JP2015216853A - Method of producing megakaryocyte progenitor cell - Google Patents
Method of producing megakaryocyte progenitor cell Download PDFInfo
- Publication number
- JP2015216853A JP2015216853A JP2014100422A JP2014100422A JP2015216853A JP 2015216853 A JP2015216853 A JP 2015216853A JP 2014100422 A JP2014100422 A JP 2014100422A JP 2014100422 A JP2014100422 A JP 2014100422A JP 2015216853 A JP2015216853 A JP 2015216853A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- cell
- cells
- progenitor cells
- megakaryocyte progenitor
- 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.)
- Granted
Links
- 210000002361 Megakaryocyte Progenitor Cell Anatomy 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 55
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims abstract description 72
- 239000003112 inhibitor Substances 0.000 claims abstract description 19
- 102000003964 Histone deacetylase Human genes 0.000 claims abstract description 10
- 108090000353 Histone deacetylase Proteins 0.000 claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims description 91
- 108090000623 proteins and genes Proteins 0.000 claims description 70
- 239000002609 medium Substances 0.000 claims description 58
- 230000006698 induction Effects 0.000 claims description 46
- 239000003276 histone deacetylase inhibitor Substances 0.000 claims description 37
- 238000012258 culturing Methods 0.000 claims description 34
- 229940121372 histone deacetylase inhibitor Drugs 0.000 claims description 32
- 210000003593 megakaryocyte Anatomy 0.000 claims description 31
- 230000004069 differentiation Effects 0.000 claims description 29
- 230000014509 gene expression Effects 0.000 claims description 29
- RTKIYFITIVXBLE-QEQCGCAPSA-N trichostatin A Chemical compound ONC(=O)/C=C/C(/C)=C/[C@@H](C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-QEQCGCAPSA-N 0.000 claims description 27
- 102000036693 Thrombopoietin Human genes 0.000 claims description 21
- 108010041111 Thrombopoietin Proteins 0.000 claims description 21
- RTKIYFITIVXBLE-UHFFFAOYSA-N Trichostatin A Natural products ONC(=O)C=CC(C)=CC(C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-UHFFFAOYSA-N 0.000 claims description 21
- WAEXFXRVDQXREF-UHFFFAOYSA-N vorinostat Chemical compound ONC(=O)CCCCCCC(=O)NC1=CC=CC=C1 WAEXFXRVDQXREF-UHFFFAOYSA-N 0.000 claims description 20
- 229960000237 vorinostat Drugs 0.000 claims description 20
- QQDIFLSJMFDTCQ-UHFFFAOYSA-N MC1568 Chemical compound CN1C(C=CC(=O)NO)=CC=C1C=CC(=O)C1=CC=CC(F)=C1 QQDIFLSJMFDTCQ-UHFFFAOYSA-N 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 19
- FWZRWHZDXBDTFK-ZHACJKMWSA-N panobinostat Chemical compound CC1=NC2=CC=C[CH]C2=C1CCNCC1=CC=C(\C=C\C(=O)NO)C=C1 FWZRWHZDXBDTFK-ZHACJKMWSA-N 0.000 claims description 16
- 229960005184 panobinostat Drugs 0.000 claims description 16
- 108700020796 Oncogene Proteins 0.000 claims description 14
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 14
- 108700042657 p16 Genes Proteins 0.000 claims description 11
- 210000000130 stem cell Anatomy 0.000 claims description 11
- 230000006907 apoptotic process Effects 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 108700025695 Suppressor Genes Proteins 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 78
- 210000001772 blood platelet Anatomy 0.000 description 75
- 239000003814 drug Substances 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 15
- 239000013598 vector Substances 0.000 description 14
- 150000007523 nucleic acids Chemical group 0.000 description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 11
- 229940079593 drug Drugs 0.000 description 10
- 239000013642 negative control Substances 0.000 description 10
- 238000011160 research Methods 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 108010085895 Laminin Proteins 0.000 description 7
- 102000007547 Laminin Human genes 0.000 description 7
- 210000000601 blood cell Anatomy 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 239000011435 rock Substances 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 239000007640 basal medium Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000005755 formation reaction Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 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 description 6
- 239000000126 substance Substances 0.000 description 6
- 101001070790 Homo sapiens Platelet glycoprotein Ib alpha chain Proteins 0.000 description 5
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 5
- 102100034173 Platelet glycoprotein Ib alpha chain Human genes 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 230000001093 anti-cancer Effects 0.000 description 5
- 230000003915 cell function Effects 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000012423 maintenance Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 4
- 108010043137 Actomyosin Proteins 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 229940123169 Caspase inhibitor Drugs 0.000 description 4
- 102000011727 Caspases Human genes 0.000 description 4
- 108010076667 Caspases Proteins 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000004992 fission Effects 0.000 description 4
- OBKXEAXTFZPCHS-UHFFFAOYSA-N gamma-phenylbutyric acid Natural products OC(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000035800 maturation Effects 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 150000004666 short chain fatty acids Chemical class 0.000 description 4
- 101150062914 BMI1 gene Proteins 0.000 description 3
- 101100356682 Caenorhabditis elegans rho-1 gene Proteins 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 241000711408 Murine respirovirus Species 0.000 description 3
- 108091057508 Myc family Proteins 0.000 description 3
- 101150111584 RHOA gene Proteins 0.000 description 3
- 241000714474 Rous sarcoma virus Species 0.000 description 3
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 102000004338 Transferrin Human genes 0.000 description 3
- 108090000901 Transferrin Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- -1 but not limited to Chemical compound 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 230000008602 contraction Effects 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- NIJJYAXOARWZEE-UHFFFAOYSA-N di-n-propyl-acetic acid Natural products CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- BDJRBEYXGGNYIS-UHFFFAOYSA-N nonanedioic acid Chemical compound OC(=O)CCCCCCCC(O)=O BDJRBEYXGGNYIS-UHFFFAOYSA-N 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 210000004623 platelet-rich plasma Anatomy 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- OHRURASPPZQGQM-GCCNXGTGSA-N romidepsin Chemical compound O1C(=O)[C@H](C(C)C)NC(=O)C(=C/C)/NC(=O)[C@H]2CSSCC\C=C\[C@@H]1CC(=O)N[C@H](C(C)C)C(=O)N2 OHRURASPPZQGQM-GCCNXGTGSA-N 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 230000012391 spindle elongation Effects 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- MSRILKIQRXUYCT-UHFFFAOYSA-M valproate semisodium Chemical compound [Na+].CCCC(C(O)=O)CCC.CCCC(C([O-])=O)CCC MSRILKIQRXUYCT-UHFFFAOYSA-M 0.000 description 3
- 229960000604 valproic acid Drugs 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- OBKXEAXTFZPCHS-UHFFFAOYSA-M 4-phenylbutyrate Chemical compound [O-]C(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-M 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000003951 Erythropoietin Human genes 0.000 description 2
- 108090000394 Erythropoietin Proteins 0.000 description 2
- 102100039996 Histone deacetylase 1 Human genes 0.000 description 2
- 101001035024 Homo sapiens Histone deacetylase 1 Proteins 0.000 description 2
- 101001071312 Homo sapiens Platelet glycoprotein IX Proteins 0.000 description 2
- 101000872170 Homo sapiens Polycomb complex protein BMI-1 Proteins 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- IDQPVOFTURLJPT-UHFFFAOYSA-N N,N'-dihydroxyoctanediamide Chemical compound ONC(=O)CCCCCCC(=O)NO IDQPVOFTURLJPT-UHFFFAOYSA-N 0.000 description 2
- 102100036851 Platelet glycoprotein IX Human genes 0.000 description 2
- 102100033566 Polycomb complex protein BMI-1 Human genes 0.000 description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
- 229930189037 Trapoxin Natural products 0.000 description 2
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 150000003936 benzamides Chemical class 0.000 description 2
- 108010071933 benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone Proteins 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000000368 destabilizing effect Effects 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- INVTYAOGFAGBOE-UHFFFAOYSA-N entinostat Chemical compound NC1=CC=CC=C1NC(=O)C(C=C1)=CC=C1CNC(=O)OCC1=CC=CN=C1 INVTYAOGFAGBOE-UHFFFAOYSA-N 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
- 210000004700 fetal blood Anatomy 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- WFKAJVHLWXSISD-UHFFFAOYSA-N isobutyramide Chemical compound CC(C)C(N)=O WFKAJVHLWXSISD-UHFFFAOYSA-N 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- OYKBQNOPCSXWBL-SNAWJCMRSA-N n-hydroxy-3-[(e)-3-(hydroxyamino)-3-oxoprop-1-enyl]benzamide Chemical compound ONC(=O)\C=C\C1=CC=CC(C(=O)NO)=C1 OYKBQNOPCSXWBL-SNAWJCMRSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 229950009215 phenylbutanoic acid Drugs 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 229930002330 retinoic acid Natural products 0.000 description 2
- 108010091666 romidepsin Proteins 0.000 description 2
- 229960003452 romidepsin Drugs 0.000 description 2
- OHRURASPPZQGQM-UHFFFAOYSA-N romidepsin Natural products O1C(=O)C(C(C)C)NC(=O)C(=CC)NC(=O)C2CSSCCC=CC1CC(=O)NC(C(C)C)C(=O)N2 OHRURASPPZQGQM-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229910052711 selenium Inorganic materials 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- 235000011649 selenium Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 108010060597 trapoxin A Proteins 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- PPCUBWWPGYHEJE-UHFFFAOYSA-N (3-chlorophenyl)urea Chemical compound NC(=O)NC1=CC=CC(Cl)=C1 PPCUBWWPGYHEJE-UHFFFAOYSA-N 0.000 description 1
- FKJMFCOMZYPWCO-HNNXBMFYSA-N (3s)-5-(2,6-dichlorobenzoyl)oxy-4-oxo-3-(phenylmethoxycarbonylamino)pentanoic acid Chemical compound N([C@@H](CC(=O)O)C(=O)COC(=O)C=1C(=CC=CC=1Cl)Cl)C(=O)OCC1=CC=CC=C1 FKJMFCOMZYPWCO-HNNXBMFYSA-N 0.000 description 1
- SGYJGGKDGBXCNY-QXUYBEEESA-N (3s,9s,12r)-3-benzyl-6,6-dimethyl-9-[6-[(2s)-oxiran-2-yl]-6-oxohexyl]-1,4,7,10-tetrazabicyclo[10.3.0]pentadecane-2,5,8,11-tetrone Chemical compound C([C@H]1C(=O)NC(C(N[C@@H](CC=2C=CC=CC=2)C(=O)N2CCC[C@@H]2C(=O)N1)=O)(C)C)CCCCC(=O)[C@@H]1CO1 SGYJGGKDGBXCNY-QXUYBEEESA-N 0.000 description 1
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- CXAGHAZMQSCAKJ-WAHHBDPQSA-N (4s,7s)-n-[(2r,3s)-2-ethoxy-5-oxooxolan-3-yl]-7-(isoquinoline-1-carbonylamino)-6,10-dioxo-2,3,4,7,8,9-hexahydro-1h-pyridazino[1,2-a]diazepine-4-carboxamide Chemical compound CCO[C@@H]1OC(=O)C[C@@H]1NC(=O)[C@H]1N(C(=O)[C@H](CCC2=O)NC(=O)C=3C4=CC=CC=C4C=CN=3)N2CCC1 CXAGHAZMQSCAKJ-WAHHBDPQSA-N 0.000 description 1
- QRPSQQUYPMFERG-LFYBBSHMSA-N (e)-5-[3-(benzenesulfonamido)phenyl]-n-hydroxypent-2-en-4-ynamide Chemical compound ONC(=O)\C=C\C#CC1=CC=CC(NS(=O)(=O)C=2C=CC=CC=2)=C1 QRPSQQUYPMFERG-LFYBBSHMSA-N 0.000 description 1
- BWDQBBCUWLSASG-MDZDMXLPSA-N (e)-n-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1h-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide Chemical compound C=1NC2=CC=CC=C2C=1CCN(CCO)CC1=CC=C(\C=C\C(=O)NO)C=C1 BWDQBBCUWLSASG-MDZDMXLPSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 description 1
- DHXNZYCXMFBMHE-UHFFFAOYSA-M 3-bromopropanoate Chemical compound [O-]C(=O)CCBr DHXNZYCXMFBMHE-UHFFFAOYSA-M 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229940121819 ATPase inhibitor Drugs 0.000 description 1
- 108010015262 Ac-AAVALLPAVLLALLAPYVAD-CHO Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 229940088872 Apoptosis inhibitor Drugs 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 1
- 101150017888 Bcl2 gene Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101800001318 Capsid protein VP4 Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- SGYJGGKDGBXCNY-UHFFFAOYSA-N Chlamydocin Natural products N1C(=O)C2CCCN2C(=O)C(CC=2C=CC=CC=2)NC(=O)C(C)(C)NC(=O)C1CCCCCC(=O)C1CO1 SGYJGGKDGBXCNY-UHFFFAOYSA-N 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010048623 Collagen Receptors Proteins 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 108010002156 Depsipeptides Proteins 0.000 description 1
- DLVJMFOLJOOWFS-UHFFFAOYSA-N Depudecin Natural products CC(O)C1OC1C=CC1C(C(O)C=C)O1 DLVJMFOLJOOWFS-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102100031690 Erythroid transcription factor Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 102000020897 Formins Human genes 0.000 description 1
- 108091022623 Formins Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 108010051041 HC toxin Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101001066268 Homo sapiens Erythroid transcription factor Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 1
- 101000608935 Homo sapiens Leukosialin Proteins 0.000 description 1
- 101000692455 Homo sapiens Platelet-derived growth factor receptor beta Proteins 0.000 description 1
- 101001023770 Homo sapiens Transcription factor NF-E2 45 kDa subunit Proteins 0.000 description 1
- 101000625727 Homo sapiens Tubulin beta chain Proteins 0.000 description 1
- 101000931374 Homo sapiens Zinc finger protein ZFPM1 Proteins 0.000 description 1
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 1
- 102000000507 Integrin alpha2 Human genes 0.000 description 1
- 102000012355 Integrin beta1 Human genes 0.000 description 1
- 108010022222 Integrin beta1 Proteins 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100039564 Leukosialin Human genes 0.000 description 1
- 108010089704 Lim Kinases Proteins 0.000 description 1
- 102000008020 Lim Kinases Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100268648 Mus musculus Abl1 gene Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000005604 Myosin Heavy Chains Human genes 0.000 description 1
- 108010084498 Myosin Heavy Chains Proteins 0.000 description 1
- 102000005640 Myosin Type II Human genes 0.000 description 1
- 108010045128 Myosin Type II Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 102100037369 Nidogen-1 Human genes 0.000 description 1
- JWOGUUIOCYMBPV-UHFFFAOYSA-N OT-Key 11219 Natural products N1C(=O)C(CCCCCC(=O)CC)NC(=O)C2CCCCN2C(=O)C(C(C)CC)NC(=O)C1CC1=CN(OC)C2=CC=CC=C12 JWOGUUIOCYMBPV-UHFFFAOYSA-N 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 1
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 108010002687 Survivin Proteins 0.000 description 1
- 108010008125 Tenascin Proteins 0.000 description 1
- 102000007000 Tenascin Human genes 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102100035412 Transcription factor NF-E2 45 kDa subunit Human genes 0.000 description 1
- 102100024717 Tubulin beta chain Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- GBJVAVGBSGRRKN-JYEBCORGSA-N Z-DEVD-FMK Chemical compound COC(=O)C[C@@H](C(=O)CF)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)OC)NC(=O)[C@H](CC(=O)OC)NC(=O)OCC1=CC=CC=C1 GBJVAVGBSGRRKN-JYEBCORGSA-N 0.000 description 1
- 102100020993 Zinc finger protein ZFPM1 Human genes 0.000 description 1
- IPBVNPXQWQGGJP-UHFFFAOYSA-N acetic acid phenyl ester Natural products CC(=O)OC1=CC=CC=C1 IPBVNPXQWQGGJP-UHFFFAOYSA-N 0.000 description 1
- 108010091550 acetyl-AAVALLPAVLLALLAP-DEVD-CHO Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000362 adenosine triphosphatase inhibitor Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 108010082820 apicidin Proteins 0.000 description 1
- 229930186608 apicidin Natural products 0.000 description 1
- 239000000158 apoptosis inhibitor Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- NCNRHFGMJRPRSK-MDZDMXLPSA-N belinostat Chemical compound ONC(=O)\C=C\C1=CC=CC(S(=O)(=O)NC=2C=CC=CC=2)=C1 NCNRHFGMJRPRSK-MDZDMXLPSA-N 0.000 description 1
- BKSBJYSNALKVEA-ZDUSSCGKSA-N benzyl (3s)-5-chloro-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxopentanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C(=O)CCl)CC(=O)OCC1=CC=CC=C1 BKSBJYSNALKVEA-ZDUSSCGKSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- LZAXPYOBKSJSEX-UHFFFAOYSA-N blebbistatin Chemical compound C1CC2(O)C(=O)C3=CC(C)=CC=C3N=C2N1C1=CC=CC=C1 LZAXPYOBKSJSEX-UHFFFAOYSA-N 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- DNSISZSEWVHGLH-UHFFFAOYSA-N butanamide Chemical compound CCCC(N)=O DNSISZSEWVHGLH-UHFFFAOYSA-N 0.000 description 1
- GYKLFBYWXZYSOW-UHFFFAOYSA-N butanoyloxymethyl 2,2-dimethylpropanoate Chemical compound CCCC(=O)OCOC(=O)C(C)(C)C GYKLFBYWXZYSOW-UHFFFAOYSA-N 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 108700023145 chlamydocin Proteins 0.000 description 1
- 108010060348 citron-kinase Proteins 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000021953 cytokinesis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- DLVJMFOLJOOWFS-INMLLLKOSA-N depudecin Chemical compound C[C@@H](O)[C@@H]1O[C@H]1\C=C\[C@H]1[C@H]([C@H](O)C=C)O1 DLVJMFOLJOOWFS-INMLLLKOSA-N 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002242 embryoid body Anatomy 0.000 description 1
- 230000034311 endomitotic cell cycle Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 102000013370 fibrillin Human genes 0.000 description 1
- 108060002895 fibrillin Proteins 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- GNYCTMYOHGBSBI-UHFFFAOYSA-N helminthsporium carbonum toxin Natural products N1C(=O)C(C)NC(=O)C(C)NC(=O)C2CCCN2C(=O)C1CCCCCC(=O)C1CO1 GNYCTMYOHGBSBI-UHFFFAOYSA-N 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- VBZWSGALLODQNC-UHFFFAOYSA-N hexafluoroacetone Chemical compound FC(F)(F)C(=O)C(F)(F)F VBZWSGALLODQNC-UHFFFAOYSA-N 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 229940047889 isobutyramide Drugs 0.000 description 1
- GWYFCOCPABKNJV-UHFFFAOYSA-N isovaleric acid Chemical compound CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 description 1
- 108010038862 laminin 10 Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- MXOOUCRHWJYCAL-ZETCQYMHSA-N methyl (3s)-5-fluoro-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxopentanoate Chemical compound COC(=O)C[C@@H](C(=O)CF)NC(=O)OC(C)(C)C MXOOUCRHWJYCAL-ZETCQYMHSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 150000002763 monocarboxylic acids Chemical class 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 210000005088 multinucleated cell Anatomy 0.000 description 1
- 229940028444 muse Drugs 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- AHLKSOXEVRYIRD-UHFFFAOYSA-N n-phenylquinazolin-2-amine Chemical class N=1C=C2C=CC=CC2=NC=1NC1=CC=CC=C1 AHLKSOXEVRYIRD-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 230000006911 nucleation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229940049953 phenylacetate Drugs 0.000 description 1
- WLJVXDMOQOGPHL-UHFFFAOYSA-N phenylacetic acid Chemical compound OC(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-N 0.000 description 1
- 125000003170 phenylsulfonyl group Chemical group C1(=CC=CC=C1)S(=O)(=O)* 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000010321 prolifix Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000003353 pseudopodial effect Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 108700042226 ras Genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 108010041788 rho-Associated Kinases Proteins 0.000 description 1
- 102000000568 rho-Associated Kinases Human genes 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- HBROZNQEVUILML-UHFFFAOYSA-N salicylhydroxamic acid Chemical compound ONC(=O)C1=CC=CC=C1O HBROZNQEVUILML-UHFFFAOYSA-N 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- AEQFSUDEHCCHBT-UHFFFAOYSA-M sodium valproate Chemical compound [Na+].CCCC(C([O-])=O)CCC AEQFSUDEHCCHBT-UHFFFAOYSA-M 0.000 description 1
- 108700026239 src Genes Proteins 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 101150024821 tetO gene Proteins 0.000 description 1
- 101150061166 tetR gene Proteins 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- YECWTLGLNDDPGE-PIFXLSLCSA-N trichostatin C Chemical compound C(/[C@@H](C)C(=O)C=1C=CC(=CC=1)N(C)C)=C(/C)\C=C\C(=O)NO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YECWTLGLNDDPGE-PIFXLSLCSA-N 0.000 description 1
- YECWTLGLNDDPGE-UHFFFAOYSA-N trichostatin D Natural products C=1C=C(N(C)C)C=CC=1C(=O)C(C)C=C(C)C=CC(=O)NOC1OC(CO)C(O)C(O)C1O YECWTLGLNDDPGE-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229940070710 valerate Drugs 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 229940102566 valproate Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
本発明は、造血前駆細胞から巨核球前駆細胞を製造する方法、造血前駆細胞から巨核球前駆細胞への分化誘導促進剤ならびに巨核球前駆細胞を維持培養する方法に関する。 The present invention relates to a method for producing megakaryocyte progenitor cells from hematopoietic progenitor cells, an agent for promoting differentiation induction from hematopoietic progenitor cells to megakaryocyte progenitor cells, and a method for maintaining and culturing megakaryocyte progenitor cells.
血液関連疾患の治療や、外科的な治療には、多くの血液細胞が必要とされる。血液細胞の中でも、血液凝固及び止血のために必須の細胞である血小板は特に重要な血液細胞の1つである。血小板は、白血病、骨髄移植、抗癌治療などにおいて需要が多く、安定供給の必要性は高い。これまでに、血小板は、ドナーからの献血により採取する方法の他、トロンボポエチン（TPO）様類似構造 (ミメティクス)製剤を投与する方法、臍帯血又は骨髄細胞から巨核球を分化させる方法などにより確保されてきた。最近では、ES細胞又はiPS細胞などの多能性幹細胞をインビトロにおいて分化誘導し、血小板などの血液細胞を調製する技術も開発されている。 Many blood cells are required for treatment of blood-related diseases and surgical treatment. Among blood cells, platelets, which are essential for blood clotting and hemostasis, are one of the most important blood cells. Platelets are in great demand for leukemia, bone marrow transplantation, anticancer treatment, etc., and the need for a stable supply is high. So far, platelets have been secured by methods such as collecting thrombopoietin (TPO) -like structures (mimetics), methods of differentiating megakaryocytes from umbilical cord blood or bone marrow cells, in addition to methods of collecting blood by donation from donors. I came. Recently, a technique for preparing blood cells such as platelets by inducing differentiation of pluripotent stem cells such as ES cells or iPS cells in vitro has also been developed.
発明者らは、ヒトES細胞またはヒトiPS細胞から巨核球及び血小板を分化誘導する技術を確立し、血小板のソースとしての多能性幹細胞の有効性を示している（非特許文献１、並びに特許文献１及び特許文献２）。 The inventors have established a technique for inducing differentiation of megakaryocytes and platelets from human ES cells or human iPS cells, and have shown the effectiveness of pluripotent stem cells as a source of platelets (Non-patent Document 1 and Patents). Document 1 and Patent document 2).
さらに、発明者らは、多能性幹細胞をもとに不死化した巨核球前駆細胞株の樹立方法を見いだし、インビトロにおいて血小板等を大量に調製するために重要な技術を開発した（非特許文献２、並びに特許文献３及び特許文献４）。 Furthermore, the inventors have found a method for establishing a megakaryocyte progenitor cell line immortalized based on pluripotent stem cells, and have developed an important technique for preparing platelets and the like in large quantities in vitro (Non-Patent Documents). 2, and Patent Document 3 and Patent Document 4).
生体において、巨核球は、proplatelets(血小板前駆体)と呼ばれる偽足形状（pseudopodial formation）を形成し、その細胞質を断片化して血小板を放出する。巨核球は、血小板を放出するまでに、核内分裂(endomitosis)によって多核化すると考えられている。巨核球の核内分裂は、分裂溝形成及び紡錘体伸長を伴わない、核分裂及び細胞質分裂の異常による多極性有糸分裂であり、その結果、幾つかに分葉化した核を含む細胞が形成される。このような核内分裂が繰り返し生じることで、巨核球の多核化が誘導される。 In living organisms, megakaryocytes form pseudopodial formations called proplatelets (platelet precursors), fragmenting the cytoplasm and releasing platelets. Megakaryocytes are thought to become multinucleated by endomitosis before releasing platelets. Meganuclear cell nuclear fission is multipolar mitosis due to abnormalities of fission and cytokinesis, without fission formation and spindle elongation, resulting in the formation of cells with several lobulated nuclei Is done. Such intranuclear fission occurs repeatedly, which induces multinucleation of megakaryocytes.
巨核球の多核化に関し、これまでに多くの研究結果が報告されている。Lodierらは（非特許文献３）、巨核球の核内分裂において、分裂溝は形成されるものの、非筋細胞ミオシンIIの収縮環への局在が認められず、収縮環形成及び紡錘体伸長に欠陥が生じていることを明らかにした。そして、これら収縮環や紡錘体伸長の異常は、RhoA及びRockの活性を阻害することにより、より顕著になることが示された（非特許文献４）。RhoAは分裂溝に蓄積し、Rhoキナーゼ (Rock)、シトロンキナーゼ、LIMキナーゼ及びmDia/forminsなどを含む幾つかのエフェクター因子の活性化を促進する。これらの結果から、RhoA及びRockなど収縮環形成などに関与する因子の活性を阻害することで、巨核球の核内分裂が促進されることが示唆されている。また、インテグリンα２／β１下流に位置するRhoのシグナルが増強されると、未熟な多核化していない巨核球の血小板前駆体（proplatelet）形成が阻害されるとの報告もある。 Many research results have been reported so far regarding meganuclear nucleation. Lodier et al. (Non-patent Document 3) show that, in the nuclear division of megakaryocytes, the division groove is formed, but the localization of the non-myocyte myosin II to the contractile ring is not observed, and the contraction ring formation and the spindle elongation. It was clarified that there was a defect. And it was shown that these abnormalities of contraction rings and spindle elongation become more prominent by inhibiting the activity of RhoA and Rock (Non-patent Document 4). RhoA accumulates in the division groove and promotes activation of several effector factors including Rho kinase (Rock), citron kinase, LIM kinase and mDia / formins. From these results, it is suggested that the nuclear division of megakaryocytes is promoted by inhibiting the activity of factors involved in the formation of contraction rings such as RhoA and Rock. There is also a report that when the Rho signal located downstream of the integrin α2 / β1 is enhanced, the formation of platelet precursors (proplatelet) of immature, non-nucleated megakaryocytes is inhibited.
Schweinfurthらは、転写因子であるオールトランスレチノイン酸（ATRA；all trans retinoic acid）、ヒストン脱アセチル化酵素（Histone Deacetylase (HDAC)）の阻害剤として知られるバルプロ酸がヒト巨核芽球性白血病由来の巨核球前駆細胞株から巨核球への分化に関与していることが報告されている（非特許文献４）。同様に、マウスES細胞由来の造血前駆細胞から誘導した巨核球株に対してバルプロ酸を添加することで巨核球の成熟が促進することが報告されている（非特許文献５）。 Schweinfurth et al. Found that valproic acid, an inhibitor of all-trans retinoic acid (ATRA) and histone deacetylase (HDAC), transcription factors, is derived from human megakaryoblastic leukemia. It has been reported that it is involved in the differentiation of megakaryocyte progenitor cell lines into megakaryocytes (Non-patent Document 4). Similarly, it has been reported that maturation of megakaryocytes is promoted by adding valproic acid to a megakaryocyte strain derived from hematopoietic progenitor cells derived from mouse ES cells (Non-patent Document 5).
この他にも巨核球の成熟を促進する方法として、癌抑制遺伝子産物であるp53をノックダウンする方法（非特許文献６）、または通常の培養温度より高温の39℃で培養する方法（非特許文献７）が報告されている。 In addition to this, as a method for promoting the maturation of megakaryocytes, a method of knocking down p53, which is a tumor suppressor gene product (Non-Patent Document 6), or a method of culturing at 39 ° C., which is higher than the normal culture temperature (Non-Patent Document 6) Reference 7) has been reported.
本発明者らは、造血前駆細胞から巨核球前駆細胞を誘導し、血小板製剤を製造するにあたり、一つの巨核球前駆細胞からより多くの血小板を製造する方法が必要であると考えた。 The present inventors considered that a method for producing more platelets from one megakaryocyte progenitor cell is necessary for inducing megakaryocyte progenitor cells from hematopoietic progenitor cells and producing a platelet preparation.
このように、本発明は、造血前駆細胞から巨核球前駆細胞を製造する新規な方法等を提供することを課題とする。 Thus, an object of the present invention is to provide a novel method for producing megakaryocyte progenitor cells from hematopoietic progenitor cells.
背景技術として、巨核球前駆細胞へHDAC阻害剤を添加することで多核化を促し、血小板を産生させることは知られていたが、造血前駆細胞から巨核球前駆細胞を製造する工程においてHDAC阻害剤を添加することでも効果があるのか不明であった。 As a background technology, it has been known to add HDAC inhibitor to megakaryocyte progenitor cells to promote multinucleation and produce platelets, but in the process of producing megakaryocyte progenitor cells from hematopoietic progenitor cells, HDAC inhibitor It was unclear whether the addition was effective.
そこで、本発明者らは、巨核球前駆細胞または血小板を製造する工程において、造血前駆細胞とHADC阻害剤を接触させて培養したところ、血小板がより多く得られることを見出し、本発明を完成するに至った。 Accordingly, the present inventors have found that, in the step of producing megakaryocyte progenitor cells or platelets, when hematopoietic progenitor cells and HADC inhibitors are contacted and cultured, more platelets can be obtained, and the present invention is completed. It came to.
すなわち、本発明は、以下に関する。
［１］ヒストン脱アセチル化酵素（HDAC）阻害剤と造血前駆細胞とを接触させる工程を含む、巨核球前駆細胞を製造する方法。
［２］前記造血前駆細胞が、多能性幹細胞から分化誘導された細胞である、［１］に記載の方法。
［３］前記HDAC阻害剤が、トリコスタチンA、ボリノスタット（Vorinostat）、MC1568およびパノビノスタット（Panobinostat）から成る群より選択される化合物である、［１］または［２］に記載の方法。
［４］前記接触工程が、幹細胞因子（stem cell factor (SCF)）およびトロンボポエチン（TPO）の存在下で実施される、［１］から［３］のいずれかに記載の方法。
［５］前記巨核球前駆細胞が、ヒト巨核球前駆細胞である、［１］から［４］のいずれかに記載の方法。
［６］HDAC阻害剤を含む、造血前駆細胞から巨核球前駆細胞への分化誘導促進剤。
［７］前記HDAC阻害剤が、トリコスタチンA、ボリノスタット、MC1568およびパノビノスタットから成る群より選択される化合物である、［６］に記載の分化誘導促進剤。
［８］SCFおよびTPOをさらに含む、［６］または［７］に記載の分化誘導促進剤。
［９］前記巨核球前駆細胞が、ヒト巨核球前駆細胞である、［６］から［８］のいずれかに記載の分化誘導促進剤。
［１０］［６］から［９］のいずれかに記載の分化誘導促進剤を含む培養液。
［１１］HDAC阻害剤の存在下で巨核球前駆細胞を維持培養する方法。
［１２］前記巨核球前駆細胞が、外来性の癌遺伝子、p16遺伝子又はp19遺伝子の発現を抑制する遺伝子、並びに／あるいはアポトーシス抑制遺伝子を発現する細胞である、［１１］に記載の方法。
［１３］前記HDAC阻害剤が、トリコスタチンA、ボリノスタット、MC1568およびパノビノスタットから成る群より選択される化合物である、［１１］または［１２］に記載の方法。
［１４］前記巨核球を培養する工程が、SCFおよびTPOを含む培地で培養する工程である、［１１］から［１３］のいずれかに記載の方法。
［１５］HDAC阻害剤を含む、巨核球前駆細胞を維持培養するための培養液。
［１６］前記巨核球前駆細胞が、外来性の癌遺伝子、p16遺伝子若しくはp19遺伝子の発現を抑制する遺伝子、及び／又はアポトーシス抑制遺伝子を発現する細胞である、［１５］に記載の培養液。
［１７］前記HDAC阻害剤が、トリコスタチンA、ボリノスタット、MC1568およびパノビノスタットから成る群より選択される化合物である、［１５］または［１６］に記載の培養液。
［１８］SCFおよびTPOをさらに含む、［１５］から［１７］のいずれかに記載の培養液。
［１９］前記巨核球前駆細胞が、ヒト巨核球前駆細胞である、［１５］から［１８］のいずれかに記載の培養液。
That is, the present invention relates to the following.
[1] A method for producing megakaryocyte progenitor cells, comprising a step of contacting a histone deacetylase (HDAC) inhibitor with hematopoietic progenitor cells.
[2] The method according to [1], wherein the hematopoietic progenitor cells are cells induced to differentiate from pluripotent stem cells.
[3] The method according to [1] or [2], wherein the HDAC inhibitor is a compound selected from the group consisting of trichostatin A, vorinostat, MC1568, and panobinostat.
[4] The method according to any one of [1] to [3], wherein the contacting step is performed in the presence of stem cell factor (SCF) and thrombopoietin (TPO).
[5] The method according to any one of [1] to [4], wherein the megakaryocyte progenitor cell is a human megakaryocyte progenitor cell.
[6] An agent for promoting differentiation from hematopoietic progenitor cells to megakaryocyte progenitor cells, comprising an HDAC inhibitor.
[7] The differentiation induction promoter according to [6], wherein the HDAC inhibitor is a compound selected from the group consisting of trichostatin A, vorinostat, MC1568 and panobinostat.
[8] The differentiation induction promoter according to [6] or [7], further comprising SCF and TPO.
[9] The differentiation induction promoter according to any one of [6] to [8], wherein the megakaryocyte progenitor cell is a human megakaryocyte progenitor cell.
[10] A culture solution containing the differentiation induction promoter according to any one of [6] to [9].
[11] A method of maintaining and culturing megakaryocyte progenitor cells in the presence of an HDAC inhibitor.
[12] The method according to [11], wherein the megakaryocyte progenitor cell is a cell that expresses an exogenous oncogene, a gene that suppresses expression of a p16 gene or a p19 gene, and / or an apoptosis suppressor gene.
[13] The method according to [11] or [12], wherein the HDAC inhibitor is a compound selected from the group consisting of trichostatin A, vorinostat, MC1568, and panobinostat.
[14] The method according to any one of [11] to [13], wherein the step of culturing the megakaryocyte is a step of culturing in a medium containing SCF and TPO.
[15] A culture solution for maintaining and culturing megakaryocyte progenitor cells, comprising an HDAC inhibitor.
[16] The culture medium according to [15], wherein the megakaryocyte progenitor cell is a cell that expresses an exogenous oncogene, a gene that suppresses expression of a p16 gene or a p19 gene, and / or an apoptosis suppressor gene.
[17] The culture solution according to [15] or [16], wherein the HDAC inhibitor is a compound selected from the group consisting of trichostatin A, vorinostat, MC1568, and panobinostat.
[18] The culture solution according to any one of [15] to [17], further comprising SCF and TPO.
[19] The culture solution according to any one of [15] to [18], wherein the megakaryocyte progenitor cells are human megakaryocyte progenitor cells.
本発明によれば、血小板を効率よく産生する巨核球前駆細胞を製造することが可能となる。 According to the present invention, it is possible to produce megakaryocyte progenitor cells that efficiently produce platelets.
（巨核球前駆細胞の製造方法）
本発明は、HDAC阻害剤と造血前駆細胞とを接触させる工程を含む、巨核球前駆細胞を製造する方法を提供する。好ましい態様において、造血前駆細胞はHDAC阻害剤を含む培養液中でHDAC阻害剤と接触される。本発明では、HDAC阻害剤以外の物質と接触させることを妨げない。
(Method for producing megakaryocyte progenitor cells)
The present invention provides a method for producing megakaryocyte progenitor cells comprising the step of contacting an HDAC inhibitor with hematopoietic progenitor cells. In a preferred embodiment, hematopoietic progenitor cells are contacted with an HDAC inhibitor in a culture medium containing the HDAC inhibitor. The present invention does not prevent contact with substances other than HDAC inhibitors.
本発明における「巨核球」は、多核化した細胞であってもよく、例えば、CD41a陽性／CD42a陽性/CD42b陽性として特徴付けられる細胞を含む。この他にも、巨核球とは、GATA1、FOG1、NF-E2およびβ1-チューブリンが発現している細胞として特徴づけてもよい。多核化した巨核球とは、造血前駆細胞と比較して核の数が相対的に増大した細胞又は細胞群のことをいう。例えば、本発明の方法を適用する造血前駆細胞の核が2Nの場合には、4N以上の細胞が多核化した巨核球となる。また、本発明において、巨核球は、巨核球株として不死化されていてもよく、クローン化された細胞群であってもよい。 The “megakaryocyte” in the present invention may be a multinucleated cell, and includes, for example, a cell characterized as CD41a positive / CD42a positive / CD42b positive. In addition, megakaryocytes may be characterized as cells expressing GATA1, FOG1, NF-E2 and β1-tubulin. A multinucleated megakaryocyte refers to a cell or a group of cells in which the number of nuclei is relatively increased as compared to hematopoietic progenitor cells. For example, when the nuclei of hematopoietic progenitor cells to which the method of the present invention is applied is 2N, 4N or more cells become multinucleated megakaryocytes. In the present invention, the megakaryocyte may be immortalized as a megakaryocyte strain or may be a group of cloned cells.
本発明における「巨核球前駆細胞」とは、成熟することで巨核球となる細胞であって、多核化していない細胞であり、例えば、CD41a陽性／CD42a陽性/CD42b弱陽性として特徴付けられる細胞を含む。本発明の巨核球前駆細胞は、好ましくは、拡大培養により増殖させることが可能である細胞であり、例えば、少なくとも60日以上は、適切な条件で拡大培養可能な細胞である。本発明において、巨核球前駆細胞は、クローン化されていてもされていなくても良く、特に限定されないが、クローン化されたものを巨核球前駆細胞株と呼ぶこともある。 The “megakaryocyte progenitor cell” in the present invention is a cell that becomes a megakaryocyte upon maturation and is not multinucleated, for example, a cell characterized as CD41a positive / CD42a positive / CD42b weak positive. Including. The megakaryocyte progenitor cells of the present invention are preferably cells that can be expanded by expansion culture, for example, cells that can be expanded under appropriate conditions for at least 60 days or longer. In the present invention, the megakaryocyte progenitor cell may or may not be cloned, and is not particularly limited, but the cloned cell may be referred to as a megakaryocyte progenitor cell line.
本発明において、巨核球前駆細胞を製造するにあたり、接触工程はサイトカインの存在下で行ってもよい。サイトカインは培養液中に含まれていてもよい。サイトカインとは、血球系分化を促進するタンパク質であり、例えば、血管内皮増殖因子（VEGF）、トロンボポエチン（TPO）、幹細胞因子（stem cell factor (SCF）)、インターロイキン（IL）-1、-3、-4、-6、-7、-11、顆粒球単球コロニー刺激因子（GM-CSF）、またはエリスロポエチン（EPO）などが例示される。本発明で用いる好ましいサイトカインは、TPOおよびSCFである。TPOおよびSCFを培養液に含める場合、培養液中の濃度は、TPOの場合、10〜200ng/mL、好ましくは、50〜100ng/mL程度が例示され、SCFの場合、10〜200ng/mL、好ましくは50ng/mL程度が例示される。 In the present invention, in producing megakaryocyte progenitor cells, the contacting step may be performed in the presence of cytokines. Cytokines may be contained in the culture medium. Cytokines are proteins that promote blood cell differentiation, such as vascular endothelial growth factor (VEGF), thrombopoietin (TPO), stem cell factor (SCF), interleukin (IL) -1, -3 -4, -6, -7, -11, granulocyte monocyte colony-stimulating factor (GM-CSF), erythropoietin (EPO) and the like. Preferred cytokines for use in the present invention are TPO and SCF. When TPO and SCF are included in the culture solution, the concentration in the culture solution is 10 to 200 ng / mL, preferably about 50 to 100 ng / mL in the case of TPO, and 10 to 200 ng / mL in the case of SCF. Preferably, about 50 ng / mL is exemplified.
本発明において、HDAC阻害剤は、HDACの脱アセチル化活性を阻害する物質として定義され、例えば、ヒドロキサム酸誘導体、環状テトラペプチド、短鎖脂肪酸（SCFA）誘導体、ベンズアミド誘導体、求電子ケトン誘導体、siRNA、およびその他のHDAC阻害活性を有する化合物等を含むがこれらに限定されない。 In the present invention, an HDAC inhibitor is defined as a substance that inhibits the deacetylation activity of HDAC. For example, hydroxamic acid derivatives, cyclic tetrapeptides, short chain fatty acid (SCFA) derivatives, benzamide derivatives, electrophilic ketone derivatives, siRNA , And other compounds having HDAC inhibitory activity, etc., but are not limited thereto.
ヒドロキサム酸誘導体としては、例えば、スベロイルアニリドヒドロキサム酸（SAHA）（Vorinostat）（Richon et al., Proc. Natl. Acad. Sci. USA 95,3003-3007 (1998)）；m-カルボキシケイ皮酸ビスヒドロキサミド（CBHA）（Richon et al., Proc. Natl. Acad. Sci. USA 95,3003-3007 (1998)）；ピロキサミド；トリコスタチンA（TSA）およびトリコスタチンCなどのトリコスタチン類縁体（Koghe et al. 1998. Biochem. Pharmacol. 56: 1359-1364）；サリチルヒドロキサム酸（Andrews et al., International J. Parasitology 30,761-768 (2000)）；スベロイルビスヒドロキサム酸（SBHA）（米国特許第5,608,108号）；アゼライン酸ビスヒドロキサム酸（ABHA）（Andrews et al., International J. Parasitology 30,761-768 (2000)）；アゼライン酸-1-ヒドロキサメート-9-アニリド（AAHA）（Qiu et al., Mol. Biol. Cell 11, 2069-2083 (2000)）；6-(3-クロロフェニルウレイド)カルポイックヒドロキサム酸（3Cl-UCHA）］；オキサムフラチン［(2E)-5-[3-[(フェニルスルホニル)アミノフェニル]-ペンタ-2-エン-4-イノヒドロキサム酸］（Kim et al. Oncogene, 18: 2461 2470 (1999)）；A-161906、スクリプタイド（Su et al. 2000 Cancer Research, 60: 3137-3142）；PXD-101（Prolifix）；LAQ-824；CHAP；MW2796（Andrews et al., International J. Parasitology 30,761-768 (2000)）；MW2996（Andrews et al., International J. Parasitology 30,761-768 (2000)）；LBH-589 (Panobinostat)；MC1568（Mai A, et al, J Med Chem. 48:3344-3353 (2005)）または米国特許第5,369,108号、第5,932,616号、第5,700,811号、第6,087,367号および第6,511,990号に開示されているヒドロキサム酸のいずれかなどが挙げられるがこれらに限定されない。 Examples of the hydroxamic acid derivative include suberoylanilide hydroxamic acid (SAHA) (Vorinostat) (Richon et al., Proc. Natl. Acad. Sci. USA 95,3003-3007 (1998)); m-carboxycinnamic acid Bishydroxamide (CBHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95,3003-3007 (1998)); Piroxamide; Trichostatin analogs such as trichostatin A (TSA) and trichostatin C (Koghe et al. 1998. Biochem. Pharmacol. 56: 1359-1364); salicylhydroxamic acid (Andrews et al., International J. Parasitology 30,761-768 (2000)); suberoylbishydroxamic acid (SBHA) (US patent) No. 5,608,108); azelaic acid bishydroxamic acid (ABHA) (Andrews et al., International J. Parasitology 30,761-768 (2000)); azelaic acid-1-hydroxamate-9-anilide (AAHA) (Qiu et al ., Mol. Biol. Cell 11, 2069-2083 (2000)); 6- (3-chlorophenylurea De) carpoic hydroxamic acid (3Cl-UCHA)]; oxamflatin [(2E) -5- [3-[(phenylsulfonyl) aminophenyl] -pent-2-en-4-inohydroxamic acid] (Kim et al. Oncogene, 18: 2461 2470 (1999)); A-161906, scriptoid (Su et al. 2000 Cancer Research, 60: 3137-3142); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews et al., International J. Parasitology 30,761-768 (2000)); MW2996 (Andrews et al., International J. Parasitology 30,761-768 (2000)); LBH-589 (Panobinostat); MC1568 (Mai A, et al, J Med Chem. 48: 3344-3353 (2005)) or any of the hydroxamic acids disclosed in US Pat. Nos. 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990, etc. It is not limited to.
環状テトラペプチドとしては、例えば、トラポキシン（TPX）A-環状テトラペプチド（シクロ-(L-フェニルアラニル-L-フェニルアラニル-D-ピペコリニル-L-2-アミノ-8-オキソ-9,10-エポキシデカノイル)）（Kijima et al., J Biol. Chem. 268,22429-22435 (1993)）；FR901228（FK 228、デプシペプチド）（Nakajima et al., Ex. Cell Res. 241,126-133 (1998)）；FR225497環状テトラペプチド（H. Mori et al., WO00/08048）；アピシジン環状テトラペプチド［シクロ(N-O-メチル-L-トリプトファニル-L-イソロイシニル-D-ピペコリニル-L-2-アミノ-8-オキソデカノイル)］（Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93,1314313147 (1996)）；アピシジンIa、アピシジンIb、アピシジンIc、アピシジンIIa、およびアピシジンIIb（WO1997/11366）；CHAP、HC-トキシン環状テトラペプチド（Bosch et al., Plant Cell 7, 1941-1950 (1995)）；WF27082環状テトラペプチド（WO1998/48825）；ならびにクラミドシン（Bosch et al., Plant Cell 7, 1941-1950 (1995)）などが挙げられるがこれらに限定されない。 Examples of the cyclic tetrapeptide include trapoxin (TPX) A-cyclic tetrapeptide (cyclo- (L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10 -Epoxydecanoyl)) (Kijima et al., J Biol. Chem. 268,22429-22435 (1993)); FR901228 (FK228, depsipeptide) (Nakajima et al., Ex. Cell Res. 241,126-133 (1998) FR225497 cyclic tetrapeptide (H. Mori et al., WO00 / 08048); apicidin cyclic tetrapeptide [cyclo (NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8) -Oxodecanoyl)] (Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93, 1314313147 (1996)); CHAP, HC-toxin cyclic tetrapeptide (Bosch et al., Plant Cell 7, 1941-1950 (1995)); WF27082 ring Tetrapeptide (WO1998 / 48825); and chlamydocin (. Bosch et al, Plant Cell 7, 1941-1950 (1995)). However are not limited to such.
短鎖脂肪酸（SCFA）誘導体としては、酪酸、プロピオン酸等の低分子のモノカルボン酸の誘導体、例えば、酪酸ナトリウム（NaB）（Cousens et al., J. Biol. Chem. 254,1716-1723 (1979)）；イソ吉草酸塩（McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)）；吉草酸塩（McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)）；4-フェニル酪酸塩（4-PBA）（Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)）；フェニル酪酸塩（PB）（Wang et al., Cancer Research, 59, 2766-2799 (1999)）；プロピオン酸塩（McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)）；ブチルアミド（Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)）；イソブチルアミド（Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)）；フェニル酢酸塩（Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)）；3-ブロモプロピオン酸塩（Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)）；トリブチリン（Guan et al., Cancer Research, 60,749-755 (2000)）；バルプロ酸、バルプロ酸塩およびPivanex（登録商標）などが挙げられるがこれらに限定されない。 Short chain fatty acid (SCFA) derivatives include derivatives of low molecular weight monocarboxylic acids such as butyric acid and propionic acid, such as sodium butyrate (NaB) (Cousens et al., J. Biol. Chem. 254,1716-1723 ( 1979)); isovalerate (McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)); valerate (McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)) 4-phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)); phenylbutyrate (PB) (Wang et al., Cancer Research, 59, 2766-2799 (1999) ); Propionate (McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)); butyramide (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)); isobutyramide (Lea and Tulsyan, Anticancer) Research, 15,879-873 (1995)); phenyl acetate (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)); 3-bromopropionate (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)) ; Tributyrin (Guan et al., Cancer Research, 60,749-755 (2000)); including, but not limited to, valproic acid, valproate and Pivanex®.
ベンズアミド誘導体としては、例えば、CI-994；MS-275［N-(2-アミノフェニル)-4-[N-(ピリジン-3-イルメトキシカルボニル)アミノメチル]ベンズアミド]（Saito et al., Proc. Natl. Acad. Sci. USA 96, 4592-4597 (1999)）；およびMS-275の3'-アミノ誘導体（Saito et al., Proc. Natl. Acad. Sci. USA 96, 4592-4597 (1999)）などが挙げられるがこれらに限定されない。 Examples of the benzamide derivative include CI-994; MS-275 [N- (2-aminophenyl) -4- [N- (pyridin-3-ylmethoxycarbonyl) aminomethyl] benzamide] (Saito et al., Proc Natl. Acad. Sci. USA 96, 4592-4597 (1999)); and 3'-amino derivatives of MS-275 (Saito et al., Proc. Natl. Acad. Sci. USA 96, 4592-4597 (1999). )) And the like, but are not limited thereto.
求電子ケトン誘導体としては、例えば、トリフルオロメチルケトン（Frey et al, Bioorganic & Med. Chem. Lett. (2002), 12, 3443-3447; U.S. 6,511,990）およびN-メチル-α-ケトアミドなどのα-ケトアミドなどが挙げられるがこれらに限定されない。 Examples of the electrophilic ketone derivative include trifluoromethyl ketone (Frey et al, Bioorganic & Med. Chem. Lett. (2002), 12, 3443-3447; US 6,511,990) and α such as N-methyl-α-ketoamide. -Ketoamide and the like can be mentioned, but not limited thereto.
siRNAとしては、例えば、HDAC1 siRNA Smartpool (Millipore)、HuSH 29mer shRNA Constructs against HDAC1 (OriGene) などが挙げられるがこれらに限定されない。 Examples of siRNA include, but are not limited to, HDAC1 siRNA Smartpool (Millipore) and HuSH 29mer shRNA Constructs against HDAC1 (OriGene).
その他のHDAC阻害剤としては、例えば、天然物、サマプリン、およびデプデシン（Kwon et al. 1998. PNAS 95: 3356-3361）などが挙げられるがこれらに限定されない。 Examples of other HDAC inhibitors include, but are not limited to, natural products, samapurin, and depudecin (Kwon et al. 1998. PNAS 95: 3356-3361).
本発明において、好ましいHDAC阻害剤は、トリコスタチンA、ボリノスタット（Vorinostat）（あるいは、本明細書においてはスベロイルアニリドヒドロキサム酸（SAHA）とも称する）、MC1568およびパノビノスタット（Panobinostat）（あるいは、本明細書においてはLBH-589とも称する）から成る群より選択される1つ以上の化合物である。これらの化合物は組み合わせて用いられてもよく、または単独で用いられてもよい。 In the present invention, preferred HDAC inhibitors are trichostatin A, vorinostat (also referred to herein as suberoylanilide hydroxamic acid (SAHA)), MC1568 and panobinostat (or Or one or more compounds selected from the group consisting of LBH-589). These compounds may be used in combination or may be used alone.
（分化誘導促進剤）
本発明はまた、HDAC阻害剤を含む、造血前駆細胞から巨核球前駆細胞への分化誘導促進剤を提供する。本発明において、「分化誘導促進剤」とは、HDAC阻害剤と接触させない対照と比較して、造血前駆細胞から巨核球前駆細胞へ分化誘導を有意に、例えば１．２倍以上、好ましくは１．５倍以上、より好ましくは１．７倍以上促進する物質を意味する。分化誘導促進の程度は、巨核球前駆細胞数の増大をフローサイトメトリーにより確認することで評価することができる。好ましい態様において、本発明の分化誘導促進剤は造血前駆細胞を培養する培養液に含まれる。
(Differentiation induction promoter)
The present invention also provides an agent for inducing differentiation from hematopoietic progenitor cells to megakaryocyte progenitor cells, comprising an HDAC inhibitor. In the present invention, the term “differentiation induction promoter” means that differentiation induction from a hematopoietic progenitor cell to a megakaryocyte progenitor cell is significantly, for example, 1.2 times or more, preferably 1 as compared with a control not contacted with an HDAC inhibitor. It means a substance that promotes 5 times or more, more preferably 1.7 times or more. The degree of differentiation induction promotion can be evaluated by confirming the increase in the number of megakaryocyte progenitor cells by flow cytometry. In a preferred embodiment, the differentiation induction promoter of the present invention is contained in a culture medium for culturing hematopoietic progenitor cells.
（巨核球前駆細胞機能改善剤）
本発明はまた、HDAC阻害剤を含む、造血前駆細胞から巨核球前駆細胞への分化誘導時において用いる巨核球前駆細胞機能改善剤を提供する。本発明において、「巨核球前駆細胞機能改善剤」とは、造血前駆細胞から巨核球前駆細胞への分化誘導時に当該細胞へ接触させることによって、HDAC阻害剤と接触させない対照と比較して、得られた巨核球前駆細胞からの血小板産生量を有意に、例えば１．２倍以上、好ましくは１．５倍以上、より好ましくは１．７倍以上増加させる物質を意味する。巨核球前駆細胞の機能改善の程度は産生される血小板の量を測定することによって評価することもできる。好ましい態様において、本発明の巨核球前駆細胞機能改善は造血前駆細胞を培養する培養液に含まれる。本発明において、特に断りがない場合は、巨核球前駆細胞機能改善剤は、分化誘導促進剤に包含される。
(Megakaryocyte precursor cell function improving agent)
The present invention also provides a megakaryocyte progenitor cell function improving agent for use in induction of differentiation from hematopoietic progenitor cells to megakaryocyte progenitor cells, comprising an HDAC inhibitor. In the present invention, the “megakaryocyte progenitor cell function improving agent” is obtained by contacting the cell at the time of induction of differentiation from a hematopoietic progenitor cell to a megakaryocyte progenitor cell, compared with a control not brought into contact with an HDAC inhibitor. It means a substance that significantly increases the amount of platelet production from the obtained megakaryocyte progenitor cells, for example, 1.2 times or more, preferably 1.5 times or more, more preferably 1.7 times or more. The degree of function improvement of megakaryocyte progenitor cells can also be evaluated by measuring the amount of platelets produced. In a preferred embodiment, the megakaryocyte progenitor cell function improvement of the present invention is contained in a culture medium for culturing hematopoietic progenitor cells. In the present invention, unless otherwise specified, the megakaryocyte precursor cell function improving agent is included in the differentiation induction promoter.
本発明で使用されるHDAC阻害剤の培養液中の濃度は、通常0.1nM〜100μMであり、HDAC阻害剤の添加量を変更することによって適宜調整可能である。HDAC阻害剤の濃度は、TSAを用いる場合、例えば、0.1nM〜100nM、1nM〜50nM、5nM〜20nM、10nMであり、MC1568を用いる場合、例えば、0.1μM〜100μM、1μM〜50μM、5μM〜20μM、10μMであり、LBH-589を用いる場合、例えば、0.01nM〜10nM、0.1nM〜5nM、0.5nM〜2nM、1nMであり、SAHAを用いる場合、例えば、1nM〜1μM、10nM〜500nM、50nM〜200nM、100nMである。 The concentration of the HDAC inhibitor used in the present invention in the culture solution is usually 0.1 nM to 100 μM, and can be appropriately adjusted by changing the amount of HDAC inhibitor added. The concentration of the HDAC inhibitor is, for example, 0.1 nM to 100 nM, 1 nM to 50 nM, 5 nM to 20 nM, 10 nM when using TSA, and when using MC1568, for example, 0.1 μM to 100 μM, 1 μM to 50 μM, 5 μM to 20 μM When LBH-589 is used, for example, 0.01 nM to 10 nM, 0.1 nM to 5 nM, 0.5 nM to 2 nM, 1 nM, and when SAHA is used, for example, 1 nM to 1 μM, 10 nM to 500 nM, 50 nM to 200nM and 100nM.
本発明において用いる培養液は、特に限定されないが、動物細胞の培養に用いられる培地を基礎培地として調製することができる。基礎培地の定義には、例えばIscove's Modified Dulbecco's Medium（IMDM）培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium（ライフテクノロジーズ）およびこれらの混合培地などが包含される。培地には、血清が含有されていてもよいし、あるいは無血清を使用してもよい。必要に応じて、基礎培地は、例えば、アルブミン、インスリン、トランスフェリン、セレン、脂肪酸、微量元素、2-メルカプトエタノール、チオールグリセロール、脂質、アミノ酸、L-グルタミン、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類、サイトカインなどの1つ以上の物質も含有し得る。 The culture solution used in the present invention is not particularly limited, but a medium used for culturing animal cells can be prepared as a basal medium. The definition of the basal medium includes, for example, Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies) and mixed media thereof are included. Serum may be contained in the medium, or serum-free may be used. If necessary, the basal medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low It may also contain one or more substances such as molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like.
本発明において好ましい基礎培地は、血清、インスリン、トランスフェリン、セリン、チオールグリセロール、アスコルビン酸を含むIMDM培地である。 A preferred basal medium in the present invention is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, and ascorbic acid.
本発明の造血前駆細胞から巨核球前駆細胞を製造する工程において、造血前駆細胞は、フィーダー細胞（例えば、哺乳類胎仔のAGM（aorta-gonad-mesonephros）領域から得られた細胞（特開２００１−３７４７１）、マウス胎仔線維芽細胞（MEF）、OP9細胞（ATCCより入手可能）またはC3H10T1/2細胞（JCRB Cell Bankより入手可能））上、あるいは細胞外基質上で培養する方法が例示される。 In the step of producing megakaryocyte progenitor cells from the hematopoietic progenitor cells of the present invention, hematopoietic progenitor cells are cells obtained from feeder cells (for example, AGM (aorta-gonad-mesonephros) region of mammalian fetus (JP 2001-37471 A). ), Mouse fetal fibroblasts (MEF), OP9 cells (available from ATCC) or C3H10T1 / 2 cells (available from JCRB Cell Bank)) or an extracellular matrix.
本発明において、細胞外基質とは、細胞の外に存在する超分子構造体であり、天然由来であっても、人工物（組換え体）であってもよい。例えば、コラーゲン、プロテオグリカン、フィブロネクチン、ヒアルロン酸、テネイシン、エンタクチン、エラスチン、フィブリリンおよびラミニンといった物質またはこれらの断片が挙げられる。これらの細胞外基質は、組み合わせて用いられてもよく、例えば、BD Matrigel（登録商標）などの細胞からの調製物であってもよい。好ましくは、細胞外基質はラミニンまたはその断片である。本発明においてラミニンとは、α鎖、β鎖、γ鎖をそれぞれ1本ずつ持つヘテロ三量体構造を有するタンパク質である。特に限定されないが、例えば、α鎖は、α１、α２、α３、α４またはα５であり、β鎖は、β１、β２またはβ３であり、γ鎖は、γ１、γ２またはγ３が例示される、より好ましいラミニンは、α５、β１およびγ１からなるラミニン511である。本発明では、ラミニンは断片であってもよく、インテグリン結合活性を有している断片であれば、特に限定されないが、例えば、エラスターゼにて消化して得られる断片であるE8フラグメントであってもよい。従って、本発明では、WO2011/043405に記載されたラミニン511E8（好ましくはヒトラミニン511E8）が例示される。 In the present invention, the extracellular matrix is a supramolecular structure existing outside the cell and may be naturally derived or an artificial product (recombinant). Examples thereof include substances such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin and laminin, or fragments thereof. These extracellular substrates may be used in combination, for example, a preparation from cells such as BD Matrigel (registered trademark). Preferably, the extracellular matrix is laminin or a fragment thereof. In the present invention, laminin is a protein having a heterotrimeric structure having one α chain, one β chain, and one γ chain. Although not particularly limited, for example, the α chain is α1, α2, α3, α4, or α5, the β chain is β1, β2, or β3, and the γ chain is exemplified by γ1, γ2, or γ3, A preferred laminin is laminin 511 consisting of α5, β1 and γ1. In the present invention, laminin may be a fragment, and is not particularly limited as long as it has an integrin-binding activity. For example, it may be an E8 fragment that is a fragment obtained by digestion with elastase. Good. Therefore, in the present invention, laminin 511E8 (preferably human laminin 511E8) described in WO2011 / 043405 is exemplified.
本発明において、巨核球前駆細胞を製造する好ましい培養条件は、C3H10T1/2細胞と造血前駆細胞を共培養する方法である。 In the present invention, a preferred culture condition for producing megakaryocyte progenitor cells is a method of co-culturing C3H10T1 / 2 cells and hematopoietic progenitor cells.
本発明において、造血前駆細胞（Hematopoietic Progenitor Cells（HPC））とは、リンパ球、好酸球、好中球、好塩基球、赤血球、巨核球等の血球系細胞に分化可能な細胞である、本発明において、造血前駆細胞と造血幹細胞は、区別されるものではなく、特に断りがなければ同一の細胞を示す。造血幹細胞／前駆細胞は、例えば、表面抗原であるCD34および/またはCD43が陽性であることによって認識できる。本発明において、造血幹細胞は、多能性幹細胞、臍帯血・骨髄血・末梢血由来の造血幹細胞及び前駆細胞などから分化誘導された造血前駆細胞に対しても適用することができる。例えば、多能性幹細胞を使用する場合、造血前駆細胞は、Takayama N., et al. J Exp Med. 2817-2830 (2010)に記載の方法にしたがって、多能性幹細胞をVEGFの存在下でC3H10T1/2上で培養することで得られるネット様構造物（ES−sac又はiPS−sacとも称する）から調製することができる。ここで、「ネット様構造物」とは、多能性幹細胞由来の立体的な嚢状（内部に空間を伴うもの）構造体で、内皮細胞集団などで形成され、内部に造血前駆細胞を含む構造体である。この他にも、多能性幹細胞からの造血前駆細胞の製造方法として、胚様体の形成とサイトカインの添加による方法（Chadwick et al. Blood 2003, 102: 906-15、Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62、Saeki et al. Stem Cells 2009, 27: 59-67）または異種由来のストローマ細胞との共培養法（Niwa A et al. J Cell Physiol. 2009 Nov;221(2):367-77.）等が例示される。本発明において、好ましい造血前駆細胞は、多能性幹細胞から誘導された造血前駆細胞である。 In the present invention, hematopoietic progenitor cells (HPC) are cells that can differentiate into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, megakaryocytes, In the present invention, hematopoietic progenitor cells and hematopoietic stem cells are not distinguished, and show the same cells unless otherwise specified. Hematopoietic stem / progenitor cells can be recognized by, for example, positive surface antigens CD34 and / or CD43. In the present invention, hematopoietic stem cells can also be applied to pluripotent stem cells, hematopoietic progenitor cells derived from cord blood, bone marrow blood, peripheral blood-derived hematopoietic stem cells and progenitor cells. For example, when pluripotent stem cells are used, hematopoietic progenitor cells are obtained by pluripotent stem cells in the presence of VEGF according to the method described in Takayama N., et al. J Exp Med. 2817-2830 (2010). It can be prepared from a net-like structure (also referred to as ES-sac or iPS-sac) obtained by culturing on C3H10T1 / 2. Here, the “net-like structure” is a three-dimensional sac-like structure (with space inside) derived from pluripotent stem cells, which is formed by an endothelial cell population and the like, and contains hematopoietic progenitor cells inside. It is a structure. In addition, other methods for producing hematopoietic progenitor cells from pluripotent stem cells include the formation of embryoid bodies and addition of cytokines (Chadwick et al. Blood 2003, 102: 906-15, Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62, Saeki et al. Stem Cells 2009, 27: 59-67) or co-culture with heterologous stromal cells (Niwa A et al. J Cell Physiol. 2009 Nov; 221 (2 ): 367-77.) And the like. In the present invention, preferred hematopoietic progenitor cells are hematopoietic progenitor cells derived from pluripotent stem cells.
本発明において、多能性幹細胞とは、生体に存在する全ての細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞であり、それには、例えば胚性幹(ES)細胞(J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848;J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165)、核移植により得られるクローン胚由来の胚性幹(ntES)細胞（T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502）、精子幹細胞（「GS細胞」）(M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012)、胚性生殖細胞（「EG細胞」）(Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551)、人工多能性幹(iPS)細胞（K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M.ら,Nat. Biotechnol. 26:101-106 (2008)；WO2007/069666）、培養線維芽細胞や骨髄幹細胞由来の多能性細胞（Muse細胞）（WO2011/007900）などが含まれる。より好ましくは、多能性幹細胞はヒト多能性幹細胞である。 In the present invention, a pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into all cells present in a living body and also has a proliferative ability, and includes, for example, an embryonic stem ( ES) cells (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165), embryos derived from cloned embryos obtained by nuclear transfer Sexual stem (ntES) cells (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502), sperm stem cells ("GS cells") (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012), embryonic germ cells ("EG cells") (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. ( 1992), Nature, 359: 550-551), human Pluripotent stem (iPS) cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); WO2007 / 069666), pluripotent cells derived from cultured fibroblasts and bone marrow stem cells (Muse cells) (WO2011 / 007900) and the like. More preferably, the pluripotent stem cell is a human pluripotent stem cell.
本発明に係る巨核球前駆細胞の製造方法は、一態様として、造血前駆細胞へ癌遺伝子、p16遺伝子又はp19遺伝子の発現を抑制する遺伝子、並びに／あるいはアポトーシス抑制遺伝子を強制発現させて該細胞を培養する工程を含んでもよい。 In one embodiment, the method for producing megakaryocyte progenitor cells according to the present invention comprises forcing the hematopoietic progenitor cells to express an oncogene, a gene that suppresses the expression of p16 gene or p19 gene, and / or an apoptosis-inhibiting gene. A step of culturing may be included.
本発明において、「癌遺伝子」とは、その発現、構造または機能等が正常細胞と異なることに起因して、正常細胞のがん化を引き起こす遺伝子であり、例えば、MYCファミリー遺伝子、Srcファミリー遺伝子、Rasファミリー遺伝子、Rafファミリー遺伝子、c-KitやPDGFR、Ablなどのプロテインキナーゼファミリー遺伝子などを挙げることができる。MYCファミリー遺伝子として、c-MYC、N-MYCおよびL-MYCが例示される。より好ましくは、癌遺伝子はc-MYC遺伝子である。c-MYC遺伝子とは、例えば、NCBIのアクセッション番号NM_002467で示される核酸配列からなる遺伝子である。また、c-MYC遺伝子には、そのホモログも含まれてよく、c-MYC遺伝子ホモログとは、そのcDNA配列が、例えば、NCBIのアクセッション番号NM_002467で示される核酸配列と実質的に同一の配列からなる遺伝子のことである。NCBIのアクセッション番号NM_002467で示される核酸配列と実質的に同一の配列からなるcDNAとは、NCBIのアクセッション番号NM_002467で表される配列からなるDNAと、約60％以上、好ましくは約70％以上、より好ましくは約80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、最も好ましくは約99％の同一性を有する配列からなるDNA、もしくは、NCBIのアクセッション番号NM_002467で表わされる核酸配列に相補的な配列からなるDNAとストリンジェントな条件下でハイブリダイズできるDNAであって、これらのDNAによってコードされるタンパク質が、造血前駆細胞など、分化段階の細胞の増幅に寄与するもののことである。 In the present invention, the term “oncogene” refers to a gene that causes canceration of normal cells due to its expression, structure or function being different from that of normal cells. For example, MYC family genes, Src family genes , Ras family genes, Raf family genes, protein kinase family genes such as c-Kit, PDGFR, and Abl. Examples of MYC family genes include c-MYC, N-MYC, and L-MYC. More preferably, the oncogene is a c-MYC gene. The c-MYC gene is, for example, a gene consisting of a nucleic acid sequence represented by NCBI accession number NM_002467. The c-MYC gene may also include homologues thereof. The c-MYC gene homologue is a sequence whose cDNA sequence is substantially the same as the nucleic acid sequence represented by NCBI accession number NM_002467, for example. It is a gene consisting of A cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown by NCBI accession number NM_002467 is about 60% or more, preferably about 70%, of the DNA consisting of the sequence shown by NCBI accession number NM_002467. More preferably, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, most preferably about 99% identity DNA or DNA complementary to the nucleic acid sequence represented by NCBI accession number NM_002467 DNAs that can hybridize under stringent conditions, and the proteins encoded by these DNAs contribute to the amplification of cells in the differentiation stage, such as hematopoietic progenitor cells.
ここで、ストリンジェントな条件とは、当業者によって容易に決定されるハイブリダイゼーションの条件のことで、一般的にプローブ長、洗浄温度、及び塩濃度に依存する経験的な実験条件である。一般に、プローブが長くなると適切なアニーリングのための温度が高くなり、プローブが短くなると温度は低くなる。ハイブリッド形成は、一般的に、相補的鎖がその融点よりやや低い環境における再アニール能力に依存する。 Here, stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical experimental conditions that depend on the probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally relies on the ability to reanneal in an environment where the complementary strand is slightly below its melting point.
例えば、低ストリンジェントな条件として、ハイブリダイゼーション後のフィルターの洗浄段階において、37℃〜42℃の温度条件下、0.1×SSC、0.1％SDS溶液中で洗浄することなどが上げられる。また、高ストリンジェントな条件として、例えば、洗浄段階において、65℃、5×SSCおよび0.1％SDS中で洗浄することなどが挙げられる。ストリンジェントな条件をより高くすることにより、相同性の高いポリヌクレオチドを得ることができる。 For example, low stringency conditions include washing in a 0.1 × SSC, 0.1% SDS solution at 37 ° C. to 42 ° C. in the filter washing step after hybridization. Examples of highly stringent conditions include washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step. By making the stringent conditions higher, a highly homologous polynucleotide can be obtained.
本発明において、c-MYCの発現量を抑制することが好ましいため、不安定化ドメインと融合させたタンパク質をコードするc-MYCであってもよい。不安定化ドメインは、ProteoTuner社またはClontech社から購入して用いることができる。 In the present invention, since it is preferable to suppress the expression level of c-MYC, c-MYC encoding a protein fused with a destabilizing domain may be used. The destabilizing domain can be purchased from ProteoTuner or Clontech.
本発明において、「p16遺伝子又はp19遺伝子の発現を抑制する遺伝子」として、BMI1またはId1などが例示される。本発明において好ましくは、p16遺伝子又はp19遺伝子の発現を抑制する遺伝子はBMI1である。BMI1遺伝子とは、例えば、NCBIのアクセッション番号NM_005180で示される核酸配列からなる遺伝子である。また、BMI1遺伝子には、そのホモログも含まれてよく、BMI1遺伝子のホモログとは、そのcDNA配列が、例えばNCBIのアクセッション番号NM_005180で示される核酸配列と実質的に同一の配列からなる遺伝子のことである。NCBIのアクセッション番号NM_005180で示される核酸配列と実質的に同一の配列からなるcDNAとは、NCBIのアクセッション番号NM_005180で表される配列からなるDNAと、約60％以上、好ましくは約70％以上、より好ましくは約80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、最も好ましくは約99％の同一性を有する配列からなるDNA、もしくは、NCBIのアクセッション番号NM_005180で表わされる核酸配列に相補的な配列からなるDNAとストリンジェントな条件下でハイブリダイズできるDNAであって、そのDNAによってコードされるタンパク質が、MYCファミリー遺伝子などの癌遺伝子が発現している細胞内で生じる癌遺伝子誘導性細胞老化を抑制し、該細胞の増幅を促進するもののことである。 In the present invention, BMI1 or Id1 is exemplified as “a gene that suppresses the expression of p16 gene or p19 gene”. In the present invention, preferably, the gene that suppresses the expression of the p16 gene or the p19 gene is BMI1. The BMI1 gene is, for example, a gene consisting of a nucleic acid sequence represented by NCBI accession number NM_005180. The BMI1 gene may also include a homologue thereof. The homologue of the BMI1 gene is a gene whose cDNA sequence is substantially the same as the nucleic acid sequence represented by NCBI accession number NM_005180, for example. That is. The cDNA consisting of a sequence substantially identical to the nucleic acid sequence shown by NCBI accession number NM_005180 is about 60% or more, preferably about 70%, of the DNA consisting of the sequence shown by NCBI accession number NM_005180. More preferably, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, most preferably about 99% identity DNA or DNA complementary to the nucleic acid sequence represented by NCBI accession number NM_005180 DNA that is capable of hybridizing under stringent conditions, and the protein encoded by the DNA suppresses oncogene-induced cell aging that occurs in cells in which oncogenes such as MYC family genes are expressed, It promotes the amplification of the cells.
本発明において、上記の遺伝子を造血前駆細胞において強制発現させる方法は、当業者の常法にしたがって行うことができる。例えば、強制発現は、これらの遺伝子を発現するベクター、これらの遺伝子がコードするタンパク質またはこれらの遺伝子をコードするRNAを造血前駆細胞へ導入することによって成し得る。さらには、これらの遺伝子の発現を誘導する低分子化合物等を造血前駆細胞と接触させることによっても強制発現を行うことができる。ここで、本発明では、一定期間、上記遺伝子を発現し続ける必要があることから、発現ベクター、タンパク質、RNAまたは発現を誘導する低分子化合物等は、必要な期間に合わせて複数回導入することによって行い得る。 In the present invention, the method for forcibly expressing the above gene in hematopoietic progenitor cells can be performed according to a conventional method of those skilled in the art. For example, forced expression can be achieved by introducing vectors expressing these genes, proteins encoded by these genes, or RNA encoding these genes into hematopoietic progenitor cells. Furthermore, forced expression can also be performed by bringing a low molecular weight compound or the like that induces expression of these genes into contact with hematopoietic progenitor cells. Here, in the present invention, since it is necessary to continue to express the gene for a certain period of time, an expression vector, protein, RNA or a low molecular weight compound that induces expression should be introduced multiple times in accordance with the necessary period. Can be done.
これらの遺伝子を発現するベクターとして、例えば、レトロウイルス、レンチウィルス、アデノウイルス、アデノ随伴ウィルス、ヘルペスウイルス及びセンダイウィルスなどのウィルスベクター、動物細胞発現プラスミド（例、pA1-11，pXT1，pRc/CMV，pRc/RSV，pcDNAI/Neo）などが用いられ得る。単回導入により実施し得るという点において、レトロウィルスベクターまたはレンチウィルスベクターが好ましいベクターである。 Examples of vectors for expressing these genes include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses, and animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV). , PRc / RSV, pcDNAI / Neo) and the like. Retroviral vectors or lentiviral vectors are preferred vectors in that they can be performed by a single introduction.
発現ベクターにおいて使用されるプロモーターの例としては、EF-αプロモーター、CAGプロモーター、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウィルス）プロモーター、MoMuLV（モロニーマウス白血病ウィルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターおよび薬剤応答性プロモーターが例示される。薬剤応答性プロモーターとは、対応する薬剤の存在下で遺伝子を発現するTREプロモーター（tetO 配列が7回連続したTet応答配列をもつCMV 最小プロモーター）が例示される。TREプロモーターを用いた場合、同一の細胞において、reverse tetR (rtetR)およびVP16ADとの融合タンパク質を同時に発現させることで、対応する薬剤（例えば、テトラサイクリンまたはドキシサイクリンが例示される）の存在下で遺伝子発現を誘導する様式を用いることが好ましい。さらに好ましいベクターは、同一ベクターにTREプロモーターならびにrtetRおよびVP16ADの融合遺伝子が配置されており、当該融合遺伝子を発現する様式を持つベクターである。 Examples of promoters used in expression vectors include EF-α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia) Examples include viral) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter and drug-responsive promoter. Examples of the drug-responsive promoter include a TRE promoter that expresses a gene in the presence of the corresponding drug (a CMV minimal promoter having a Tet response sequence in which tetO sequences are continuous seven times). When the TRE promoter is used, gene expression in the presence of the corresponding drug (eg, tetracycline or doxycycline) is achieved by simultaneously expressing a reverse tetR (rtetR) and VP16AD fusion protein in the same cell. It is preferred to use a mode that induces. A more preferred vector is a vector in which a TRE promoter and a fusion gene of rtetR and VP16AD are arranged in the same vector and has a mode of expressing the fusion gene.
発現ベクターは、プロモーターの他に、所望によりエンハンサー、ポリＡ付加シグナル、選択マーカー遺伝子、SV40複製起点などを含有していてもよい。有用な選択マーカー遺伝子としては、例えば、ジヒドロ葉酸還元酵素遺伝子、ネオマイシン耐性遺伝子、ピューロマイシン耐性遺伝子等が挙げられる。 In addition to the promoter, the expression vector may optionally contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like. Useful selection marker genes include, for example, dihydrofolate reductase gene, neomycin resistance gene, puromycin resistance gene and the like.
本発明において、Cre-loxPシステムを使用して、遺伝子をベクターから切り出すため、loxP配列にて遺伝子またはプロモーター領域もしくはその両方をはさむようにloxP配列を設置された発現ベクターを用いてもよい。 In the present invention, in order to cut out a gene from a vector using the Cre-loxP system, an expression vector in which a loxP sequence is placed so as to sandwich the gene and / or promoter region with the loxP sequence may be used.
本発明では、同時に複数の遺伝子を導入するため、遺伝子が縦に連結されてポリシストロニックなベクターを得てもよい。ポリシストロニックな発現を可能にするために、強制発現させる複数の遺伝子間に、口蹄疫ウィルスの2A自己開裂ペプチド（Science, 322, 949-953, 2008などを参照)およびIRES（Internal ribosome entry site）配列などをライゲーションしてもよい。 In the present invention, since a plurality of genes are introduced at the same time, the genes may be vertically linked to obtain a polycistronic vector. To enable polycistronic expression, a 2A self-cleaving peptide of foot-and-mouth disease virus (see Science, 322, 949-953, 2008, etc.) and IRES (Internal ribosome entry site) An array or the like may be ligated.
本発明において、発現ベクターを造血前駆細胞に導入する方法は、ウィルスベクターの場合、該核酸を含むプラスミドを適当なパッケージング細胞（例、Plat-E細胞）や補完細胞株（例、293細胞）に導入して、培養上清中に産生されたウィルスを回収し造血前駆細胞と接触させ感染させることによって成し得る。非ウィルスベクターの場合、リポフェクション法、リポソーム法、エレクトロポレーション法、リン酸カルシウム共沈殿法、DEAEデキストラン法、マイクロインジェクション法、遺伝子銃法などを用いてプラスミドベクターを細胞に導入することができる。 In the present invention, the method of introducing an expression vector into a hematopoietic progenitor cell is as follows. In the case of a viral vector, a plasmid containing the nucleic acid is appropriately packaged (eg, Plat-E cell) or a complementary cell line (eg, 293 cell) The virus produced in the culture supernatant can be collected and contacted with hematopoietic progenitor cells for infection. In the case of a non-viral vector, a plasmid vector can be introduced into a cell using a lipofection method, a liposome method, an electroporation method, a calcium phosphate coprecipitation method, a DEAE dextran method, a microinjection method, a gene gun method, or the like.
本発明に係る巨核球前駆細胞の製造方法の一態様として、造血前駆細胞へ癌遺伝子、あるいはp16遺伝子又はp19遺伝子の発現を抑制する遺伝子を強制発現させた後に、さらに、アポトーシス抑制遺伝子を強制発現させてもよい。アポトーシス抑制遺伝子の強制発現も、上述と同様に、発現ベクター、またはこれらの遺伝子がコードするタンパク質またはこれらの遺伝子をコードするRNAを造血前駆細胞へ導入することによって成し得る。 As one embodiment of the method for producing megakaryocyte progenitor cells according to the present invention, after forcibly expressing an oncogene, or a gene that suppresses the expression of p16 gene or p19 gene in hematopoietic progenitor cells, and further forcibly expressing an apoptosis-suppressing gene You may let them. In the same manner as described above, forced expression of apoptosis-suppressing genes can also be achieved by introducing expression vectors, proteins encoded by these genes, or RNAs encoding these genes into hematopoietic progenitor cells.
本発明において、さらにアポトーシス抑制遺伝子を強制発現させる場合、特に限定しないが、癌遺伝子、およびp16遺伝子又はp19遺伝子の発現を抑制する遺伝子を少なくとも7日以上、8日以上、9日以上、10日以上、11日以上、12日以上、13日以上または14日間以上強制発現した後にアポトーシス抑制遺伝子の強制発現が行われ、より好ましくは、14日以上である。 In the present invention, when the apoptosis-suppressing gene is forcibly expressed, it is not particularly limited, but the gene that suppresses the expression of the oncogene and the p16 gene or the p19 gene is at least 7 days, 8 days, 9 days, 10 days. As described above, the forced expression of the apoptosis-suppressing gene is performed after forced expression for 11 days or more, 12 days or more, 13 days or more, or 14 days or more, and more preferably 14 days or more.
本発明において、「アポトーシス抑制遺伝子」とは、アポトーシスを抑制する遺伝子であれば特に限定されず、例えば、BCL2遺伝子、BCL−XL遺伝子、Survivin、MCL1などが挙げられる。好ましくは、アポトーシス抑制遺伝子はBCL-XL遺伝子である。BCL−XL遺伝子とは、例えば、NCBIのアクセッション番号NM_001191またはNM_138578で示される核酸配列からなる遺伝子である。また、BCL−XL遺伝子には、そのホモログも含まれてよく、BCL−XL遺伝子のホモログとは、そのcDNA配列が、例えば、NCBIのアクセッション番号NM_001191またはNM_138578で示される核酸配列と実質的に同一の配列からなる遺伝子のことである。NCBIのアクセッション番号NM_001191またはNM_138578で示される核酸配列と実質的に同一の配列からなるcDNAとは、NCBIのアクセッション番号NM_001191またはNM_138578で表される配列からなるDNAと、約60％以上、好ましくは約70％以上、より好ましくは約80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、最も好ましくは約99％の同一性を有する配列からなるDNA、もしくは、NCBIのアクセッション番号NM_001191またはNM_138578で表わされる核酸配列に相補的な配列からなるDNAとストリンジェントな条件下でハイブリダイズできるDNAであって、そのDNAによってコードされるタンパク質が、アポトーシスを抑制する効果を有するもののことである。 In the present invention, the “apoptosis suppressor gene” is not particularly limited as long as it is a gene that suppresses apoptosis, and examples thereof include BCL2 gene, BCL-XL gene, Survivin, MCL1, and the like. Preferably, the apoptosis inhibitor gene is a BCL-XL gene. The BCL-XL gene is, for example, a gene consisting of a nucleic acid sequence represented by NCBI accession number NM_001191 or NM_138578. The BCL-XL gene may also include a homologue thereof. The homologue of the BCL-XL gene is substantially the same as the nucleic acid sequence represented by the NCBI accession number NM_001191 or NM_138578, for example. A gene consisting of the same sequence. The cDNA comprising substantially the same sequence as the nucleic acid sequence represented by NCBI accession number NM_001191 or NM_138578 is approximately 60% or more, preferably the DNA comprising the sequence represented by NCBI accession number NM_001191 or NM_138578. Is more than about 70%, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, most preferably about 99% identity DNA or complementary to the nucleic acid sequence represented by NCBI accession number NM_001191 or NM_138578 DNA which can hybridize with DNA consisting of a typical sequence under stringent conditions, and the protein encoded by the DNA has an effect of suppressing apoptosis.
本発明では、アポトーシス抑制遺伝子を造血前駆細胞において強制発現させることに代わって、カスパーゼ阻害剤を細胞と接触させても良い。本発明において、カスパーゼ阻害剤は、ペプチド性化合物、非ペプチド性化合物、あるいは、生物由来のタンパク質のいずれであってもよい。ペプチド性化合物としては、例えば人工的に化学合成された下記（１）〜（１０）のペプチド性化合物を挙げることができる。
（１）Z-Asp-CH2-DCB（分子量４５４．２６）
（２）Boc-Asp(OMe)-FMK（分子量２６３．３）
（３）Boc-Asp(OBzl)-CMK（分子量３５５．８）
（４）Ac-AAVALLPAVLLALLAP-YVAD-CHO（分子量１９９０．５）（配列番号１）
（５）Ac-AAVALLPAVLLALLAP-DEVD-CHO（分子量２０００．４）（配列番号２）
（６）Ac-AAVALLPAVLLALLAP-LEVD-CHO（分子量１９９８．５）（配列番号３）
（７）Ac-AAVALLPAVLLALLAP-IETD-CHO（分子量２０００．５）（配列番号４）
（８）Ac-AAVALLPAVLLALLAP-LEHD-CHO（分子量２０３６．５）（配列番号５）
（９）Z-DEVD-FMK （Z-Asp-Glu-Val-Asp-fluoromethylketone）（配列番号６）
（１０）Z-VAD FMK
In the present invention, a caspase inhibitor may be brought into contact with cells instead of forcing the apoptosis-suppressing gene to be expressed in hematopoietic progenitor cells. In the present invention, the caspase inhibitor may be a peptidic compound, a non-peptidic compound, or a biological protein. Examples of the peptide compound include the following peptide compounds (1) to (10) which are artificially chemically synthesized.
(1) Z-Asp-CH2-DCB (molecular weight 454.26)
(2) Boc-Asp (OMe) -FMK (molecular weight 263.3)
(3) Boc-Asp (OBzl) -CMK (molecular weight 355.8)
(4) Ac-AAVALLPAVLLALLAP-YVAD-CHO (molecular weight 1990.5) (SEQ ID NO: 1)
(5) Ac-AAVALLPAVLLALLAP-DEVD-CHO (molecular weight 2000.4) (SEQ ID NO: 2)
(6) Ac-AAVALLPAVLLALLAP-LEVD-CHO (molecular weight 19988.5) (SEQ ID NO: 3)
(7) Ac-AAVALLPAVLLALLAP-IETD-CHO (molecular weight 2000.5) (SEQ ID NO: 4)
(8) Ac-AAVALLPAVLLALLAP-LEHD-CHO (molecular weight 2036.5) (SEQ ID NO: 5)
(9) Z-DEVD-FMK (Z-Asp-Glu-Val-Asp-fluoromethylketone) (SEQ ID NO: 6)
(10) Z-VAD FMK
例えば、ペプチド性化合物のカスパーゼ阻害剤として、（１）VX-740 - Vertex Pharmaceuticals (Leung-Toung et al., Curr. Med. Chem. 9, 979-1002 (2002))、（２）HMR-3480 - Aventis Pharma AG (Randle et al., Expert Opin. Investig. Drugs 10, 1207-1209 (2001))、を挙げることができる。 For example, as caspase inhibitors of peptide compounds, (1) VX-740-Vertex Pharmaceuticals (Leung-Toung et al., Curr. Med. Chem. 9, 979-1002 (2002)), (2) HMR-3480 -Aventis Pharma AG (Randle et al., Expert Opin. Investig. Drugs 10, 1207-1209 (2001)).
非ペプチド性化合物のカスパーゼ阻害剤としては、（１）アニリノキナゾリン（anilinoquinazolines (AQZs)）-AstraZeneca Pharmaceuticals （Scott et al., J. Pharmacol. Exp. Ther. 304, 433-440 (2003)）、（２）Ｍ８２６ - Merck Frosst （Han et al., J. Biol. Chem. 277, 30128-30136 (2002)）、（３）Ｍ８６７ - Merck Frosst （Methot et al., J.Exp. Med. 199, 199-207 (2004)）、（４）ニコチニルアスパチルケトンズ（Nicotinyl aspartyl ketones）- Merck Frosst (Isabel et al., Bioorg. Med. Chem. Lett. 13, 2137-2140 (2003))、などを例示することができる。 Examples of caspase inhibitors of non-peptide compounds include (1) anilinoquinazolines (AQZs) -AstraZeneca Pharmaceuticals (Scott et al., J. Pharmacol. Exp. Ther. 304, 433-440 (2003)), (2) M826-Merck Frosst (Han et al., J. Biol. Chem. 277, 30128-30136 (2002)), (3) M867-Merck Frosst (Methot et al., J. Exp. Med. 199, 199-207 (2004)), (4) Nicotinyl aspartyl ketones-Merck Frosst (Isabel et al., Bioorg. Med. Chem. Lett. 13, 2137-2140 (2003)), etc. Can be illustrated.
また、その他の非ペプチド性化合物のカスパーゼ阻害剤として、（１）IDN-6556 - Idun Pharmaceuticals (Hoglen et al., J.Pharmacol. Exp. Ther. 309, 634-640 (2004))、（２）MF-286 and MF-867 - Merck Frosst (Los et al., Drug Discov. Today 8, 67-77 (2003))、（３）IDN-5370 - Idun Pharmaceuticals (Deckwerth et al., Drug Dev. Res. 52, 579-586 (2001))、（４）IDN-1965 - Idun Pharmaceuticals (Hoglen et al., J. Pharmacol. Exp. Ther. 297, 811-818 (2001))、（５）VX-799 - Vertex Pharmaceuticals (Los et al., Drug Discov. Today 8, 67-77 (2003))、などを挙げることができる。このほかに、M-920 and M-791 - Merck Frosst (Hotchkiss et al., Nat. Immunol. 1, 496-501 (2000))などもカスパーゼ阻害剤として挙げることができる。 Further, as caspase inhibitors of other non-peptidic compounds, (1) IDN-6556-Idun Pharmaceuticals (Hoglen et al., J. Pharmacol. Exp. Ther. 309, 634-640 (2004)), (2) MF-286 and MF-867-Merck Frosst (Los et al., Drug Discov. Today 8, 67-77 (2003)), (3) IDN-5370-Idun Pharmaceuticals (Deckwerth et al., Drug Dev. Res. 52, 579-586 (2001)), (4) IDN-1965-Idun Pharmaceuticals (Hoglen et al., J. Pharmacol. Exp. Ther. 297, 811-818 (2001)), (5) VX-799- Vertex Pharmaceuticals (Los et al., Drug Discov. Today 8, 67-77 (2003)). In addition, M-920 and M-791-Merck Frosst (Hotchkiss et al., Nat. Immunol. 1, 496-501 (2000)) can also be mentioned as caspase inhibitors.
本発明において、好ましいカスパーゼ阻害剤は、Z-VAD FMKである。カスパーゼ阻害剤としてZ-VAD FMKを用いる場合、その添加は造血前駆細胞を培養する培地に対し行われる。培地中でのZ-VAD FMKの好ましい濃度は、例えば、10μM以上、20μM以上、30μM以上、40μM以上、および50μM以上が挙げられ、好ましくは、30μM以上である。 In the present invention, a preferred caspase inhibitor is Z-VAD FMK. When Z-VAD FMK is used as a caspase inhibitor, its addition is performed on a medium for culturing hematopoietic progenitor cells. Preferred concentrations of Z-VAD FMK in the medium include, for example, 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, and 50 μM or more, and preferably 30 μM or more.
本発明において、培養する際の温度条件は、特に限定されないが、37℃以上の温度で造血前駆細胞を培養することにより、巨核球前駆細胞への分化を促進することが確認されている。ここで、37℃以上の温度とは、細胞にダメージを与えない程度の温度が適当であることから、例えば、約37℃〜約42℃程度、約37〜約39℃程度が好ましい。また、37℃以上の温度における培養期間については、当業者であれば巨核球前駆細胞の数などをモニターしながら、適宜決定することが可能である。所望の巨核球前駆細胞が得られる限り、日数は特に限定されないが、例えば、少なくとも6日間以上、12日以上、18日以上、24日以上、30日以上、42日以上、48日以上、54日以上、60日以上であり、好ましくは60日以上である。培養期間が長いことについては、巨核球前駆細胞の製造においては問題とされない。また、培養期間中は、適宜、継代を行うことが望ましい。 In the present invention, the temperature conditions for culturing are not particularly limited, and it has been confirmed that culturing hematopoietic progenitor cells at a temperature of 37 ° C. or higher promotes differentiation into megakaryocyte progenitor cells. Here, the temperature of 37 ° C. or higher is appropriate as a temperature that does not damage cells, and is preferably about 37 ° C. to about 42 ° C., and preferably about 37 to about 39 ° C., for example. In addition, the culture period at a temperature of 37 ° C. or higher can be appropriately determined by those skilled in the art while monitoring the number of megakaryocyte progenitor cells and the like. The number of days is not particularly limited as long as the desired megakaryocyte progenitor cell is obtained.For example, at least 6 days, 12 days, 18 days, 24 days, 30 days, 42 days, 48 days, 54 days, 54 More than 60 days and more preferably 60 days or more. The long culture period is not a problem in the production of megakaryocyte progenitor cells. In addition, it is desirable to perform subculture as appropriate during the culture period.
（巨核球前駆細胞の維持培養方法）
本発明はまた、HDAC阻害剤の存在下で巨核球前駆細胞を維持培養する方法を提供する。本明細書において「維持培養」とは、巨核球前駆細胞の巨核球への分化・成熟を抑制しながら巨核球前駆細胞を増殖させることをいう。維持培養においては、培養細胞の数が全体として増加を続ける限り、巨核球前駆細胞の一部が巨核球に分化してもよい。
(Maintenance culture method of megakaryocyte progenitor cells)
The present invention also provides a method of maintaining and culturing megakaryocyte progenitor cells in the presence of an HDAC inhibitor. As used herein, “maintenance culture” refers to proliferating megakaryocyte progenitor cells while suppressing differentiation / maturation of megakaryocyte progenitor cells into megakaryocytes. In maintenance culture, as long as the number of cultured cells continues to increase as a whole, some megakaryocyte progenitor cells may differentiate into megakaryocytes.
本発明において、巨核球前駆細胞の維持培養は、巨核球前駆細胞と同様の培養条件によって行い得る。巨核球前駆細胞が「癌遺伝子」、「p16遺伝子又はp19遺伝子の発現を抑制する遺伝子」および／または「アポトーシス抑制遺伝子」を発現させている場合、維持培養の間、これらの遺伝子は発現を維持されることが望ましい。 In the present invention, maintenance culture of megakaryocyte progenitor cells can be performed under the same culture conditions as megakaryocyte progenitor cells. When megakaryocyte progenitor cells express “oncogene”, “gene that suppresses expression of p16 gene or p19 gene” and / or “apoptosis suppressor gene”, these genes maintain expression during maintenance culture It is desirable that
（血小板の製造方法）
本発明は、上述の方法で得られた巨核球前駆細胞からさらに巨核球細胞および/または血小板を製造する方法を提供する。「癌遺伝子」、「p16遺伝子又はp19遺伝子の発現を抑制する遺伝子」および／または「アポトーシス抑制遺伝子」を強制発現させている場合、当該強制発現を停止して培養することによって巨核球細胞および/または血小板が製造され得る。強制発現の停止は、例えば、薬剤応答性ベクターを用いて強制発現をしている場合、対応する薬剤と当該細胞と接触させないことによって達成してもよい。この他にも、上記のLoxPを含むベクターを用いた場合は、Creリコンビナーゼを当該細胞に導入することによって強制発現を停止してもよい。さらに、一過性発現ベクター、およびRNAまたはタンパク質導入を用いた場合は、当該ベクター等との接触を止めることによって強制発現を停止してもよい。強制発現の停止において用いられる培地は、上記と同一の培地を用いて行うことができる。
(Platelet production method)
The present invention further provides a method for producing megakaryocyte and / or platelets from megakaryocyte progenitor cells obtained by the method described above. When “oncogene”, “gene that suppresses expression of p16 gene or p19 gene” and / or “apoptosis suppressor gene” is forcibly expressed, megakaryocytes and / or Or platelets can be produced. For example, when forced expression is carried out using a drug-responsive vector, the forced expression may be stopped by not bringing the corresponding drug into contact with the cell. In addition, when the above-mentioned vector containing LoxP is used, forced expression may be stopped by introducing Cre recombinase into the cell. Further, when a transient expression vector and RNA or protein introduction are used, forced expression may be stopped by stopping contact with the vector or the like. The medium used for stopping the forced expression can be performed using the same medium as described above.
強制発現を停止して培養する際の温度条件は、特に限定されないが、例えば、約37℃〜約42℃程度、約37〜約39℃程度が好ましい。また、37℃以上の温度における培養期間については、当業者であれば巨核球の数などをモニターしながら、適宜決定することが可能であるが、例えば、2日間〜10日間、好ましくは3日間〜7日間程度である。少なくとも3日以上であることが望ましい。また、培養期間中は、適宜、継代を行うことが望ましい。 There are no particular limitations on the temperature conditions when culturing with forced expression stopped, but for example, about 37 ° C to about 42 ° C, preferably about 37 ° C to about 39 ° C. The culture period at a temperature of 37 ° C. or higher can be appropriately determined by those skilled in the art while monitoring the number of megakaryocytes, etc., for example, 2 days to 10 days, preferably 3 days About 7 days. Desirably at least 3 days. In addition, it is desirable to perform subculture as appropriate during the culture period.
本発明では、上述の方法で得られた巨核球前駆細胞を凍結保存することができる。巨核球前駆細胞は、凍結保存した状態で流通させることができる。 In the present invention, megakaryocyte progenitor cells obtained by the above method can be cryopreserved. Megakaryocyte progenitor cells can be distributed in a cryopreserved state.
本発明において、巨核球細胞および/または血小板の製造方法の一態様では、培地にROCK阻害剤および／またはアクトミオシン複合体機能阻害剤が加えられる。ROCK阻害剤としては、例えばY27632が挙げられる。アクトミオシン複合体機能阻害剤としては、ミオシン重鎖II ATPase阻害剤である、ブレビスタチンが挙げられる。培地には、ROCK阻害剤を単独で加えてもよく、ROCK阻害剤とアクトミオシン複合体機能阻害剤を異なるタイミングでそれぞれ単独で加えてもよいし、これらを組み合わせて加えてもよい。 In the present invention, in one embodiment of the method for producing megakaryocytes and / or platelets, a ROCK inhibitor and / or an actomyosin complex function inhibitor is added to the medium. Examples of the ROCK inhibitor include Y27632. Examples of the actomyosin complex function inhibitor include blebbistatin, which is a myosin heavy chain II ATPase inhibitor. A ROCK inhibitor may be added to the medium alone, or a ROCK inhibitor and an actomyosin complex function inhibitor may be added individually at different timings, or a combination thereof may be added.
ROCK阻害剤および／またはアクトミオシン複合体機能阻害剤は、0.1μM〜30μMで培地に加えることが好ましく、より具体的には、阻害剤の濃度を例えば0.5μM〜25μM、5μM〜20μM等としてもよい。 The ROCK inhibitor and / or the actomyosin complex function inhibitor is preferably added to the medium at 0.1 μM to 30 μM. More specifically, the inhibitor concentration may be 0.5 μM to 25 μM, 5 μM to 20 μM, or the like. Good.
本明細書中に記載される「細胞」の由来は、ヒト及び非ヒト動物（例えば、マウス、ラット、ウシ、ウマ、ブタ、ヒツジ、サル、イヌ、ネコ、トリなど）であり、特に限定はされないが、ヒト由来の細胞が特に好ましい。 The origin of “cells” described herein is human and non-human animals (eg, mice, rats, cows, horses, pigs, sheep, monkeys, dogs, cats, birds, etc.), with particular limitations Although not, human-derived cells are particularly preferred.
以下に実施例を示してさらに詳細に説明するが、本発明は実施例により何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.
1) ES/iPS細胞からの造血前駆細胞の調製
ヒトES細胞（khES3：京都大学より入手、H1：WiCell Research Instituteより入手可能）およびiPS細胞（TKDN SeV2およびTKDN SeV5：センダイウィルスを用いて樹立されたヒト胎児皮膚繊維芽細胞由来iPS細胞、TKPB SeV8およびTKPB SeV9：センダイウィルスを用いて樹立されたヒト末梢血単核球由来iPS細胞、EP1（585A1）、EP2（585B1）およびEP6（692D2）：Okita K, et al, Stem Cells 31, 458-66, 2013に記載のエピソーマルベクターを用いて樹立されたヒト末梢血単核球由来iPS細胞）から、Takayama N., et al. Blood, 5298-5306 (2008)に記載の方法に従って、血球細胞への分化培養を実施した。即ち、ヒトES/iPS細胞コロニーを20ng/mL VEGF (R&D SYSTEMS)を含む基本培地（15% Fetal Bovine Serum (GIBCO)、1% Penicillin-Streptomycin-Glutamine (GIBCO)、1% Insulin, Transferrin, Selenium Solution (ITS -G) (GIBCO)、0.45mM 1-Thioglycerol (Sigma-Aldrich)、50μg/mL L-Ascorbic Acid (Sigma-Aldrich)を含有するIMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich)）中でC3H10T1/2細胞（RIKEN BioResource Center）と14日間共培養して造血前駆細胞（Hematopoietic Progenitor Cells（HPC））を作製した。培養は20% O2、5% CO2で実施した（特に記載がない限り、以下同条件）。
1) Preparation of hematopoietic progenitor cells from ES / iPS cells Human ES cells (khES3: obtained from Kyoto University, H1: available from WiCell Research Institute) and iPS cells (TKDN SeV2 and TKDN SeV5: established using Sendai virus) Human fetal skin fibroblast-derived iPS cells, TKPB SeV8 and TKPB SeV9: Human peripheral blood mononuclear cell-derived iPS cells established using Sendai virus, EP1 (585A1), EP2 (585B1) and EP6 (692D2): From human peripheral blood mononuclear cell-derived iPS cells established using the episomal vector described in Okita K, et al, Stem Cells 31, 458-66, 2013), Takayama N., et al. Blood, 5298- According to the method described in 5306 (2008), differentiation culture into blood cells was performed. That is, human ES / iPS cell colonies were basal medium containing 20ng / mL VEGF (R & D SYSTEMS) (15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45 mM 1-Thioglycerol (Sigma-Aldrich), in IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich) containing 50 μg / mL L-Ascorbic Acid (Sigma-Aldrich)) Hematopoietic progenitor cells (HPC) were prepared by co-culture with C3H10T1 / 2 cells (RIKEN BioResource Center) for 14 days. The culture was carried out in 20% O 2 and 5% CO 2 (the same conditions unless otherwise stated).
2)ヒト血小板
ドナーより同意を得て得られた全血に１／９量のデキストロース溶液を添加し、900rpm,で１０分間遠心した後、回収した上清を多血小板血漿（platelet rich plasma （PRP））として用いた。
2) Human platelets After adding 1/9 volume of dextrose solution to whole blood obtained with the consent of the donor and centrifuging at 900 rpm for 10 minutes, the collected supernatant was treated with platelet rich plasma (PRP). )).
3)血小板の誘導
上記1)の共培養14日目に上記HPCを回収し、6well plate中のC3H10 T1/2細胞上に3x104/wellで播種し、50ng/mlのSCF (R&D SYSTEMS)、50ng/mlのTPO (R&D SYSTEMS)およびHDAC 阻害剤（10nM TSA（トリコスタチンA）（Sigma-Aldrich）、100nM SAHA（Vorinostat）（Sigma-Aldrich）、10μM MC1568（Sigma-Aldrich）、または1nM LBH-589 (Panobinostat) （Sigma-Aldrich））を含む基本培地で10日間培養した。陰性対照として、50ng/mlのSCF、50ng/mlのTPOおよびDMSOを含む基本培地で10日間 HPCを培養した。
3) Induction of platelets On the 14th day of co-culture of 1) above, the HPC was collected, seeded at 3 × 10 4 / well on C3H10 T1 / 2 cells in a 6-well plate, 50 ng / ml SCF (R & D SYSTEMS), 50 ng / ml TPO (R & D SYSTEMS) and HDAC inhibitors (10 nM TSA (trichostatin A) (Sigma-Aldrich), 100 nM SAHA (Vorinostat) (Sigma-Aldrich), 10 μM MC1568 (Sigma-Aldrich), or 1 nM LBH- The cells were cultured in a basic medium containing 589 (Panobinostat) (Sigma-Aldrich) for 10 days. As a negative control, HPC was cultured for 10 days in basal medium containing 50 ng / ml SCF, 50 ng / ml TPO and DMSO.
10日目に回収した培養上清に、１／９量のデキストロース溶液（85ｍM クエン酸ナトリウム、104 mM グルコース、および65mM クエン酸）、抗CD41a抗体（BioLegend）および抗CD42b抗体（BioLegend）を添加した後、フローサイトメーターを用いて大きさ（FSCおよびSSC）によって、2)で得られた多血小板血漿と同等の大きさの粒子を選択し（図１AおよびB）、当該選択した粒子を細胞表面抗原（CD41aおよびCD42b）によって血小板として計数した（図１C）。CD41a陽性/CD42b陽性画分のパーティクル数をBD Trucountチューブを用いて補正し、１wellあたりの血小板の総数として算出した。得られた血小板数は、HDAC 阻害剤の代わりにDMSOを添加した陰性対照の血小板数に対する比率として算出し、当該比率を各iPS細胞株からの血小板産出量として図２から図５に示した。 1/9 volume of dextrose solution (85 mM sodium citrate, 104 mM glucose, and 65 mM citric acid), anti-CD41a antibody (BioLegend) and anti-CD42b antibody (BioLegend) were added to the culture supernatant collected on day 10 Then, using a flow cytometer, select the size (FSC and SSC) of the same size as the platelet-rich plasma obtained in 2) (FIGS. 1A and B), and select the selected particles on the cell surface. Counted as platelets by antigen (CD41a and CD42b) (FIG. 1C). The number of particles of the CD41a positive / CD42b positive fraction was corrected using a BD Trucount tube and calculated as the total number of platelets per well. The obtained platelet count was calculated as a ratio with respect to the platelet count of the negative control to which DMSO was added instead of the HDAC inhibitor, and the ratio was shown in FIGS. 2 to 5 as the amount of platelet production from each iPS cell line.
その結果、各iPS細胞株からTSA、SAHA、MC1568、またはLBH-589を添加することで得られた血小板数は、添加しなかった場合と比較して約１．７倍から５．０倍程度増加した。 As a result, the number of platelets obtained by adding TSA, SAHA, MC1568, or LBH-589 from each iPS cell line is about 1.7 to 5.0 times that in the case of not adding them. Increased.
以上の結果より、HDAC阻害剤を添加した培地中で造血前駆細胞を培養することで、血小板の産生量を増加させることができることが確認された。 From the above results, it was confirmed that the amount of platelet production can be increased by culturing hematopoietic progenitor cells in a medium supplemented with an HDAC inhibitor.
HDAC阻害剤の添加時期の検討
実施例１に記載した方法でTKDN SeV5およびEP2から得られたHPCを、6well plate中のC3H10 T1/2細胞上に3x104/wellで播種し、次の５つの条件のいずれかで培養した；
（条件１）DMSOを添加した血小板誘導培地（50ng/ml SCFおよび50ng/ml TPOを添加した基本培地）で１０日間培養（DMSO）、
（条件２）10nM TSAを添加した血小板誘導培地で１０日間培養（Day 0-10）、
（条件３）10nM TSAを添加した血小板誘導培地で４日間培養した後、DMSOを添加した血小板誘導培地に交換しさらに６日間培養（Day 0-4）、
（条件４）DMSOを添加した血小板誘導培地で４日間培養後、10nM TSAを添加した血小板誘導培地に交換し４日間培養した後、DMSOを添加した血小板誘導培地に交換し２日間培養（Day 4-8）、または
（条件５）DMSOを添加した血小板誘導培地で８日間培養後、10nM TSAを添加した血小板誘導培地に交換し２日間培養（Day 8-10）。
Examination of HDAC inhibitor addition time HPC obtained from TKDN SeV5 and EP2 by the method described in Example 1 was seeded at 3 × 10 4 / well on C3H10 T1 / 2 cells in a 6-well plate. Cultured under any of the conditions;
(Condition 1) Culture for 10 days in DMSO-added platelet induction medium (basic medium supplemented with 50 ng / ml SCF and 50 ng / ml TPO) (DMSO),
(Condition 2) Culture for 10 days in a platelet induction medium supplemented with 10 nM TSA (Day 0-10),
(Condition 3) After culturing in a platelet induction medium supplemented with 10 nM TSA for 4 days, the medium was replaced with a platelet induction medium supplemented with DMSO and further cultured for 6 days (Day 0-4).
(Condition 4) After culturing for 4 days in a platelet induction medium supplemented with DMSO, replaced with a platelet induction medium supplemented with 10 nM TSA and cultured for 4 days, then replaced with a platelet induction medium supplemented with DMSO and cultured for 2 days (Day 4 -8) or (Condition 5) After culturing in a platelet induction medium supplemented with DMSO for 8 days, the medium was replaced with a platelet induction medium supplemented with 10 nM TSA and cultured for 2 days (Day 8-10).
各条件で得られた血小板産出量を実施例１の方法に従って算出した。TKDN SeV5における結果を図６に示し、EP2における結果を図７に示した。 The amount of platelet production obtained under each condition was calculated according to the method of Example 1. The result in TKDN SeV5 is shown in FIG. 6, and the result in EP2 is shown in FIG.
続いて、実施例１に記載した方法でEP6から得られたHPCを、6well plate中のC3H10 T1/2細胞上に3x104/wellで播種し、次の５つの条件のいずれかで培養した；
（条件１）DMSOを添加した血小板誘導培地で１０日間培養（DMSO）、
（条件２）10nM TSAを添加した血小板誘導培地で１０日間培養（Day 0-10）、
（条件３）10nM TSAを添加した血小板誘導培地で４日間培養した後、DMSOを添加した血小板誘導培地に交換しさらに６日間培養（Day 0-4）、
（条件４）DMSOを添加した血小板誘導培地で４日間培養後、10nM TSAを添加した血小板誘導培地に交換し３日間培養した後、DMSOを添加した血小板誘導培地に交換し３日間培養（Day 4-8）、または
（条件５）DMSOを添加した血小板誘導培地で７日間培養後、10nM TSAを添加した血小板誘導培地に交換し３日間培養（Day 8-10）。
Subsequently, HPC obtained from EP6 by the method described in Example 1 was seeded at 3 × 10 4 / well on C3H10 T1 / 2 cells in a 6-well plate and cultured under any of the following five conditions;
(Condition 1) Culture for 10 days in a platelet induction medium supplemented with DMSO (DMSO),
(Condition 2) Culture for 10 days in a platelet induction medium supplemented with 10 nM TSA (Day 0-10),
(Condition 3) After culturing in a platelet induction medium supplemented with 10 nM TSA for 4 days, the medium was replaced with a platelet induction medium supplemented with DMSO and further cultured for 6 days (Day 0-4).
(Condition 4) After culturing for 4 days in a platelet induction medium supplemented with DMSO, replaced with a platelet induction medium supplemented with 10 nM TSA and cultured for 3 days, then replaced with a platelet induction medium supplemented with DMSO and cultured for 3 days (Day 4 -8) or (Condition 5) After culturing in a platelet induction medium supplemented with DMSO for 7 days, the medium was replaced with a platelet induction medium supplemented with 10 nM TSA and cultured for 3 days (Day 8-10).
この条件で得られた血小板産出量を実施例１の方法に従って算出し、結果を図８に示した。 The amount of platelet production obtained under these conditions was calculated according to the method of Example 1, and the results are shown in FIG.
さらに、実施例１に記載した方法でEP2から得られたHPCを、6well plate中のC3H10 T1/2細胞上に3x104/wellで播種し、次の５つの条件のいずれかで培養した；
（条件１）DMSOを添加した血小板誘導培地（50ng/ml SCFおよび50ng/ml TPOを添加した基本培地）で１０日間培養（DMSO）、
（条件２）10μM MC1568を添加した血小板誘導培地で１０日間培養（Day 0-10）、
（条件３）10μM MC1568を添加した血小板誘導培地で４日間培養した後、DMSOを添加した血小板誘導培地に交換しさらに６日間培養（Day 0-4）、
（条件４）DMSOを添加した血小板誘導培地で４日間培養後、10μM MC1568を添加した血小板誘導培地に交換し４日間培養した後、DMSOを添加した血小板誘導培地に交換し２日間培養（Day 4-8）、または（条件５）DMSOを添加した血小板誘導培地で８日間培養後、10μM MC1568を添加した血小板誘導培地に交換し２日間培養（Day 8-10）。
Furthermore, HPC obtained from EP2 by the method described in Example 1 was seeded at 3 × 10 4 / well on C3H10 T1 / 2 cells in a 6-well plate and cultured under any of the following five conditions;
(Condition 1) Culture for 10 days in DMSO-added platelet induction medium (basic medium supplemented with 50 ng / ml SCF and 50 ng / ml TPO) (DMSO),
(Condition 2) Culture for 10 days in a platelet induction medium supplemented with 10 μM MC1568 (Day 0-10),
(Condition 3) After culturing for 4 days in a platelet induction medium supplemented with 10 μM MC1568, the medium was replaced with a platelet induction medium supplemented with DMSO and further cultured for 6 days (Day 0-4).
(Condition 4) After culturing for 4 days in a platelet induction medium supplemented with DMSO, replaced with a platelet induction medium supplemented with 10 μM MC1568 for 4 days, then replaced with a platelet induction medium supplemented with DMSO and cultured for 2 days (Day 4 -8) or (Condition 5) After culturing in a platelet induction medium supplemented with DMSO for 8 days, the medium was replaced with a platelet induction medium supplemented with 10 μM MC1568 and cultured for 2 days (Day 8-10).
この条件で得られた血小板産出量を実施例１の方法に従って算出し、結果を図９に示した。 The amount of platelet production obtained under these conditions was calculated according to the method of Example 1, and the results are shown in FIG.
以上の結果より、HDAC阻害剤は、HPCの分化誘導初期に作用させることで効率よく血小板を誘導することができることが示唆された。この結果は、巨核球の多核化にHDAC阻害剤が有用であるとの従来の知見とは異なり、新たな血小板誘導技術を提供するものである。 From the above results, it was suggested that the HDAC inhibitor can induce platelets efficiently by acting at the early stage of differentiation induction of HPC. This result is different from the conventional knowledge that HDAC inhibitors are useful for multinucleation of megakaryocytes, and provides a new platelet induction technique.
Claims (19)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014100422A JP6551963B2 (en) | 2014-05-14 | 2014-05-14 | Method for producing megakaryocyte precursor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014100422A JP6551963B2 (en) | 2014-05-14 | 2014-05-14 | Method for producing megakaryocyte precursor cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015216853A true JP2015216853A (en) | 2015-12-07 |
| JP6551963B2 JP6551963B2 (en) | 2019-07-31 |
Family
ID=54776858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014100422A Expired - Fee Related JP6551963B2 (en) | 2014-05-14 | 2014-05-14 | Method for producing megakaryocyte precursor cells |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6551963B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019526245A (en) * | 2016-08-04 | 2019-09-19 | アイカーン スクール オブ メディシン アット マウント サイナイ | Therapies for the manufacture of megakaryocyte compositions and the treatment of thrombocytopenia |
| CN110373387A (en) * | 2019-08-01 | 2019-10-25 | 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) | Histon deacetylase (HDAC) inhibitor promotes the purposes in the product that pluripotent stem cell differentiation is hematopoietic stem/progenitor cells in preparation |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002527101A (en) * | 1998-10-16 | 2002-08-27 | ノバルティス アクチエンゲゼルシャフト | Improved transduction of hematopoietic stem cells and promotion of self-renewal by histone deacetylase inhibitors |
| JP2005512961A (en) * | 2001-09-18 | 2005-05-12 | ゲー・ツヴァイ・エム キャンサー ドラッグス アー・ゲー | Valproic acid and its derivatives for combination therapy treatment of human cancer and treatment of tumor metastasis and minimal residual disease |
| JP2012065644A (en) * | 2010-06-14 | 2012-04-05 | Nissan Chem Ind Ltd | Method of producing hematopoietic stem cell |
| WO2012157586A1 (en) * | 2011-05-13 | 2012-11-22 | 国立大学法人東京大学 | Polynucleated megakaryocytic cell and method for manufacturing platelets |
| WO2013051625A1 (en) * | 2011-10-03 | 2013-04-11 | 日産化学工業株式会社 | Method for producing megakaryocytes and/or platelets from pluripotent stem cells |
| JP2013538810A (en) * | 2010-09-01 | 2013-10-17 | ノバルティス アーゲー | Combination of HDAC inhibitor and thrombocytopenia |
| JP2014003975A (en) * | 2012-05-30 | 2014-01-16 | Nissan Chem Ind Ltd | Method for producing hematopoietic stem cells using pyrazole compound |
| JP2015528284A (en) * | 2012-08-15 | 2015-09-28 | ボストン メディカル センター コーポレーション | Production of red blood cells and platelets from stem cells |
-
2014
- 2014-05-14 JP JP2014100422A patent/JP6551963B2/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002527101A (en) * | 1998-10-16 | 2002-08-27 | ノバルティス アクチエンゲゼルシャフト | Improved transduction of hematopoietic stem cells and promotion of self-renewal by histone deacetylase inhibitors |
| JP2005512961A (en) * | 2001-09-18 | 2005-05-12 | ゲー・ツヴァイ・エム キャンサー ドラッグス アー・ゲー | Valproic acid and its derivatives for combination therapy treatment of human cancer and treatment of tumor metastasis and minimal residual disease |
| JP2012065644A (en) * | 2010-06-14 | 2012-04-05 | Nissan Chem Ind Ltd | Method of producing hematopoietic stem cell |
| JP2013538810A (en) * | 2010-09-01 | 2013-10-17 | ノバルティス アーゲー | Combination of HDAC inhibitor and thrombocytopenia |
| WO2012157586A1 (en) * | 2011-05-13 | 2012-11-22 | 国立大学法人東京大学 | Polynucleated megakaryocytic cell and method for manufacturing platelets |
| WO2013051625A1 (en) * | 2011-10-03 | 2013-04-11 | 日産化学工業株式会社 | Method for producing megakaryocytes and/or platelets from pluripotent stem cells |
| JP2014003975A (en) * | 2012-05-30 | 2014-01-16 | Nissan Chem Ind Ltd | Method for producing hematopoietic stem cells using pyrazole compound |
| JP2015528284A (en) * | 2012-08-15 | 2015-09-28 | ボストン メディカル センター コーポレーション | Production of red blood cells and platelets from stem cells |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019526245A (en) * | 2016-08-04 | 2019-09-19 | アイカーン スクール オブ メディシン アット マウント サイナイ | Therapies for the manufacture of megakaryocyte compositions and the treatment of thrombocytopenia |
| US11713447B2 (en) | 2016-08-04 | 2023-08-01 | Icahn School Of Medicine At Mount Sinai | Production of megakaryoctye compositions and therapies for treatment of thrombocytopenia |
| CN110373387A (en) * | 2019-08-01 | 2019-10-25 | 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) | Histon deacetylase (HDAC) inhibitor promotes the purposes in the product that pluripotent stem cell differentiation is hematopoietic stem/progenitor cells in preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6551963B2 (en) | 2019-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220017866A1 (en) | Production methods for megakaryocytes and platelets | |
| JP7215994B2 (en) | Immune cells derived from induced pluripotent stem cells | |
| KR102600772B1 (en) | Method for producing highly functional platelets | |
| JP2016525882A (en) | Human erythroblasts (ESRE) that self-regenerate extensively | |
| US20240191195A1 (en) | Method for producing platelets | |
| JP6959135B2 (en) | A method for producing a culture containing megakaryocytes and a method for producing platelets using the same. | |
| JP6551963B2 (en) | Method for producing megakaryocyte precursor cells | |
| US20240026304A1 (en) | Method for Producing Stem Cell Clones Suitable for Induction of Differentiation into Somatic Cells | |
| JPWO2017047492A1 (en) | Method for producing platelets by rotary stirring culture method | |
| JP6385340B2 (en) | Megakaryocyte maturation promoter | |
| JP6112733B2 (en) | Method for inducing erythropoietin-producing cells | |
| JP2018058768A (en) | Composition for maintaining and/or enhancing functions of platelet |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20151028 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20170512 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20180314 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20180412 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20180608 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20180731 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20181220 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190218 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190604 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20190627 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6551963 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |