CN115322958B - 胚胎干细胞培养用的培养基添加剂及其应用 - Google Patents
胚胎干细胞培养用的培养基添加剂及其应用 Download PDFInfo
- Publication number
- CN115322958B CN115322958B CN202210949539.6A CN202210949539A CN115322958B CN 115322958 B CN115322958 B CN 115322958B CN 202210949539 A CN202210949539 A CN 202210949539A CN 115322958 B CN115322958 B CN 115322958B
- Authority
- CN
- China
- Prior art keywords
- embryonic stem
- embryo
- stem cells
- medium
- inhibitor
- 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
- 210000001671 embryonic stem cell Anatomy 0.000 title claims abstract description 46
- 239000000654 additive Substances 0.000 title claims abstract description 32
- 230000000996 additive effect Effects 0.000 title claims abstract description 30
- 239000001963 growth medium Substances 0.000 title claims abstract description 20
- 238000012258 culturing Methods 0.000 title claims description 13
- 208000035199 Tetraploidy Diseases 0.000 claims abstract description 43
- 239000003112 inhibitor Substances 0.000 claims abstract description 39
- 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 34
- 239000011435 rock Substances 0.000 claims abstract description 18
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229930003268 Vitamin C Natural products 0.000 claims abstract description 16
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 16
- 239000011718 vitamin C Substances 0.000 claims abstract description 16
- 210000001161 mammalian embryo Anatomy 0.000 claims description 42
- 239000002609 medium Substances 0.000 claims description 39
- 241001465754 Metazoa Species 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 22
- 230000008569 process Effects 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims description 6
- 239000007640 basal medium Substances 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- 239000006143 cell culture medium Substances 0.000 claims description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- 238000010362 genome editing Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 4
- 235000020776 essential amino acid Nutrition 0.000 claims description 3
- 239000003797 essential amino acid Substances 0.000 claims description 3
- 230000000379 polymerizing effect Effects 0.000 claims description 2
- ISZXEMUWHQLLTC-LSIVYLFASA-N 2-[(2r,5s,6s)-6-[(2e,4e,6s)-7-[(2r,3r)-3-[(2r,3r,4r)-4-hydroxy-3-methoxypentan-2-yl]-2-methyloxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-5-methyloxan-2-yl]acetic acid Chemical compound CO[C@@H]([C@@H](C)O)[C@@H](C)[C@H]1O[C@]1(C)C[C@H](C)\C=C\C=C(/C)[C@@H]1[C@@H](C)CC[C@H](CC(O)=O)O1 ISZXEMUWHQLLTC-LSIVYLFASA-N 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 26
- 210000002257 embryonic structure Anatomy 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000002993 trophoblast Anatomy 0.000 description 6
- 102100032352 Leukemia inhibitory factor Human genes 0.000 description 5
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 5
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 5
- 210000002459 blastocyst Anatomy 0.000 description 5
- 238000003169 complementation method Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 210000003101 oviduct Anatomy 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000282898 Sus scrofa Species 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000001900 endoderm Anatomy 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 210000003716 mesoderm Anatomy 0.000 description 4
- 210000001324 spliceosome Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 206010011732 Cyst Diseases 0.000 description 3
- ISZXEMUWHQLLTC-UHFFFAOYSA-N Herboxidiene Natural products COC(C(C)O)C(C)C1OC1(C)CC(C)C=CC=C(C)C1C(C)CCC(CC(O)=O)O1 ISZXEMUWHQLLTC-UHFFFAOYSA-N 0.000 description 3
- 208000031513 cyst Diseases 0.000 description 3
- 210000002969 egg yolk Anatomy 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 210000000287 oocyte Anatomy 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- 210000001325 yolk sac Anatomy 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000282994 Cervidae Species 0.000 description 2
- 206010068051 Chimerism Diseases 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 102000001267 GSK3 Human genes 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 2
- 241000282575 Gorilla Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000009328 Perro Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102000004389 Ribonucleoproteins Human genes 0.000 description 2
- 108010081734 Ribonucleoproteins Proteins 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 230000003169 placental effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 210000004340 zona pellucida Anatomy 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 1
- 102100022975 Glycogen synthase kinase-3 alpha Human genes 0.000 description 1
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 101000669917 Homo sapiens Rho-associated protein kinase 1 Proteins 0.000 description 1
- 101000669921 Homo sapiens Rho-associated protein kinase 2 Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000691459 Homo sapiens Serine/threonine-protein kinase N2 Proteins 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 1
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 1
- 102100039313 Rho-associated protein kinase 1 Human genes 0.000 description 1
- 102100039314 Rho-associated protein kinase 2 Human genes 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 102100026180 Serine/threonine-protein kinase N2 Human genes 0.000 description 1
- 108700025832 Serum Response Element Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- SDOUORKJIJYJNW-QHOUZYGJSA-N [(2s,3s,4e,6s,7r,10r)-7,10-dihydroxy-2-[(2e,4e,6s)-7-[(2r,3r)-3-[(2r,3s)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] acetate Chemical compound CC[C@H](O)[C@@H](C)[C@H]1O[C@@H]1C[C@H](C)\C=C\C=C(/C)[C@@H]1[C@@H](C)/C=C/[C@H](OC(C)=O)[C@](C)(O)CC[C@@H](O)CC(=O)O1 SDOUORKJIJYJNW-QHOUZYGJSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 210000001643 allantois Anatomy 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003627 anti-cholesterol Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000625 blastula Anatomy 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000021953 cytokinesis Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 210000002242 embryoid body Anatomy 0.000 description 1
- 210000001650 focal adhesion Anatomy 0.000 description 1
- 108010049611 glycogen synthase kinase 3 alpha Proteins 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002350 laparotomy Methods 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000000472 morula Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 231100000208 phytotoxic Toxicity 0.000 description 1
- 230000000885 phytotoxic effect Effects 0.000 description 1
- 229930001119 polyketide Natural products 0.000 description 1
- 150000003881 polyketide derivatives Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108010041788 rho-Associated Kinases Proteins 0.000 description 1
- 102000000568 rho-Associated Kinases Human genes 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000016160 smooth muscle contraction Effects 0.000 description 1
- 210000003518 stress fiber Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000001262 western blot Methods 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
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- 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
- C12N5/0604—Whole embryos; Culture medium therefor
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal 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
- C12N2500/30—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
- C12N2500/38—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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种培养基添加剂及其应用。本发明提供的培养基添加剂包括剪切体抑制剂、维生素C和ROCK抑制剂。本发明还提供了包含该培养基添加剂的培养基。采用本发明的培养基培养胚胎干细胞(ESC)可显著提高ESC四倍体效率。
Description
技术领域
本发明属于细胞生物学技术领域,具体涉及一种胚胎干细胞培养用的培养基添加剂及其应用。
背景技术
四倍体是指细胞中含有的染色体是最小染色体组数的四倍,即细胞中具有四个完整的染色体组的生物个体或者细胞。在自然条件下,哺乳动物四倍体胚胎的发生率极低而且普遍不能正常发育成为一个单独个体。
将一定数量的胚胎干细胞(ES细胞)和四倍体胚胎嵌合,在其嵌合体的发育过程中,ES细胞和四倍体胚胎不是随机分布的,即其四倍体胚胎部分仅参与卵黄囊内胚层和胎盘滋养层细胞(如绒毛膜外胚层、滋养层细胞等)等胚外组织的形成,而ES细胞则广泛的参与胚体、尿囊、羊膜、卵黄囊中胚层和绒毛膜中胚层部分的生成,而不参与生成卵黄囊内胚层和胎盘滋养层细胞谱系,即二者的发育潜能具有互补性,这种现象称之为四倍体互补技术。
由这项技术克隆出的动物个体,完全是由ES细胞发育而来,称之为ES动物。四倍体补偿技术,即将ESC细胞系注射入囊胚期的四倍体胚胎载体中,在现有技术中效率大多在1%左右,小于3%,其中ESC的质量多能性性能至关重要。
发明内容
为了解决现有技术中存在的技术问题,本发明提供了一种用于胚胎干细胞培养的培养基添加剂以及包含该培养基添加剂的培养基。采用本发明的培养基培养胚胎干细胞(ESC)可显著提高胚胎干细胞的四倍体效率。
为了实现本发明的目的,采用如下技术方案。
在第一方面,本发明提供了一种培养基添加剂,包括剪切体抑制剂、维生素C和ROCK抑制剂。
在一些实施方式中,所述剪切体抑制剂选自plaB、FR901464、E7107和GEX1A中的至少一种,优选为plaB。
在一些实施方式中,所述ROCK抑制剂选自Y-27632和H-1152中的至少一种,优选为Y-27632。
在一些实施方式中,所述培养基添加剂包括:0.5-5nM剪切体抑制剂;20-100μg/ml维生素C;和0.5-5μM ROCK抑制剂。
在一些实施方式中,所述培养基添加剂中,剪切体抑制剂的浓度可以为0.5nM、1nM、1.5nM、2nM、2.5nM、3nM、3.5nM、4nM、4.5nM、5nM或它们之间的任意值。
在一些实施方式中,所述培养基添加剂中,维生素C的浓度可以为20μg/ml、30μg/ml、40μg/ml、50μg/ml、60μg/ml、70μg/ml、80μg/ml、90μg/ml、100μg/ml或它们之间的任意值。
在一些实施方式中,所述培养基添加剂中,ROCK抑制剂的浓度可以为0.5μM、1μM、1.5μM、2μM、2.5μM、3μM、3.5μM、4μM、4.5μM、5μM或它们之间的任意值。
在一些实施方式中,所述培养基添加剂包括:0.5-2nM剪切体抑制剂;40-60μg/ml维生素C;和0.5-2μM ROCK抑制剂。
在一些实施方式中,所述培养基添加剂包括1nM剪切体抑制剂、50μg/ml维生素C和1μMROCK抑制剂。
在一些实施方式中,所述培养基添加剂包括1nM剪切体抑制剂plaB、50μg/ml维生素C和1μM Y27632。
在第二方面,本发明提供了第一方面所述的培养基添加剂在胚胎干细胞培养或制备动物胚胎中的应用。
在一些实施方式中,所述培养基添加剂用于四倍体胚胎互补法或四倍体补偿法中的胚胎干细胞培养。
在一些实施方式中,所述胚胎干细胞为经基因编辑的胚胎干细胞。
在一些实施方式中,所述动物胚胎为哺乳动物四倍体胚胎。在一些实施方式中,所述动物为基因编辑动物。
在一些实施方式中,所述动物为哺乳动物。在一些实施例中,所述动物为非人类哺乳动物。在一些实施例中,所述动物选自猪、大鼠、小鼠、仓鼠、兔、猪、牛、鹿、绵羊、山羊、小鸡、猫、马、狗、猩猩、猴。
在第三方面,本发明提供了一种胚胎干细胞培养基,其包括基础培养基和第一方面所述的培养基添加剂。
本发明对基础培养基的种类不做特别限定,满足胚胎干细胞的培养即可,包括但不限于DMEM培养基、DMEM/F12培养基、N2B27培养基、F-12培养基等。
在一些实施方式中,所述培养基还包括胎牛血清(FBS)、Glutamax、非必需氨基酸(NEAA)、β-巯基乙醇(β-me)、LIF、pD0325901和Chir99021中的一种或多种。
在一些实施方式中,所述培养基还包括:DMEM培养基、15%胎牛血清、Glutamax、非必需氨基酸、β-巯基乙醇、LIF、1μM pD0325901和3μM Chir99021。
在一些实施方式中,所述基础培养基包括KnockoutDMEM、15%FBS(Gibco)、Glutamax(Gibco,100x)、NEAA(Gibco,100x)、β-me+LIF(Millipore)、1μM pD0325901(Selleck,S1036)和3um Chir99021(selleck)。
在一些实施方式中,所述培养基用于胚胎干细胞培养或制备动物胚胎。
在一些实施方式中,所述培养基用于四倍体胚胎互补法或四倍体补偿法中的胚胎干细胞培养;
在一些实施方式中,所述胚胎干细胞为经基因编辑的胚胎干细胞;
在一些实施方式中,所述动物胚胎为哺乳动物四倍体胚胎;
在一些实施方式中,所述动物为基因编辑动物。
在一些实施方式中,所述动物为哺乳动物。在一些实施例中,所述动物为非人类哺乳动物。在一些实施例中,所述动物选自猪、大鼠、小鼠、仓鼠、兔、猪、牛、鹿、绵羊、山羊、小鸡、猫、马、狗、猩猩、猴。
在一些实施方式中,所述培养基用于制备动物四倍体胚胎。
在第五方面,本发明提供了一种四倍体胚胎或嵌合体胚胎的制备方法,其包括使用第一方面所述的培养基添加剂或第四方面所述的培养基培养胚胎干细胞。
在一些实施方式中,所述制备方法包括将四倍体胚胎与所述胚胎干细胞聚合得到嵌合体胚胎。
一些实施例中,所述制备方法使用了四倍体补偿法或四倍体胚胎互补法。
相比于现有技术,采用本发明提供的含有剪切体抑制剂plaB、维生素C和Y27632的培养基可显著提升了ESC四倍体动物克隆的效率,可达10-30%。
下面提供实施例以帮助理解本发明。但应理解,这些实施例仅用于说明本发明,但不构成任何限制。本发明的实际保护范围在权利要求书中进行阐述。应理解,在不脱离本发明精神的情况下,可以进行任何修改和改变。
附图说明
图1显示了根据本申请实施例1得到的四倍体成活率的结果图。
具体实施方式
定义
除非另有定义,否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的,将应用以下定义,并且在适当时,以单数形式使用的术语也将包括复数形式,反之亦然。
除非上下文另有明确说明,否则本文所用的表述“一种”和“一个”包括复数指代。例如,提及“一个细胞”包括多个这样的细胞及本领域技术人员可知晓的等同物等等。
本文所用术语“包含”、“包括”将被理解为是指包括所述的步骤或要素或步骤和要素的集合,但并不排除任何其它的步骤或要素或步骤和要素的集合;即开放式限定。
如本文所用的术语“剪切体抑制剂(splicing inhibitor)”,也称“剪接体抑制剂”,是指能够抑制剪切体活性的任何分子。术语“剪切体/剪接体”是具有五个核心亚单位和几个辅因子的大分子核糖核蛋白(RNP)复合物,并且是mRNA剪接和成熟的动态分子机器。剪接体也可以直接控制转录的起始、延伸和终止。本文所用术语“plaB(pladienolide B)”是一种大环内酯的天然抗肿瘤类似物,可与SF3B复合物特异性结合以抑制剪切体和剪切的功能。因为SF3B亚基(SF3B2–SF3B5)的敲除激活了全能标记基因,推测PlaB可以驱动多能细胞向全能细胞的转变。本文所用术语“FR901464”是一种有效的剪接体抑制剂,具有显着的抗肿瘤和抗癌作用。本文所用术语“E7107”是pladienolide的衍生物,靶向剪切体的SF3B亚基,在体外和体内均显示出显着的抗肿瘤活性。本文所用术语“GEX1A”,也称Herboxidiene,是一种有效的植物毒性聚酮类化合物,来源于链霉菌A7847,具有多种活性,包括除草、抗胆固醇、抗肿瘤等。GEX1A通过与剪接体相关蛋白(SAP)155结合抑制前mRNA剪接(pre-mRNAsplicing)过程,SAP是剪接体中SF3B的一个亚单位。
如本文所用的术语“ROCK抑制剂”是指能够抑制ROCK活性的任何分子,如通过抑制ROCK磷酸化水平确定的(通过蛋白质印迹分析检测)。术语“ROCK”是指Rho相关卷曲螺旋形成蛋白激酶,其具有丝氨酸/苏氨酸激酶活性,并调节胞质***、平滑肌收缩、肌动蛋白应力纤维和粘着斑的形成以及c-fos血清反应元件的激活。如本文所用的术语“Y-27632”是一种ROCK Rho激酶的小分子抑制剂。本文所用术语“Y27632”是ROCK的特异性抑制剂,包括ROCK1和ROCK2,同时也抑制PRK2,其抑制作用是通过与ATP竞争结合催化位点来实现的。Y-27632可以阻止胚胎干细胞的细胞凋亡。如本文所用术语“H-1152”是一种可透过膜的选择性ROCK抑制剂。
本文所用术语“维生素C”,也称L-抗坏血酸。
pD0325901是一种特异性的MEK(mitogen-activated protein kinase kinase)小分子抑制剂。MEK是RAS/RAF/MEK/ERK信号通路的关键组成部分。MEK/ERK调控细胞外信号刺激的细胞增殖、存活和分化。Chir99021是一种有效且高度选择性的糖原合酶激酶3(GSK-3)抑制剂,包括GSK-3β和GSK-3α。LIF是白血病抑制因子。
本文所用术语“四倍体互补技术”与“四倍体胚胎补偿技术”或“四倍体互补方法”或“四倍体胚胎互补方法”可以等同,其指的是将一定数量的胚胎干细胞(ES)细胞和四倍体胚胎嵌合,在其嵌合体的发育过程中,ES细胞和四倍体胚胎不是随机分布的,即其四倍体胚胎部分仅参与卵黄囊内胚层和胎盘滋养层细胞(如绒毛膜外胚层、滋养层细胞等)等胚外组织的形成,而ES细胞则广泛的参与胚体、尿囊、羊膜、卵黄囊中胚层和绒毛膜中胚层部分的生成,而不参与生成卵黄囊内胚层和胎盘滋养层细胞谱系,即二者的发育潜能具有互补性,这种现象称之为四倍体互补技术。
在一些情况中,术语“胚胎”是指来源于出生前任意时间的动物受试者或来源于出生前任意时间的动物受试者体内的组织和细胞。
在一些情况中,“胚胎”被用于描述在受精后八周变成胎儿前移植入子宫后的受精***。根据该定义,受精的***在进行移植前通常被称为胚前期(pre-embryo)。然而,贯穿该专利申请,我们将使用更宽的术语胚胎定义,其包括胚前期阶段。因此其包括从***受精到桑葚胚、胚泡期孵化和植入的所有发育阶段。
本文中使用的术语“嵌合体囊胚”或“嵌合体胚胎”指的是包含胚胎干细胞的处于嵌合体状态的囊胚或胚胎。该嵌合体囊胚或胚胎除了通过注射方法生产以外,还可使用例如所谓“聚集方法”生产,在该“聚集方法”中,令胚胎+胚胎、或胚胎+细胞在有盖培养皿中彼此紧密粘附,以生产嵌合体囊胚。
本文所用术语“胚胎干细胞”或"ES细胞”包括在引入胚胎中后能够促进发育胚胎的任何组织的源自胚胎的全能或多潜能细胞。本文所述的胚胎干细胞可以为普通的胚胎干细胞,也可以为基因编辑的胚胎干细胞,例如人源化基因的动物胚胎干细胞等,具体不限。
为使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明,并不用于构成对本发明的任何限制。此外,在以下说明中,省略了对公知结构和技术的描述,以避免不必要地混淆本公开的概念。这样的结构和技术在许多出版物中也进行了描述。
实施例
1、小鼠胚胎干细胞培养
将C57小鼠胚胎干细胞弃掉原有培养液,加入500μL PBS(12孔)洗一遍细胞,然后加入200μL Accutase(12孔板)消化,晃匀。放入37℃培养箱消化5-8min。5min后取出培养板在镜下观察消化情况,当克隆消化完全呈葡萄状时加入200μL(12孔板)ESC培养基(mES)终止消化;力度柔和并快速得吹打细胞,切勿吹出气泡。将细胞悬液收集到1.5mL EP管中,250g离心5min。弃上清,加入200μL ESC培养基重悬细胞。细胞计数,12孔种1万细胞,把细胞种到提前种有CF-1的饲养层细胞上,分别采用如下的培养基进行培养。其中,
①号培养基(mES)配方:KnockoutDMEM+15%FBS(Gibco)+Glutamax(Gibco,100x)+NEAA(Gibco,100x)+β-me+LIF(Millipore)+1μM pD0325901(Selleck,S1036)+3umChir99021(selleck)。
②号培养基(TetrESC)配方:mES培养基+1nM剪切体抑制剂plaB+50μg/ml维生素C+1μM Y27632(Selleck,S1049)。
③号培养基(TetrESC-plaB)配方:mES培养基+50μg/ml维生素C+1μM Y27632(Selleck,S1049)。
④号培养基(TetrESC-Vc)配方:mES培养基+1nM剪切体抑制剂plaB+1μM Y27632(Selleck,S1049)。
⑤号培养基(TetrESC-γ)配方:mES培养基+1nM剪切体抑制剂plaB+50μg/ml维生素C。
2、四倍体小鼠的形成
1)、腹腔注射7.5单位孕马血清***(PMSG)到4-10周龄B6C3F1雌鼠,48小时后注射人绒毛膜***(hCG)并与CD1种公鼠合笼,次日早上检查雌鼠阴栓并将有阴栓的雌鼠挑出,记录雌鼠相应的受精时间。
2)、下一日将孕鼠进行颈椎脱臼法安乐死,70%酒精消毒小鼠腹部,用辅料镊子和眼科剪刀剪开腹部皮肤和肌肉层,打开腹腔。用镊子抓住一个子宫角的上部,用剪刀在靠近输卵管的膜上开一个小口,将输卵管和卵巢连接处剪断,将输卵管和附带的子宫移至35mm培养皿中,用镊子固定好输卵管伞口端,用装填好M2培养液(Hogan,1994)的冲洗针轻轻***伞口,用0.1mL的M2培养液冲洗输卵管,用移卵管收集冲出的胚胎并用M2清洗3次,收集E1.5的小鼠2-细胞胚胎。
3)、对收集到的小鼠胚胎放入含0.1mM MgSO4、0.1mM CaCl2和0.3%牛血清白蛋白的0.3M甘露醇(Sigma-Aldrich Inc.,St.Louis,MO)中,用Cellfusion CF-150/B电融合仪和250-um融合槽(BLS Ltd.,Budapest,Hungary)进行60V 50微秒直流电融合,获得4倍体胚胎;放入KSOM培养基,于CO2培养箱中培养24小时后用酸性台氏液(Sigma-Aldrich,T1788)去掉透明带,与上述步骤1获得的胚胎干细胞聚集,形成嵌合体胚胎。
4)、于CO2培养箱中培养过夜,移植到E2.5天的假孕鼠子宫,17天后在将***鼠颈椎脱臼法安乐死后进行剖腹手术,统计仔鼠出生率。ESC四倍体出生情况如图1所示。
结果显示,采用含有本发明的培养基添加剂的培养基培养四倍体胚胎干细胞显著提升了四倍体的出生率,可达25%以上。
本发明的技术方案不限于上述具体实施例的限制,凡是根据本发明的技术方案做出的技术变形,均落入本发明的保护范围之内。
Claims (15)
1.一种胚胎干细胞培养用的培养基添加剂,所述培养基添加剂为剪切体抑制剂、维生素C和ROCK抑制剂,所述剪切体抑制剂选自plaB、FR901464、E7107和GEX1A中的至少一种;所述ROCK抑制剂选自Y-27632和H-1152中的至少一种。
2. 根据权利要求1所述的培养基添加剂,其特征在于,所述培养基添加剂为:0.5-5nM剪切体抑制剂;20-100μg/ml 维生素C;和0.5-5μM ROCK抑制剂。
3. 根据权利要求1所述的培养基添加剂,其特征在于,所述培养基添加剂为:0.5-2nM剪切体抑制剂;40-60μg/ml 维生素C;和0.5-2μM ROCK抑制剂。
4. 根据权利要求1所述的培养基添加剂,其特征在于,所述培养基添加剂为1nM 剪切体抑制剂、50μg/ml 维生素C和1μM ROCK抑制剂。
5.权利要求1-4中任一项所述的培养基添加剂在非人哺乳动物的胚胎干细胞培养或制备非人哺乳动物的动物胚胎中的应用,所述培养基添加剂用于四倍体胚胎互补法或四倍体补偿法中的胚胎干细胞培养,所述动物胚胎为非人哺乳动物四倍体胚胎。
6.根据权利要求5所述的应用,其特征在于,所述胚胎干细胞为经基因编辑的胚胎干细胞。
7.根据权利要求5所述的应用,其特征在于,所述动物为基因编辑动物。
8.一种胚胎干细胞培养基,其包括基础培养基和权利要求1-4中任一项所述的培养基添加剂。
9.根据权利要求8所述的胚胎干细胞培养基,其特征在于,所述胚胎干细胞培养基还包括胎牛血清、Glutamax、非必需氨基酸、β-巯基乙醇、LIF、pD0325901和Chir99021中的一种或多种。
10. 根据权利要求8所述的胚胎干细胞培养基,其特征在于,所述胚胎干细胞培养基还包括:15%胎牛血清、Glutamax、非必需氨基酸、β-巯基乙醇、LIF、1μM pD0325901和3μMChir99021,其中所述基础培养基为DMEM基础培养基。
11.权利要求8-10中任一项所述的胚胎干细胞培养基在非人哺乳动物的胚胎干细胞培养或制备非人哺乳动物的动物胚胎中的应用,所述胚胎干细胞培养基用于四倍体胚胎互补法或四倍体补偿法中的胚胎干细胞培养,所述动物胚胎为非人哺乳动物四倍体胚胎。
12.根据权利要求11所述的应用,其特征在于,所述胚胎干细胞为经基因编辑的胚胎干细胞。
13.根据权利要求11所述的应用,其特征在于,所述动物为基因编辑动物。
14.一种非人哺乳动物的四倍体胚胎或嵌合体胚胎的制备方法,其包括使用权利要求1-4中任一项所述的培养基添加剂或权利要求8-10中任一项所述的培养基培养胚胎干细胞。
15.根据权利要求14所述的方法,其特征在于,所述制备方法包括将四倍体胚胎与所述胚胎干细胞聚合得到嵌合体胚胎。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210949539.6A CN115322958B (zh) | 2022-08-09 | 2022-08-09 | 胚胎干细胞培养用的培养基添加剂及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210949539.6A CN115322958B (zh) | 2022-08-09 | 2022-08-09 | 胚胎干细胞培养用的培养基添加剂及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115322958A CN115322958A (zh) | 2022-11-11 |
CN115322958B true CN115322958B (zh) | 2023-08-01 |
Family
ID=83921723
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210949539.6A Active CN115322958B (zh) | 2022-08-09 | 2022-08-09 | 胚胎干细胞培养用的培养基添加剂及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115322958B (zh) |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2001294842A1 (en) * | 2000-09-28 | 2002-04-08 | Atairgin Technologies, Inc. | A method of determining tumor characteristics by determining abnormal copy number or expression level of lipid-associated genes |
CN1852974A (zh) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | 用于治疗和诊断癌症的组合物和方法 |
CA2682439A1 (en) * | 2008-10-17 | 2010-04-17 | F. Hoffmann-La Roche Ag | Cell monitoring and molecular analysis |
WO2014066848A1 (en) * | 2012-10-25 | 2014-05-01 | Whitehead Institute For Biomedical Research | Super-enhancers and methods of use thereof |
US20180156780A1 (en) * | 2015-05-26 | 2018-06-07 | Children's Medical Center Corporation | Compositions and methods for modulating oncogenic mirna |
IL262658A (en) * | 2018-10-28 | 2020-04-30 | Memorial Sloan Kettering Cancer Center | Prevention of age related clonal hematopoiesis and diseases associated therewith |
-
2022
- 2022-08-09 CN CN202210949539.6A patent/CN115322958B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN115322958A (zh) | 2022-11-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11773368B2 (en) | Culture method for differentiating primordial germ cells into functionally mature oocytes | |
Brevini et al. | Porcine embryonic stem cells: Facts, challenges and hopes | |
De Paepe et al. | Totipotency and lineage segregation in the human embryo | |
Irie et al. | Germ cell specification and pluripotency in mammals: a perspective from early embryogenesis | |
Oestrup et al. | From zygote to implantation: morphological and molecular dynamics during embryo development in the pig | |
Hall et al. | Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals | |
Behboodi et al. | Establishment of goat embryonic stem cells from in vivo produced blastocyst‐stage embryos | |
JP2022529589A (ja) | 哺乳類の拡大ポテンシャル幹細胞のための培地、その組成物および方法 | |
KR20100014504A (ko) | 분화된 세포를 리프로그래밍하고, 리프로그래밍된 세포로부터 동물 및 배아 줄기세포를 생성시키기 위한 고도로 효율적인 방법 | |
Huang et al. | Establishment of bovine trophoblast stem-like cells from in vitro-produced blastocyst-stage embryos using two inhibitors | |
BR112020013546A2 (pt) | derivação eficiente de células-tronco embrionárias pluripotentes estáveis de bovinos | |
US20070298496A1 (en) | Method of deriving pluripotent stem cells from a single blastomere | |
CN115322958B (zh) | 胚胎干细胞培养用的培养基添加剂及其应用 | |
Botigelli et al. | In vitro gametogenesis from embryonic stem cells in livestock species: recent advances, opportunities, and challenges to overcome | |
Renard et al. | Nuclear reprogramming and pluripotency of embryonic cells: Application to the isolation of embryonic stem cells in farm animals | |
Familari et al. | The potential for derivation of embryonic stem cells in vertebrates | |
Lee et al. | Aggregation of cloned embryos in empty zona pellucida improves derivation efficiency of pig ES-like cells | |
US20170283777A1 (en) | Mammalian chimeric complementation | |
US20080044395A1 (en) | In Vitro Method for Isolating, Proliferating and Differentiating Germ-Line Stem Cells | |
CN115466718B (zh) | 雌性胚胎干细胞及其制备方法和应用 | |
US20200157496A1 (en) | Method of differentiating hair follicle cell into germline stem cell and use thereof | |
KR101374190B1 (ko) | 돼지배아줄기세포 및 돼지 미성숙 난자로부터 돼지배아줄기세포를 제조하는 방법 | |
WO2015152146A1 (ja) | 1倍体胚性幹細胞の培養方法 | |
KR102140149B1 (ko) | 비근교계 마우스 유래 배아줄기세포주 및 이의 제조방법 | |
TWI392736B (zh) | 由單一***球取得多能幹細胞之方法 |
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 |