细胞及其制备方法和用途 |
| CN114181968A (zh) * | 2022-02-11 | 2022-03-15 | 北京干细胞与再生医学研究院 | 一种具备b谱系分化潜能的人造血祖细胞的制备方法及其应用 |
| CN115029314A (zh) * | 2022-06-06 | 2022-09-09 | 北京呈诺医学科技有限公司 | Cd34+细胞分化培养基、方法及应用 |
| CN115247151A (zh) * | 2022-09-21 | 2022-10-28 | 呈诺再生医学科技(北京)有限公司 | 制备造血内皮细胞的方法及制备造血干细胞或造血干祖细胞的方法 |
| CN115772496A (zh) * | 2023-02-10 | 2023-03-10 | 山东兴瑞生物科技有限公司 | 一种诱导性多能干细胞制备通用血小板的方法 |
Non-Patent Citations (1)
| Title |
|---|
| A protocol describing the use of a recombinant protein-based, animal product-free medium （APEL） for human embryonic stem cell differentiation as spin embryoid bodies;Elizabeth S Ng等;《NATURE PROTOCOLS》;第3卷(第5期);第770页，表1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116574672A (zh) | 2023-08-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2018148921A (ja) | 赤血球の産生 | |
| EP2865749A1 (en) | High-concentration stem cell production method | |
| CN103857789A (zh) | 制备间充质干细胞基础培养基和利用间充质干细胞基础培养基制备细胞治疗产品的方法及用该培养基得到的分化产品 | |
| Tarle et al. | Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs | |
| CN116574672B (zh) | 一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 | |
| CN109370985A (zh) | 一种人脐带间充质干细胞大规模培养无血清培养基 | |
| WO2023221438A1 (zh) | 一种通过调节NGN3表达促进iPSC向胰岛细胞分化的方法 | |
| CN113692282A (zh) | 一种生物活性物质组合物、包含所述组合物的无血清培养基及其用途 | |
| KR20110097064A (ko) | 무혈청 줄기 세포 배양 조성물, 이를 함유한 조직 재생용 조성물 및 이를 이용한 조직 재생 방법 | |
| KR101753557B1 (ko) | 줄기세포 증식 향상 배지 조성물 및 줄기세포의 배양방법 | |
| JP2011211956A (ja) | 幹細胞の未分化維持剤及び増殖促進剤 | |
| JP2012044914A (ja) | 幹細胞の未分化維持剤及び増殖促進剤 | |
| CN108148808B (zh) | 有助于诱导生成神经前体细胞的诱导培养基 | |
| CN114807028B (zh) | 一种无血清间充质干细胞培养基及干细胞培养方法 | |
| CN114990064B (zh) | 一种造血干细胞、其制备方法及其应用 | |
| CN117778315A (zh) | 一种t细胞的制备方法 | |
| KR20060114381A (ko) | 배아줄기세포용 피더세포 작성 배지 및 피더세포 | |
| CN116478914A (zh) | 无血清细胞培养体系及其细胞消化终止液 | |
| JP2016192962A (ja) | ヒトｉＰＳ細胞から、ヒト歯原性上皮細胞やヒト歯原性間葉細胞を製造する方法 | |
| Ji et al. | Multilineage differentiation capability comparison between mesenchymal stem cells and multipotent adult progenitor cells | |
| CN115449503A (zh) | 一种体外诱导ips细胞分化生成人肝实质细胞的方法 | |
| EP3564363A1 (en) | Production of megakaryocytes in bioreactors | |
| CN115975926A (zh) | 一种造血干细胞无血清培养基及其应用 | |
| JP2024024550A (ja) | 未分化幹細胞の細胞死誘導剤、及び分化細胞の純化方法 | |
| Donta | Growth of functional glial cells in a serumless medium |
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 |