CN114369577B - 牛诱导扩展多能性成体干细胞、建系方法及培养液 - Google Patents
牛诱导扩展多能性成体干细胞、建系方法及培养液 Download PDFInfo
- Publication number
- CN114369577B CN114369577B CN202011105359.7A CN202011105359A CN114369577B CN 114369577 B CN114369577 B CN 114369577B CN 202011105359 A CN202011105359 A CN 202011105359A CN 114369577 B CN114369577 B CN 114369577B
- Authority
- CN
- China
- Prior art keywords
- bovine
- adult stem
- induced expansion
- multipotent adult
- culture solution
- 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
- 241000283690 Bos taurus Species 0.000 title claims abstract description 69
- 210000004504 adult stem cell Anatomy 0.000 title claims abstract description 42
- 238000010276 construction Methods 0.000 title abstract description 12
- 210000000130 stem cell Anatomy 0.000 claims abstract description 18
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 41
- 239000002609 medium Substances 0.000 claims description 17
- 150000001413 amino acids Chemical class 0.000 claims description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 8
- 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 claims description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 8
- ZGSXEXBYLJIOGF-ALFLXDJESA-N IWR-1-endo Chemical compound C=1C=CC2=CC=CN=C2C=1NC(=O)C(C=C1)=CC=C1N1C(=O)[C@@H]2[C@H](C=C3)C[C@H]3[C@@H]2C1=O ZGSXEXBYLJIOGF-ALFLXDJESA-N 0.000 claims description 8
- KLGQSVMIPOVQAX-UHFFFAOYSA-N XAV939 Chemical compound N=1C=2CCSCC=2C(O)=NC=1C1=CC=C(C(F)(F)F)C=C1 KLGQSVMIPOVQAX-UHFFFAOYSA-N 0.000 claims description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 8
- 102000004127 Cytokines Human genes 0.000 claims description 7
- 108090000695 Cytokines Proteins 0.000 claims description 7
- 101100239628 Danio rerio myca gene Proteins 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 6
- 101150037203 Sox2 gene Proteins 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims description 6
- 101001109685 Homo sapiens Nuclear receptor subfamily 5 group A member 2 Proteins 0.000 claims description 5
- 101000984042 Homo sapiens Protein lin-28 homolog A Proteins 0.000 claims description 5
- 101001132658 Homo sapiens Retinoic acid receptor gamma Proteins 0.000 claims description 5
- 102100022669 Nuclear receptor subfamily 5 group A member 2 Human genes 0.000 claims description 5
- 102100025460 Protein lin-28 homolog A Human genes 0.000 claims description 5
- 102100033912 Retinoic acid receptor gamma Human genes 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 4
- 229930003268 Vitamin C Natural products 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 229960005322 streptomycin Drugs 0.000 claims description 4
- 235000019154 vitamin C Nutrition 0.000 claims description 4
- 239000011718 vitamin C Substances 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 229960003722 doxycycline Drugs 0.000 claims description 2
- XQTWDDCIUJNLTR-CVHRZJFOSA-N doxycycline monohydrate Chemical compound O.O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O XQTWDDCIUJNLTR-CVHRZJFOSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 abstract description 7
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 4
- 241001465754 Metazoa Species 0.000 abstract description 3
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000010367 cloning Methods 0.000 abstract description 2
- 238000009509 drug development Methods 0.000 abstract description 2
- 238000010362 genome editing Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000001850 reproductive effect Effects 0.000 abstract description 2
- 230000029087 digestion Effects 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 210000001671 embryonic stem cell Anatomy 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- CRDNMYFJWFXOCH-YPKPFQOOSA-N (3z)-3-(3-oxo-1h-indol-2-ylidene)-1h-indol-2-one Chemical compound N/1C2=CC=CC=C2C(=O)C\1=C1/C2=CC=CC=C2NC1=O CRDNMYFJWFXOCH-YPKPFQOOSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 108010019160 Pancreatin Proteins 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229940055695 pancreatin Drugs 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 206010043276 Teratoma Diseases 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 230000008672 reprogramming Effects 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 210000000689 upper leg Anatomy 0.000 description 3
- CRDNMYFJWFXOCH-BUHFOSPRSA-N Couroupitine B Natural products N\1C2=CC=CC=C2C(=O)C/1=C1/C2=CC=CC=C2NC1=O CRDNMYFJWFXOCH-BUHFOSPRSA-N 0.000 description 2
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 description 2
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 2
- 102100022975 Glycogen synthase kinase-3 alpha Human genes 0.000 description 2
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 2
- -1 IQ-L Chemical compound 0.000 description 2
- 239000002067 L01XE06 - Dasatinib Substances 0.000 description 2
- 239000002137 L01XE24 - Ponatinib Substances 0.000 description 2
- 229930192392 Mitomycin Natural products 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 230000004156 Wnt signaling pathway Effects 0.000 description 2
- 108010023082 activin A Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 229960002448 dasatinib Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 108010049611 glycogen synthase kinase 3 alpha Proteins 0.000 description 2
- CRDNMYFJWFXOCH-UHFFFAOYSA-N isoindigotin Natural products N1C2=CC=CC=C2C(=O)C1=C1C2=CC=CC=C2NC1=O CRDNMYFJWFXOCH-UHFFFAOYSA-N 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229960001131 ponatinib Drugs 0.000 description 2
- PHXJVRSECIGDHY-UHFFFAOYSA-N ponatinib Chemical compound C1CN(C)CCN1CC(C(=C1)C(F)(F)F)=CC=C1NC(=O)C1=CC=C(C)C(C#CC=2N3N=CC=CC3=NC=2)=C1 PHXJVRSECIGDHY-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000002993 trophoblast Anatomy 0.000 description 2
- HQWTUOLCGKIECB-XZWHSSHBSA-N (6S,9aS)-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-3,6,9,9a-tetrahydro-2H-pyrazino[1,2-a]pyrimidine-1-carboxamide Chemical compound C1=CC(O)=CC=C1C[C@H]1C(=O)N(CC=2C3=CC=CC=C3C=CC=2)C[C@H]2N1C(=O)CCN2C(=O)NCC1=CC=CC=C1 HQWTUOLCGKIECB-XZWHSSHBSA-N 0.000 description 1
- AXNUEXXEQGQWPA-UHFFFAOYSA-N 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide Chemical compound CC1=CC([N+]([O-])=O)=CC=C1NS(=O)(=O)C1=CC(Cl)=CC=C1Cl AXNUEXXEQGQWPA-UHFFFAOYSA-N 0.000 description 1
- QTRXIFVSTWXRJJ-UHFFFAOYSA-N 2-(1,3-dimethyl-2,6-dioxopurin-7-yl)-n-(6-phenylpyridazin-3-yl)acetamide Chemical compound C1=2C(=O)N(C)C(=O)N(C)C=2N=CN1CC(=O)NC(N=N1)=CC=C1C1=CC=CC=C1 QTRXIFVSTWXRJJ-UHFFFAOYSA-N 0.000 description 1
- JJAXTFSPCLZPIW-UHFFFAOYSA-N 2-(2,3,4-trihydroxyphenyl)chromen-4-one Chemical compound OC1=C(O)C(O)=CC=C1C1=CC(=O)C2=CC=CC=C2O1 JJAXTFSPCLZPIW-UHFFFAOYSA-N 0.000 description 1
- QESQGTFWEQMCMH-UHFFFAOYSA-N 2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)sulfanyl]-n-(5-phenylpyridin-2-yl)acetamide Chemical compound C=1C=C(C=2C=CC=CC=2)C=NC=1NC(=O)CSC1=NC=2CCSC=2C(=O)N1C1=CC=CC=C1 QESQGTFWEQMCMH-UHFFFAOYSA-N 0.000 description 1
- RNUXIZKXJOGYQP-UHFFFAOYSA-N 2-[3-[[4-(4-methoxyphenyl)-5-pyridin-4-yl-1,2,4-triazol-3-yl]sulfanyl]propyl]benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(OC)=CC=C1N1C(C=2C=CN=CC=2)=NN=C1SCCCN(C1=O)C(=O)C2=C3C1=CC=CC3=CC=C2 RNUXIZKXJOGYQP-UHFFFAOYSA-N 0.000 description 1
- KHZOJCQBHJUJFY-UHFFFAOYSA-N 2-[4-(2-methylpyridin-4-yl)phenyl]-n-(4-pyridin-3-ylphenyl)acetamide Chemical compound C1=NC(C)=CC(C=2C=CC(CC(=O)NC=3C=CC(=CC=3)C=3C=NC=CC=3)=CC=2)=C1 KHZOJCQBHJUJFY-UHFFFAOYSA-N 0.000 description 1
- JSQPAEVPPSCQMV-UHFFFAOYSA-N 2-[4-(2-methylsulfonylphenyl)piperazin-1-yl]-3,5,7,8-tetrahydrothiopyrano[4,3-d]pyrimidin-4-one Chemical compound CS(=O)(=O)C1=CC=CC=C1N1CCN(C=2NC=3CCSCC=3C(=O)N=2)CC1 JSQPAEVPPSCQMV-UHFFFAOYSA-N 0.000 description 1
- RGTAEYDIDMGJLX-UHFFFAOYSA-N 3-(3-aminophenyl)-4-(1-methylindol-3-yl)pyrrole-2,5-dione Chemical compound C12=CC=CC=C2N(C)C=C1C(C(NC1=O)=O)=C1C1=CC=CC(N)=C1 RGTAEYDIDMGJLX-UHFFFAOYSA-N 0.000 description 1
- VPVLEBIVXZSOMQ-UHFFFAOYSA-N 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol Chemical compound NC1=CC=CC(C=2NC3=NC=NC(OC=4C=C(O)C=CC=4)=C3C=2)=C1 VPVLEBIVXZSOMQ-UHFFFAOYSA-N 0.000 description 1
- ZUQIFHLBPBLRRM-QPJJXVBHSA-N 4-[(e)-3-phenylprop-2-enyl]-n-(4-sulfamoylphenyl)piperazine-1-carbothioamide Chemical compound C1=CC(S(=O)(=O)N)=CC=C1NC(=S)N1CCN(C\C=C\C=2C=CC=CC=2)CC1 ZUQIFHLBPBLRRM-QPJJXVBHSA-N 0.000 description 1
- FYWRWBSYRGSWIQ-UHFFFAOYSA-N 4-[2-(3H-benzimidazol-5-ylamino)quinazolin-8-yl]oxycyclohexan-1-ol Chemical compound C1CC(O)CCC1OC(C1=N2)=CC=CC1=CN=C2NC1=CC=C(N=CN2)C2=C1 FYWRWBSYRGSWIQ-UHFFFAOYSA-N 0.000 description 1
- HIWVLHPKZNBSBE-OUKQBFOZSA-N 4-[5-[(e)-2-[4-(2-chlorophenyl)-5-(5-methylsulfonylpyridin-2-yl)-1,2,4-triazol-3-yl]ethenyl]-1,3,4-oxadiazol-2-yl]benzonitrile Chemical compound N1=CC(S(=O)(=O)C)=CC=C1C(N1C=2C(=CC=CC=2)Cl)=NN=C1\C=C\C1=NN=C(C=2C=CC(=CC=2)C#N)O1 HIWVLHPKZNBSBE-OUKQBFOZSA-N 0.000 description 1
- ZCCPLJOKGAACRT-UHFFFAOYSA-N 4-methyl-3-[[1-methyl-6-(3-pyridinyl)-4-pyrazolo[3,4-d]pyrimidinyl]amino]-N-[3-(trifluoromethyl)phenyl]benzamide Chemical compound CC1=CC=C(C(=O)NC=2C=C(C=CC=2)C(F)(F)F)C=C1NC(C=1C=NN(C)C=1N=1)=NC=1C1=CC=CN=C1 ZCCPLJOKGAACRT-UHFFFAOYSA-N 0.000 description 1
- PQXINDBPUDNMPE-UHFFFAOYSA-N 5-(furan-2-yl)-n-(3-imidazol-1-ylpropyl)-1,2-oxazole-3-carboxamide Chemical compound C1=C(C=2OC=CC=2)ON=C1C(=O)NCCCN1C=CN=C1 PQXINDBPUDNMPE-UHFFFAOYSA-N 0.000 description 1
- BLTVBQXJFVRPFK-UHFFFAOYSA-N AZD1080 Chemical compound OC=1NC2=CC=C(C#N)C=C2C=1C(N=C1)=CC=C1CN1CCOCC1 BLTVBQXJFVRPFK-UHFFFAOYSA-N 0.000 description 1
- FHCSBLWRGCOVPT-UHFFFAOYSA-N AZD2858 Chemical compound C1CN(C)CCN1S(=O)(=O)C1=CC=C(C=2N=C(C(N)=NC=2)C(=O)NC=2C=NC=CC=2)C=C1 FHCSBLWRGCOVPT-UHFFFAOYSA-N 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- 102000006277 CDX2 Transcription Factor Human genes 0.000 description 1
- 108010083123 CDX2 Transcription Factor Proteins 0.000 description 1
- MDZCSIDIPDZWKL-UHFFFAOYSA-N CHIR-98014 Chemical compound C1=C([N+]([O-])=O)C(N)=NC(NCCNC=2N=C(C(=CN=2)N2C=NC=C2)C=2C(=CC(Cl)=CC=2)Cl)=C1 MDZCSIDIPDZWKL-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 1
- 102100026818 Inhibin beta E chain Human genes 0.000 description 1
- XXYGTCZJJLTAGH-UHFFFAOYSA-N LGK974 Chemical compound C1=NC(C)=CC(C=2C(=CC(CC(=O)NC=3N=CC(=CC=3)C=3N=CC=NC=3)=CN=2)C)=C1 XXYGTCZJJLTAGH-UHFFFAOYSA-N 0.000 description 1
- HRJWTAWVFDCTGO-UHFFFAOYSA-N LY-2090314 Chemical compound C1CN(C=23)C=C(C=4C(NC(=O)C=4C=4N5C=CC=CC5=NC=4)=O)C3=CC(F)=CC=2CN1C(=O)N1CCCCC1 HRJWTAWVFDCTGO-UHFFFAOYSA-N 0.000 description 1
- WRKPZSMRWPJJDH-UHFFFAOYSA-N N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thio]acetamide Chemical compound S1C2=CC(C)=CC=C2N=C1NC(=O)CSC1=NC=2CCSC=2C(=O)N1C1=CC=CC=C1 WRKPZSMRWPJJDH-UHFFFAOYSA-N 0.000 description 1
- 238000011789 NOD SCID mouse Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- PQCXVIPXISBFPN-UHFFFAOYSA-N SB 415286 Chemical compound C1=C(Cl)C(O)=CC=C1NC1=C(C=2C(=CC=CC=2)[N+]([O-])=O)C(=O)NC1=O PQCXVIPXISBFPN-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- JDSJDASOXWCHPN-UHFFFAOYSA-N TDZD-8 Chemical compound O=C1N(C)SC(=O)N1CC1=CC=CC=C1 JDSJDASOXWCHPN-UHFFFAOYSA-N 0.000 description 1
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 1
- HUDSYNWJCPDHLL-CJLVFECKSA-N [(E)-[2-(6-bromo-2-hydroxy-1H-indol-3-yl)indol-3-ylidene]amino] acetate Chemical compound CC(=O)O\N=C1\C(=Nc2ccccc12)c1c(O)[nH]c2cc(Br)ccc12 HUDSYNWJCPDHLL-CJLVFECKSA-N 0.000 description 1
- VHOZWHQPEJGPCC-AZXNYEMZSA-N [4-[[(6s,9s,9as)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-(quinolin-8-ylmethyl)-3,6,9,9a-tetrahydropyrazino[2,1-c][1,2,4]triazin-6-yl]methyl]phenyl] dihydrogen phosphate Chemical compound C([C@@H]1N2[C@@H](N(N(C)CC2=O)C(=O)NCC=2C=CC=CC=2)[C@@H](N(C1=O)CC=1C2=NC=CC=C2C=CC=1)C)C1=CC=C(OP(O)(O)=O)C=C1 VHOZWHQPEJGPCC-AZXNYEMZSA-N 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960003736 bosutinib Drugs 0.000 description 1
- UBPYILGKFZZVDX-UHFFFAOYSA-N bosutinib Chemical compound C1=C(Cl)C(OC)=CC(NC=2C3=CC(OC)=C(OCCCN4CCN(C)CC4)C=C3N=CC=2C#N)=C1Cl UBPYILGKFZZVDX-UHFFFAOYSA-N 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- YAEMHJKFIIIULI-UHFFFAOYSA-N n-(4-methoxybenzyl)-n'-(5-nitro-1,3-thiazol-2-yl)urea Chemical compound C1=CC(OC)=CC=C1CNC(=O)NC1=NC=C([N+]([O-])=O)S1 YAEMHJKFIIIULI-UHFFFAOYSA-N 0.000 description 1
- LXFKEVQQSKQXPR-UHFFFAOYSA-N n-(6-chloro-1,3-benzothiazol-2-yl)-3-(3,4-dimethoxyphenyl)propanamide Chemical compound C1=C(OC)C(OC)=CC=C1CCC(=O)NC1=NC2=CC=C(Cl)C=C2S1 LXFKEVQQSKQXPR-UHFFFAOYSA-N 0.000 description 1
- JJEDWBQZCRESJL-DEDYPNTBSA-N n-[(e)-(5-methylfuran-2-yl)methylideneamino]-2-phenoxybenzamide Chemical compound O1C(C)=CC=C1\C=N\NC(=O)C1=CC=CC=C1OC1=CC=CC=C1 JJEDWBQZCRESJL-DEDYPNTBSA-N 0.000 description 1
- PHUNRLYHXGMOLG-WQRRWHLMSA-M potassium 3-[(5Z)-5-[[5-(4-nitrophenyl)furan-2-yl]methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]propanoate Chemical compound [K+].[O-]C(=O)CCN1C(=S)S\C(=C/c2ccc(o2)-c2ccc(cc2)[N+]([O-])=O)C1=O PHUNRLYHXGMOLG-WQRRWHLMSA-M 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- OUKYUETWWIPKQR-UHFFFAOYSA-N saracatinib Chemical compound C1CN(C)CCN1CCOC1=CC(OC2CCOCC2)=C(C(NC=2C(=CC=C3OCOC3=2)Cl)=NC=N2)C2=C1 OUKYUETWWIPKQR-UHFFFAOYSA-N 0.000 description 1
- 229950009919 saracatinib Drugs 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- PMJIHLSCWIDGMD-UHFFFAOYSA-N tideglusib Chemical compound O=C1SN(C=2C3=CC=CC=C3C=CC=2)C(=O)N1CC1=CC=CC=C1 PMJIHLSCWIDGMD-UHFFFAOYSA-N 0.000 description 1
- 229950005284 tideglusib Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-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
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
-
- 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
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/602—Sox-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
- C12N2501/60—Transcription factors
- C12N2501/603—Oct-3/4
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/604—Klf-4
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/605—Nanog
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/606—Transcription factors c-Myc
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/608—Lin28
-
- 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
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- 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)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Transplantation (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种牛诱导扩展多能性成体干细胞、建系方法及培养液，通过从牛成纤维体细胞中转基因、诱导培养得到，并能够在体外稳定传代培养。该干细胞具有全能性、稳定性和安全性，能在体内外向胚胎和胚胎外组织分化，可用于育种繁育、基因编辑、动物克隆、医学模型、药物开发载体和诱导生产牛生殖配子等多种生命科学以及医学领域的应用。
Description
技术领域
本发明属于细胞生物学和分子生物学技术领域，具体涉及一种牛诱导扩展多能性成体干细胞、建系方法及培养液。
背景技术
诱导多能干细胞(iPSCs)是将特定转录因子导入到体细胞，诱导其细胞核重编程而得到的干细胞，是通过在分化的细胞中表达特定的几个转录因子，诱导体细胞的重编程而获得的可不断自我更新且具有多向分化潜能的细胞。诱导多能干细胞(iPSCs)除了可以无限的自我更新并能分化为包括三个胚层在内的所有细胞类型外，最大的特点是不需使用胎儿或胚胎，而是将分化细胞通过基因表达的调整直接重编程到多能性阶段，在研究体细胞重编程和再生医学方面具有深远影响。
目前研究已经报道了小鼠(Establishment in culture of pluripotentialcells from mouse embryos[J].Nature，1981，292(5819):154-156.)、人(Embryonic StemCell Lines Derived from Human Blastocysts[J].Science，1998，282(5391):1145-1147.)、大鼠(Germline Competent Embryonic Stem Cells Derived from RatBlastocysts[J].Cell，2008，135(7):1299-1310.)、猪(Establishment of porcine andhuman expanded potential stem cells[J].Nature Cell Biology，2019，21(6):687-699.)、羊、马、牛的iPS细胞，但大动物iPS的多能性特别是嵌合体形成和生殖细胞传代还没有得到确认。李荣风等(Establishment of bovine trophoblast stem-Like cells fromIn vitro-produced blastocyst-stage embryos using two inhibitors[J].Stem Cellsand Development，2014，23(13):1501-1514.)研究报道从附植前的胚胎细胞中获得牛滋养层干细胞(BTSCs)，该细胞注射NOD-SCID小鼠能形成畸胎瘤，并在体外分化成胎盘样细胞。吴侠等(Establishment of bovine embryonic stem cells after knockdown of CDX2[J].Scientific Reports，2016，6(1):28343-28343.)从CDX2敲出的胚胎中获得牛扩展多能性胚胎干细胞(CDX2-KD bESCs)，具有体内外分化能力，但没有形成嵌合体。赵丽霞等(Characterization of the single-cell derived bovine induced pluripotent stemcells[J].Tissue&Cell，2017，49(5):521-527)转入牛源四因子Oct4、Sox2、Klf4、cMyc到牛体细胞中获得具有三胚层分化能力的牛诱导多功能干细胞。Yanina Soledad Bogliotti等(Efficient derivation of stable primed pluripotent embryonic stem cells frombovine blastocysts[J].Proceedings of the National Academy of Sciences of theUnited States of America，2018，115(9):2090-2095.)从牛胚胎中获得了激发态胚胎干细胞(primed bESCs)，该干细胞没有报道形成嵌合体，其全能性不如小鼠原始态胚胎干细胞(naive mESCs)。
发明内容
本研究首次从牛成纤维体细胞中转基因、诱导培养得到牛诱导扩展多能性成体干细胞(bEPSCiPS)，该干细胞能在体内外中向胚胎和胚胎外组织分化，具有全能性、稳定性和安全性。
上述技术问题，本发明通过以下技术方案实现：
一种牛诱导扩展多能性成体干细胞的建系方法，包括以下步骤:
(a)解冻牛成纤维细胞(BEF)，在牛成纤维细胞培养液上培养，待细胞汇合度达到80％以上，收集细胞计数1×106；
(b)转入外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG，在提前解冻的饲养层细胞上培养，培养条件为M15+DOX培养液；
(c)待干细胞克隆长出后，挑入细胞因子培养液培养，隔天换液，1到5天构建成牛诱导扩展多能性成体干细胞系。
优选地，所述外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG的核苷酸序列如SEQ.ID.NO：1-8所示。
优选地，所述饲养层细胞的制备方法为：解冻牛成纤维细胞，在牛成纤维细胞培养液上培养，待细胞汇合度达到80％以上，按照1：16进行细胞传代，待细胞的汇合度再次达到80％时，添加10μg/ml的丝裂霉素于培养箱中培养2.5-3h，胰酶消化后用BEF培养液中止消化，离心并去上清，经含10％DMSO、10％胎牛血清和80％牛成纤维细胞培养液的培养液重悬并冻存为饲养层细胞。
优选地，所述步骤(b)中的提前解冻的饲养层细胞为实验前一天，解冻冻存的饲养层细胞并将其接种于铺有0.1％明胶的培养皿中用牛成纤维细胞培养液培养。
优选地，所述BEF培养液是在DMEM培养基中，添加10％FBS，1×谷氨酰胺，1×非必需氨基酸，1×青链霉素。
优选地，所述M15+DOX培养液是在DMEM培养基中，添加15％FBS、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、强力霉素Dox。
一种牛诱导扩展多能性成体干细胞建系用细胞因子培养液，所述细胞因子培养液包括：(1)有效量的成纤维细胞生长因子或其等效物；(2)有效量的一种或多种Wnt信号通路抑制剂；(3)有效量的一种或多种GSK-3α/β信号通路抑制剂；(4)有效量的一种或多种Lck/Src信号通路抑制剂；(5)有效量的Activin A蛋白或其等效物。
优选地，所述Wnt信号通路抑制剂为IWR-1、XAV-939、ICG-001、Wnt-C59、LGK-974、LF3、CP21R7、NCB-0846、PNU-74654、SKL2001、KY02111、IWP-2、IWP-L6、FH535、WIKI4、PRI-724、IQ-l、KYA1797K、C59、ETC-159、G007-LK、G244-LM中的一种或多种。
优选地，所述GSK-3α/β信号通路抑制剂为CHIR99021、B216763、AT7519、CHIR-98014、TWS119、Tideglusib、SB415286、AZD2858、AZD1080、AR-A014418、TDZD-8、LY2090314、2-D08、6-溴靛玉红-3'-丙酮肟(BIO-acetoxime)、IM-12、1-氮杂坎帕罗酮(1-Azakenpaullone)、靛玉红(Indirubin)、Bikinin中的一种或多种。
优选地，所述Lck/Src信号通路抑制剂为WH-4-023、达沙替尼(Dasatinib)、塞卡替尼(Saracatinib)、普纳替尼(Ponatinib)、SKI-606、KX2-391(Tirbanibulin)、NVP-BHG712、PP2、PP121中的一种或多种；
优选地，所述细胞因子培养液组成为：
1×谷氨酰胺
1×非必需氨基酸
1×青链霉素
0.1mM 2-巯基乙醇
1μM CHIR99021
0.3μM WH-4-023
5μM XAV939 or IWR-1
50μg/ml vitamin C
10ng/ml LIF
20.0ng/ml Activin A
0.3％FBS
mTeSRTM1培养基
其中，IWR-1结构式为：
CHIR99021结构式为：
WH-4-023结构式为：
XAV939结构式为：
一种牛诱导扩展多能性成体干细胞的建系方法得到的牛诱导扩展多能性成体干细胞。
本发明的有益效果是：
(1)首次从牛成纤维体细胞中转基因、诱导培养得到牛诱导扩展多能性成体干细胞(bEPSCiPS)；
(2)构建的牛诱导扩展多能性成体干细胞，具有全能性、稳定性和安全性；
(3)利用该方法的牛诱导扩展多能性成体干细胞建系效率快并能够在体外稳定传代；
(4)该干细胞能在体内外中向胚胎和胚胎外组织分化；
(5)该方法制备的牛诱导扩展多能性成体干细胞能够用于育种繁育、基因编辑、动物克隆、医学模型、药物开发载体和诱导生产牛生殖配子等多种生命科学以及医学领域的应用。
附图说明
图1是本发明所述的牛诱导扩展多能性成体干细胞(克隆形态良好界限清晰，符合干细胞典型形态结构)；
图2是本发明所述的牛诱导扩展多能性成体干细胞AP染色图片(干细胞特有的特异性蛋白阳性表达)；
图3是本发明所述的牛诱导扩展多能性成体干细胞多能基因检测图片(关键多能基因阳性表达)；
图4是本发明所述的牛诱导扩展多能性成体干细胞畸胎瘤图片(具有异种三胚层分化能力)；
具体实施方式
实施例1
饲养层细胞的制备及培养
1.1饲养层细胞的制备及培养：
解冻牛成纤维细胞(BEF)，BEF培养液为含有10％FBS(BI)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素的Knockout DMEM培养基。待细胞汇合度达到80％以上，按照1：16进行细胞传代。待细胞的汇合度再次达到80％时，添加10μg/ml的丝裂霉素于培养箱中培养2.5-3h。胰酶消化后用BEF培养液中止消化，离心并去上清，经含10％DMSO、10％胎牛血清和80％BEF培养液的培养液重悬并冻存为饲养层细胞。
1.2饲养层细胞的培养：
实验前一天，解冻饲养层细胞并将其接种于铺有0.1％明胶的培养皿中用BEF培养液培养。
实施例2
牛诱导扩展多能性成体干细胞的建系、传代及冻存
2.1牛诱导扩展多能性成体干细胞的建系：
解冻牛成纤维细胞(BEF)，BEF培养液为含有10％FBS(BI)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素的Knockout DMEM培养基。待细胞汇合度达到80％以上，收集细胞计数1×106。转入外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG。转入的外源基因序列见SEQUENCE LISTING。在提前解冻好的饲养层细胞上培养，培养条件为M15+DOX培养液，M15+DOX培养液15％FBS(BI)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、强力霉素Dox的Knockout DMEM培养基。待干细胞克隆长出后挑入细胞因子培养液，培养条件为培养液含有1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、0.1mM 2-巯基乙醇、1μM CHIR99021(Tocris，cat.no.4423)、0.3μM WH-4-023(Tocris，cat.no.5413)、5μM XAV939(Sigma，cat.no.X3004)or 5μM IWR-1(Tocris，cat.no.3532)、50μg ml-1vitamin C(Sigma，cat.no.49752-100G)、10ng ml-1LIF(Millipore)，20.0ng ml-1Activin A(R&D)、0.3％FBS(Gibco，cat.no.10270)和mTeSRTM1(STEMCELL)培养基。
其中，IWR-1结构式为：
CHIR99021结构式为：
WH-4-023结构式为：
XAV939结构式为：
隔天换液，第1到5天牛诱导扩展多能性成体干细胞建系成功。细胞建系形态结果见图1，本发明所述的牛诱导扩展多能性成体干细胞克隆形态良好界限清晰，符合干细胞典型形态结构。
2.2牛诱导扩展多能性成体干细胞的传代培养：
待牛诱导扩展多能性成体干细胞的汇合度达到80％时，胰酶消化后用K10培养液中止消化。K10培养液含有10％KSR(GIBCO)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素的F12 DMEM培养基。按1：2或1:4隔日传代。
2.3牛诱导扩展多能性成体干细胞的冻存：
待牛诱导扩展多能性成体干细胞的汇合度达到80％时，胰酶消化后用K10培养液中止消化。
离心并去上清，经含10％DMSO、90％胎牛血清和培养液重悬并冻存。
实施例3
牛诱导扩展多能性成体干细胞的检测
3.1牛诱导扩展多能性成体干细胞AP染色：
牛诱导扩展多能性成体干细胞培养汇合度达到80％时，将样品细胞经柠檬酸盐(citrate)-丙酮(acetone)–甲醛(formaldehyde)固定后，按照碱性磷酸酶染色试剂盒[theAlkaline Phosphatase Kit(Sigma-Aldrich)]的操作说明严格进行。室温避光反应30-40分钟，镜检。检测结果见图2，AP染色结果显示碱性磷酸酶活性很强，该干细胞处于未分化状态。
3.2牛诱导扩展多能性成体干细胞多能基因检测：
参照Qiagen公司的RNeasy Mini Kit提取方法获得牛诱导扩展多能性成体干细胞的总RNA，并用RNase-Free水溶解。取其中1.8μl用于测量RNA的浓度和纯度后，吸取1μg RNA利用Qiagen公司的QuantiTect Reverse Transcription Kit试剂盒反转录合成20μl(50ng/μl)cDNA并用于定量PCR检测牛诱导扩展多能性成体干细胞多能基因OCT4，SOX2和Nanog的表达。
实时定量PCR(Q-PCR)的反应条件如下：94℃反应30s，60℃反应30s，and 68℃反应30s，循环反应30次。在所有的Q-PCR实验中，用到的Taqman Probe(Assay ID:Mm01232884_m1)和荧光定量PCR仪(9700HT Fast Real-Time PCR System)均购于Applied Biosciences公司。基因表达量的计算我们应用的是ΔCt算法，内参为GAPDH。检测结果见图3，如图所示牛诱导扩展多能性成体干细胞多能性基因的检测表达高于牛囊胚。
3.3牛诱导扩展多能性成体干细胞畸胎瘤实验
收集牛诱导扩展多能性成体干细胞1×106个到15ml离心管，1300rpm离心3min，弃上清，加500μlPBS吹悬，将细胞悬液吸入到1ml注射器。将免疫缺陷鼠从鼠房取出，请专业人员将小鼠用手固定抓牢，露出大腿，在大腿外侧先擦碘酒，再擦酒精，然后大腿外侧皮下注射细胞悬液。放回IVC***照看小鼠，一个月后观察小鼠大腿是否长出肿瘤，当肿瘤有豌豆大时，将小鼠解剖，肿瘤取出，用固定液固定好后做组织切片苏木精-伊红染色(Hematoxylin-eosin staining，HE)，判断细胞是否具有向三胚层组织细胞分化的潜能。如图4所示该干细胞具有异种三胚层分化能力。
本发明提供了一种牛诱导扩展多能性成体干细胞、建系方法及培养液，结合实施例加以具体说明，实施例中所述的原料均为市售原料，相关领域的人员完全可以根据本发明提供的方法进行适当改动或变更与组合，来实现该技术。需要特别说明的是，所有这些通过对本发明提供的***进行相类似的改动或变更与重新组合，都被视为在本发明的保护范围和内容中。
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
Claims (4)
1.一种牛诱导扩展多能性成体干细胞的建系方法，其特征在于，包括以下步骤:
(a)解冻牛成纤维细胞，在牛成纤维细胞培养液上培养，待细胞汇合度达到80％以上，收集细胞计数1×106；
(b)转入外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG，在提前解冻的饲养层细胞上培养，培养条件为M15+DOX培养液；
所述M15+DOX培养液是在DMEM培养基中，添加15％FBS、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素及强力霉素Dox；
(c)待干细胞克隆长出后，挑入细胞因子培养液培养，隔天换液，1到5天构建成牛诱导扩展多能性成体干细胞系；
所述细胞因子培养液的具体组分为：1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、0.1mM 2-巯基乙醇、1μM CHIR99021、0.3μM WH-4-023、5μM XAV939或5μM IWR-1、50μg/mlvitamin C、10ng/ml LIF、20.0ng/mlActivinA、mTeSRTM1培养基。
2.根据权利要求1所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG的核苷酸序列如SEQ.ID.NO：1-8所示。
3.根据权利要求1所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于：所述牛成纤维细胞培养液是在DMEM培养基中，添加10％FBS，1×谷氨酰胺，1×非必需氨基酸，1×青链霉素。
4.一种权利要求1-3任一所述牛诱导扩展多能性成体干细胞建系方法用细胞因子培养液，其特征在于，所述培养液组成为：
1×谷氨酰胺
1×非必需氨基酸
1×青链霉素
0.1mM 2-巯基乙醇
1μM CHIR99021
0.3μM WH-4-023
5μM XAV939或IWR-1
50μg/ml vitamin C
10ng/ml LIF
20.0ng/ml ActivinA
mTeSRTM1培养基
其中，IWR-1结构式为：
CHIR99021结构式为：
WH-4-023结构式为：
XAV939结构式为：
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011105359.7A CN114369577B (zh) | 2020-10-15 | 2020-10-15 | 牛诱导扩展多能性成体干细胞、建系方法及培养液 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011105359.7A CN114369577B (zh) | 2020-10-15 | 2020-10-15 | 牛诱导扩展多能性成体干细胞、建系方法及培养液 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114369577A CN114369577A (zh) | 2022-04-19 |
| CN114369577B true CN114369577B (zh) | 2024-02-06 |
Family
ID=81138006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011105359.7A Active CN114369577B (zh) | 2020-10-15 | 2020-10-15 | 牛诱导扩展多能性成体干细胞、建系方法及培养液 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114369577B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114752625A (zh) * | 2022-05-25 | 2022-07-15 | 内蒙古大学 | 一种山羊源oskm及其构建方法与应用 |
| CN116064660B (zh) * | 2022-08-22 | 2023-10-17 | 山西农业大学 | 绵羊诱导性多能干细胞及其制备方法 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016079146A1 (en) * | 2014-11-17 | 2016-05-26 | Genome Research Limited | In vitro production of expanded potential stem cells |
-
2020
- 2020-10-15 CN CN202011105359.7A patent/CN114369577B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016079146A1 (en) * | 2014-11-17 | 2016-05-26 | Genome Research Limited | In vitro production of expanded potential stem cells |
Non-Patent Citations (5)
| Title |
|---|
| Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts;Yanina Soledad Bogliotti等;《PNAS》;第115卷(第9期);摘要 * |
| Establishment of bovine expanded potential stem cells;Lixia Zhao等;《PNAS》;第118卷(第15期);第8页Culturing bEPSCs部分 * |
| Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment;Juan Du等;《Scientific Reports》(第10期);摘要 * |
| 利用piggyBac转座子制备牛成体多能干细胞诱导技术研究;赵丽霞;张金吨;张健;李云霞;苏杰;孙伟;赵高平;戴雁峰;郭继彤;胡树香;WANG Wei;LIU Pen-tao;李喜和;;中国生物工程杂志;33(02);摘要，第78页"1.2 方法"部分，第80页左栏最后一段 * |
| 赵丽霞 ; 张金吨 ; 张健 ; 李云霞 ; 苏杰 ; 孙伟 ; 赵高平 ; 戴雁峰 ; 郭继彤 ; 胡树香 ; WANG Wei ; LIU Pen-tao ; 李喜和 ; .利用piggyBac转座子制备牛成体多能干细胞诱导技术研究.中国生物工程杂志.2013,33(02),摘要，第78页"1.2 方法"部分，第80页左栏最后一段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114369577A (zh) | 2022-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5456467B2 (ja) | ラットおよび他種由来の多能性細胞 | |
| Vassiliev et al. | In vitro and in vivo characterization of putative porcine embryonic stem cells | |
| US7910369B2 (en) | Culture systems for ex vivo development | |
| Bodnar et al. | Propagation and maintenance of undifferentiated human embryonic stem cells | |
| WO2009021151A1 (en) | Isolation, characterization and propagation of germline stem cells | |
| CN114369577B (zh) | 牛诱导扩展多能性成体干细胞、建系方法及培养液 | |
| Zhang et al. | Generation of artificial gamete and embryo from stem cells in reproductive medicine | |
| JP2009502124A (ja) | 生殖系列幹細胞の療法的再プログラミング | |
| Moraveji et al. | Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis | |
| Segunda et al. | Sertoli cell-mediated differentiation of bovine fetal mesenchymal stem cells into germ cell lineage using an in vitro co-culture system | |
| Xu et al. | Fish pluripotent stem-like cell line induced by small-molecule compounds from caudal fin and its developmental potentiality | |
| Jiang et al. | Transcriptome profile of bovine iPSCs derived from Sertoli Cells | |
| US20220235327A1 (en) | Pancreatic differentiation | |
| CN114369567B (zh) | 牛扩展多能性胚胎干细胞的建系方法及培养液 | |
| Wu et al. | Induced multilineage differentiation of chicken embryonic germ cells via embryoid body formation | |
| JP2023550680A (ja) | 人工多能性幹細胞を作製する方法、人工多能性幹細胞、および人工多能性幹細胞を使用する方法 | |
| Heo et al. | Production of somatic chimera chicks by injection of bone marrow cells into recipient blastoderms | |
| Yu et al. | Isolation and characterization of parthenogenetic embryonic stem (pES) cells containing genetic background of the Kunming mouse strain | |
| US20040043482A1 (en) | Method of producing stem cell lines | |
| Nehlin et al. | Strategies for future histocompatible stem cell therapy | |
| Procópio et al. | Development of Artificial gametes | |
| Bui et al. | Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative Medicine | |
| Mohamed | Generation and Characterization of Clinical Grade Induced Pluripotent Stem Cells (iPSCs) from Human Umbilical Cord Tissue Mesenchymal Stromal Cells (CT-MSCs) | |
| Zhang et al. | Peptide‐coating 2D and small chemical molecules prolong the passage of porcine spermatogonia stem cells | |
| JP2024006674A (ja) | 精巣体細胞様細胞の製造方法、精巣体細胞様細胞、***の形成方法、中期中胚葉様細胞を精巣体細胞様細胞に分化誘導する方法、精巣体細胞様細胞の製造用培地サプリメント、及び精巣体細胞様細胞の製造用培地キット |
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 |