JPWO2005033298A1 - 幹細胞から心筋細胞を分化誘導する方法 - Google Patents
幹細胞から心筋細胞を分化誘導する方法 Download PDFInfo
- Publication number
- JPWO2005033298A1 JPWO2005033298A1 JP2005514483A JP2005514483A JPWO2005033298A1 JP WO2005033298 A1 JPWO2005033298 A1 JP WO2005033298A1 JP 2005514483 A JP2005514483 A JP 2005514483A JP 2005514483 A JP2005514483 A JP 2005514483A JP WO2005033298 A1 JPWO2005033298 A1 JP WO2005033298A1
- Authority
- JP
- Japan
- Prior art keywords
- cells
- cell
- cardiomyocytes
- culture
- noggin
- 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
- 210000004413 cardiac myocyte Anatomy 0.000 title claims abstract description 153
- 238000000034 method Methods 0.000 title claims abstract description 136
- 230000004069 differentiation Effects 0.000 title claims abstract description 102
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 41
- 230000001939 inductive effect Effects 0.000 title claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims abstract description 342
- 230000006698 induction Effects 0.000 claims abstract description 49
- 239000000126 substance Substances 0.000 claims abstract description 47
- 230000011664 signaling Effects 0.000 claims abstract description 25
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 108700007229 noggin Proteins 0.000 claims description 136
- 102000045246 noggin Human genes 0.000 claims description 135
- 210000002242 embryoid body Anatomy 0.000 claims description 64
- 108090000623 proteins and genes Proteins 0.000 claims description 55
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 46
- 239000005557 antagonist Substances 0.000 claims description 34
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 11
- 238000000338 in vitro Methods 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 8
- 230000002776 aggregation Effects 0.000 claims description 6
- 238000004220 aggregation Methods 0.000 claims description 6
- 210000004602 germ cell Anatomy 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 5
- -1 chodin Proteins 0.000 claims description 5
- 241000202252 Cerberus Species 0.000 claims description 4
- 102000016970 Follistatin Human genes 0.000 claims description 4
- 108010014612 Follistatin Proteins 0.000 claims description 4
- 102100038367 Gremlin-1 Human genes 0.000 claims description 4
- 101001032872 Homo sapiens Gremlin-1 Proteins 0.000 claims description 4
- 102000019307 Sclerostin Human genes 0.000 claims description 3
- 108050006698 Sclerostin Proteins 0.000 claims description 3
- 102000013361 fetuin Human genes 0.000 claims description 3
- 108060002885 fetuin Proteins 0.000 claims description 3
- 101710010675 Cerberus Proteins 0.000 claims description 2
- 101150079463 NBL1 gene Proteins 0.000 claims description 2
- 101150118520 dan gene Proteins 0.000 claims description 2
- 230000002107 myocardial effect Effects 0.000 description 50
- 238000004114 suspension culture Methods 0.000 description 48
- 239000002609 medium Substances 0.000 description 47
- 238000011282 treatment Methods 0.000 description 41
- 230000000694 effects Effects 0.000 description 40
- 230000014509 gene expression Effects 0.000 description 32
- 230000000541 pulsatile effect Effects 0.000 description 28
- 241000699666 Mus <mouse, genus> Species 0.000 description 20
- 239000003814 drug Substances 0.000 description 20
- 208000019622 heart disease Diseases 0.000 description 16
- 239000003550 marker Substances 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 13
- 238000011161 development Methods 0.000 description 13
- 230000018109 developmental process Effects 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 230000010349 pulsation Effects 0.000 description 13
- 238000012136 culture method Methods 0.000 description 12
- 102000003505 Myosin Human genes 0.000 description 10
- 108060008487 Myosin Proteins 0.000 description 10
- 230000019491 signal transduction Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 102000002723 Atrial Natriuretic Factor Human genes 0.000 description 9
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 9
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 9
- 101100013973 Mus musculus Gata4 gene Proteins 0.000 description 9
- 102000013394 Troponin I Human genes 0.000 description 9
- 108010065729 Troponin I Proteins 0.000 description 9
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 102000010825 Actinin Human genes 0.000 description 8
- 108010063503 Actinin Proteins 0.000 description 8
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 description 8
- 102000001893 Bone Morphogenetic Protein Receptors Human genes 0.000 description 8
- 108010040422 Bone Morphogenetic Protein Receptors Proteins 0.000 description 8
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 8
- 206010019280 Heart failures Diseases 0.000 description 8
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 8
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 108010084498 Myosin Heavy Chains Proteins 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000002441 reversible effect Effects 0.000 description 8
- 102000005604 Myosin Heavy Chains Human genes 0.000 description 7
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 7
- 238000003501 co-culture Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000002054 transplantation Methods 0.000 description 7
- 101000597041 Gallus gallus Transcriptional enhancer factor TEF-3 Proteins 0.000 description 6
- 101000653735 Homo sapiens Transcriptional enhancer factor TEF-1 Proteins 0.000 description 6
- 102100026925 Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Human genes 0.000 description 6
- 101710105127 Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Proteins 0.000 description 6
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 210000004165 myocardium Anatomy 0.000 description 6
- 230000000638 stimulation Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000009471 action Effects 0.000 description 5
- 230000001747 exhibiting effect Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 5
- 210000005003 heart tissue Anatomy 0.000 description 5
- 230000000984 immunochemical effect Effects 0.000 description 5
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 4
- 101000588969 Homo sapiens Putative uncharacterized protein MYH16 Proteins 0.000 description 4
- 101150114527 Nkx2-5 gene Proteins 0.000 description 4
- 102100032974 Putative uncharacterized protein MYH16 Human genes 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 101100460507 Xenopus laevis nkx-2.5 gene Proteins 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 230000001605 fetal effect Effects 0.000 description 4
- 238000007667 floating Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000007758 minimum essential medium Substances 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 230000001172 regenerating effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- NMUSYJAQQFHJEW-UHFFFAOYSA-N 5-Azacytidine Natural products O=C1N=C(N)N=CN1C1C(O)C(O)C(CO)O1 NMUSYJAQQFHJEW-UHFFFAOYSA-N 0.000 description 3
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 3
- 102100039819 Actin, alpha cardiac muscle 1 Human genes 0.000 description 3
- 101710170648 Actin, alpha cardiac muscle 1 Proteins 0.000 description 3
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 3
- 108020005544 Antisense RNA Proteins 0.000 description 3
- 108090000994 Catalytic RNA Proteins 0.000 description 3
- 102000053642 Catalytic RNA Human genes 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 241000269370 Xenopus <genus> Species 0.000 description 3
- 238000004115 adherent culture Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000074 antisense oligonucleotide Substances 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 229960002756 azacitidine Drugs 0.000 description 3
- 230000000747 cardiac effect Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 210000000038 chest Anatomy 0.000 description 3
- 235000013330 chicken meat Nutrition 0.000 description 3
- 102000006533 chordin Human genes 0.000 description 3
- 108010008846 chordin Proteins 0.000 description 3
- 239000003184 complementary RNA Substances 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 210000002458 fetal heart Anatomy 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 108091092562 ribozyme Proteins 0.000 description 3
- 238000009331 sowing Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- OXEUETBFKVCRNP-UHFFFAOYSA-N 9-ethyl-3-carbazolamine Chemical compound NC1=CC=C2N(CC)C3=CC=CC=C3C2=C1 OXEUETBFKVCRNP-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229920001342 Bakelite® Polymers 0.000 description 2
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 2
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 description 2
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 2
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 208000020446 Cardiac disease Diseases 0.000 description 2
- 206010007558 Cardiac failure chronic Diseases 0.000 description 2
- 206010007559 Cardiac failure congestive Diseases 0.000 description 2
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- 102000014429 Insulin-like growth factor Human genes 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000009525 Myocarditis Diseases 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 108010051583 Ventricular Myosins Proteins 0.000 description 2
- 235000018936 Vitellaria paradoxa Nutrition 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000004637 bakelite Substances 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- OROGSEYTTFOCAN-DNJOTXNNSA-N codeine Chemical compound C([C@H]1[C@H](N(CC[C@@]112)C)C3)=C[C@H](O)[C@@H]1OC1=C2C3=CC=C1OC OROGSEYTTFOCAN-DNJOTXNNSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 210000001900 endoderm Anatomy 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 210000001654 germ layer Anatomy 0.000 description 2
- 230000004217 heart function Effects 0.000 description 2
- OROGSEYTTFOCAN-UHFFFAOYSA-N hydrocodone Natural products C1C(N(CCC234)C)C2C=CC(O)C3OC2=C4C1=CC=C2OC OROGSEYTTFOCAN-UHFFFAOYSA-N 0.000 description 2
- 206010020871 hypertrophic cardiomyopathy Diseases 0.000 description 2
- 238000000760 immunoelectrophoresis Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 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
- 239000005541 ACE inhibitor Substances 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 1
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- 208000022621 Caronte Diseases 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 240000001879 Digitalis lutea Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101100175482 Glycine max CG-3 gene Proteins 0.000 description 1
- 101100456626 Homo sapiens MEF2A gene Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 101000934639 Mus musculus Bone morphogenetic protein receptor type-1A Proteins 0.000 description 1
- 101000943784 Mus musculus Chordin Proteins 0.000 description 1
- 101100079042 Mus musculus Myef2 gene Proteins 0.000 description 1
- 102100021148 Myocyte-specific enhancer factor 2A Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 230000010757 Reduction Activity Effects 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102100038126 Tenascin Human genes 0.000 description 1
- 108010008125 Tenascin Proteins 0.000 description 1
- 206010043276 Teratoma Diseases 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 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 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 229940097320 beta blocking agent Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229940112869 bone morphogenetic protein Drugs 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000000496 cardiotonic agent Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000009391 cell specific gene expression Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000012649 demethylating agent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000004039 endoderm cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 101150014102 mef-2 gene Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 210000002235 sarcomere Anatomy 0.000 description 1
- 210000002107 sheath cell Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 210000002820 sympathetic nervous system Anatomy 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Cardiology (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Hospice & Palliative Care (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
(1)幹細胞から心筋細胞を分化誘導する方法において、幹細胞を、BMPシグナル伝達を抑制する物質の存在下で分化誘導のための培養をすることを特徴とする当該方法。
(2)分化誘導のための幹細胞の培養が、浮遊凝集培養し、胚様体を形成させる工程を含む上記(1)に記載の方法。
(3)分化誘導のための幹細胞の培養が、フィーダー細胞と共培養する工程を含む上記(1)に記載の方法。
(4)分化誘導のための幹細胞の培養が、培養容器上で平面培養する工程を含む上記(1)に記載の方法。
(5)BMPシグナル伝達を抑制する物質を、分化誘導期の最初の数日間のみ添加する上記(1)〜(4)の何れか1項に記載の方法。
(6)BMPシグナル伝達を抑制する物質を、分化誘導期前の幹細胞に前もって処理しておく工程を含む上記(1)〜(4)の何れか1項に記載の方法。
(7)BMPシグナル伝達を抑制する物質を分化誘導期前の幹細胞に前もって処理しておく工程を含み、かつ、BMPシグナル伝達を抑制する物質を分化誘導期の最初の数日間のみ添加する、上記(1)〜(4)の何れか1項に記載の方法。
(8)BMPシグナル伝達を抑制する物質がBMPアンタゴニストである上記(1)〜(7)の何れか1項に記載の方法。
(9)BMPアンタゴニストが、ノギン、コーディン、フェチュイン、フォリスタチン、スクレロスチン、ダン、サーベラス、グレムリン、及びダンテ、並びにそれらの関連蛋白質からなる群から1つ又は複数選択されたものである、上記(8)に記載の方法。
(10)幹細胞が、試験管内培養下で心筋細胞へ分化し得る能力を有した哺乳動物由来の細胞である上記(1)〜(9)の何れか1項に記載の方法。
(11)心筋細胞へ分化し得る能力を有した哺乳動物由来の細胞が、多能性幹細胞又は当該細胞に由来する細胞である上記(10)に記載の方法。
(12)多能性幹細胞が、胚性幹細胞、胚性幹細胞に類似した形質を有する細胞、胚性生殖細胞又は成人型多能性幹細胞である上記(11)に記載の方法。
(13)多能性幹細胞が、胚性幹細胞である上記(12)に記載の方法。
(14)幹細胞がヒト由来である、上記(1)〜(13)に記載の方法。
(15)上記(1)〜(14)の何れか1項に記載の方法を含む、心筋細胞の製造方法。(16)上記(1)〜(14)の何れか1項に記載の方法により得られる心筋細胞。
(17)心筋細胞が衰弱、機能停止又は死滅したことに起因する心疾患の治療方法において、上記(16)に記載の心筋細胞を心筋細胞が衰弱、機能停止又は死滅した部位又はその周辺に投与(移植)することからなる当該心疾患の治療方法。
(18)心筋細胞が衰弱、機能停止又は死滅したことに起因する心疾患の治療に有用な物質のスクリーニング方法において、被験物質を上記(16)に記載の心筋細胞と接触させて、当該細胞の心筋細胞への分化効率又はその細胞機能の質的若しくは量的な変化を測定することからなる当該スクリーニング方法。
(19)上記(16)に記載の心筋細胞を有効成分として含有する、心筋細胞が衰弱、機能停止又は死滅したことに起因する心疾患治療用の医薬組成物又は支持体。
ES細胞を浮遊凝集培養(以下、本願実施例においては単に浮遊培養と記す)によりEBを形成させ、心筋細胞に分化誘導する実験系を用い、培地中へのノギン蛋白質添加が心筋細胞の出現率に及ぼす影響を検討した。
実施例1で示す様に、ノギン処理を行うことにより、ES細胞から作製したEBの拍動性が有意に高まったが、この拍動性EBにおいて、その拍動性細胞が心筋細胞であることを確認するため、各種心筋特異的マーカー分子の遺伝子発現、ならびに蛋白質産生を検討した。
GATA-4(順方向)5'-CTGTCATCTC ACTATGGGCA-3'(SEQ ID NO:1)、
GATA-4(逆方向)5'- CCAAGTCCGA GCAGGAATTT -3'(SEQ ID NO:2);
TEF-1(順方向)5'-AAGACGTCAA GCCCTTTGTG-3'(SEQ ID NO:3)、
TEF-1(逆方向)5'-AAAGGAGCAC ACTTTGGTGG-3'(SEQ ID NO:4);
Tbx-5(順方向)5'-GGAGCCTGAT TCCAAAGACA-3'(SEQ ID NO:5)、
Tbx-5(逆方向)5'-TTCAGCCACA GTTCACGTTC-3'(SEQ ID NO:6);
MEF-2c(順方向)5'-AGCAAGAATA CGATGCCATC-3'(SEQ ID NO:7)、
MEF-2c(逆方向)5'-GAAGGGGTGG TGGTACGGTC-3'(SEQ ID NO:8);
αMHC(順方向)5'-GGAAGAGTGA GCGGCCATCA AGG-3'(SEQ ID NO:9)、
αMHC(逆方向)5'-CTGCTGGAGA GGTTATTCCT CG-3'(SEQ ID NO:10);
MLC-2v(順方向)5'-GCCAAGAAGC GGATAGAAGG-3'(SEQ ID NO:11)、
MLC-2v(逆方向)5'-CTGTGGTTCA GGGCTCAGTC-3'(SEQ ID NO:12);
α-cardiac actin(順方向)5'-CTGAGATGTC TCTCTCTCTC TTAG-3' (SEQ ID NO:13)、
α-cardiac actin(逆方向)5'-ACAATGACTG ATGAGAGATG-3' (SEQ ID NO:14);
GAPDH(順方向)5'-TTCAACGGCA CAGTCAAGG-3' (SEQ ID NO:15)、
GAPDH(逆方向)5'-CATGGACTGT GGTCATGAG-3' (SEQ ID NO:16)。
ノギン処理の最適時期及び期間を検討するため、上記浮遊培養系においてノギン蛋白質の添加時期を変え、その後の心筋分化誘導効果に及ぼす影響を検討した。
ノギン処理によるES細胞の心筋分化誘導促進効果が、ノギンのBMPアンタゴニストとしての活性を介している可能性を確認するため、ノギン処理と同時にBMP-2を培地中に添加し、その効果を検討した。
ES細胞を、培養プレート上に前もって播種したST2細胞やOP9細胞等のストローマ系細胞を支持細胞(フィーダー細胞)として共培養することにより、浮遊培養法や懸滴培養法の様な三次元培養を必要とせずに、心筋細胞への分化を誘導できることが報告されている(Yamaneら、Methods Mol. Biol. 184:261, 2002;Schroederら、Proc. Natl. Acad. Sci. USA 100:4018, 2003)。そこで、ST2細胞(理研・細胞バンクから購入)をフィーダー細胞として用いた分化誘導系において、ノギン処理の効果を検討した。ST2細胞は、市販の細胞培養用6ウェルプレート(CORNING社製)に播種し、Dulbecco MEM培地(Invitrogen/GIBCO社製)に10%の牛胎児血清(Invitrogen/GIBCO社製)を加えた培地を用いてコンフルエント状態にまで培養したものをフィーダー細胞として使用した。上記と同様の方法で単一細胞状態としたES細胞の懸濁液を調製し、フィーダーをPBSで2回洗浄後、2000細胞/2mL培地/1ウェルで播種した。その後、経時的に拍動性心筋細胞の出現を顕鏡観察するとともに、培養後8日目に一部の細胞を70%エタノール溶液で固定し、1次抗体として抗サルコメア・ミオシン抗体(MF20;American Type Culture Collection社製)、引き続きHorseradish Peroxidase標識2次抗体(ヒストファイン シンプルステインPO(M);ニチレイ社製)と順次反応させ、最後にACE(3-amino-9-ethylcarbazole)基質液(ニチレイ社製)を用いた呈色反応を行った後、光学顕微鏡下にて観察を行った。
ノギンと同様、BMPアンタゴニストとしての作用を示すことが知られているコーディンの心筋誘導効果について、ノギンの効果と比較・検討した。コーディン蛋白質としては、市販のリコンビナント・コーディン−Fc キメラ蛋白質(Recombinant Mouse Chordin/Fc Chimera; R&D systems社)(以下、単に「コーディン(蛋白質)」と称する)を使用した。ノギン(500 ng/mL)、又はコーディン(500 ng/mL)を添加した培地、又はどちらの因子も添加しない培地で3日間培養したES細胞を用い、実施例1と同様の方法で懸滴培養を行った。懸滴培養4日目にEBを回収した後、細胞培養用ディッシュ上に播種し、引き続き付着培養により心筋細胞への分化を誘導した。その結果、コーディン処理したES細胞では、ノギン処理したES細胞とほぼ匹敵する程度の拍動性心筋細胞の出現が認められ(図8)、コーディンはES細胞に対し、ノギンと同様の心筋分化誘導効果を示すことが確認できた。
引き続き、BMPアンタゴニストして、フォリスタチン、ダン、カロンティ(Caronte;ニワトリのサーベラス様因子)、グレムリンの4種のBMPアンタゴニスト(何れもR&D systems社製のFcキメラ型リコンビナント蛋白質を使用)の効果を検討した。上記の各種BMPアンタゴニスト(150 ng/mL)を添加した培地、およびBMPアンタゴニストを添加しない培地で3日間培養したES細胞を、市販の浮遊培養用プレート(96穴マルチプレート;住友ベークライト社製)に播種してEBを形成させ、心筋細胞への分化を誘導した。
Claims (14)
- 幹細胞から心筋細胞を分化誘導する方法において、幹細胞を、BMPシグナル伝達を抑制する物質の存在下で分化誘導のための培養をすることを特徴とする当該方法。
- 分化誘導のための幹細胞の培養が、浮遊凝集培養し、胚様体を形成させる工程を含む請求項1に記載の方法。
- 分化誘導のための幹細胞の培養が、フィーダー細胞と共培養する工程を含む請求項1に記載の方法。
- 分化誘導のための幹細胞の培養が、培養容器上で平面培養する工程を含む請求項1に記載の方法。
- BMPシグナル伝達を抑制する物質を、分化誘導期の最初の数日間のみ添加する請求項1〜4の何れか1項に記載の方法。
- BMPシグナル伝達を抑制する物質を、分化誘導期前の幹細胞に前もって処理しておく工程を含む請求項1〜4の何れか1項に記載の方法。
- BMPシグナル伝達を抑制する物質を分化誘導期前の幹細胞に前もって処理しておく工程を含み、かつ、BMPシグナル伝達を抑制する物質を分化誘導期の最初の数日間のみ添加する、請求項1〜4の何れか1項に記載の方法。
- BMPシグナル伝達を抑制する物質がBMPアンタゴニストである請求項1〜7の何れか1項に記載の方法。
- BMPアンタゴニストが、ノギン、コーディン、フェチュイン、フォリスタチン、スクレロスチン、ダン、サーベラス、グレムリン、及びダンテ、並びにそれらの関連蛋白質からなる群から1つ又は複数選択されたものである、請求項8に記載の方法。
- 幹細胞が、試験管内培養下で心筋細胞へ分化し得る能力を有した哺乳動物由来の細胞である請求項1〜9の何れか1項に記載の方法。
- 心筋細胞へ分化し得る能力を有した哺乳動物由来の細胞が、多能性幹細胞又は当該細胞に由来する細胞である請求項10に記載の方法。
- 多能性幹細胞が、胚性幹細胞、胚性幹細胞に類似した形質を有する細胞、胚性生殖細胞又は成人型多能性幹細胞である請求項11に記載の方法。
- 多能性幹細胞が、胚性幹細胞である請求項12に記載の方法。
- 請求項1〜13の何れか1項に記載の方法により得られる心筋細胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2005514483A JP4684108B2 (ja) | 2003-10-03 | 2004-10-04 | 幹細胞から心筋細胞を分化誘導する方法 |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003346248 | 2003-10-03 | ||
JP2003346248 | 2003-10-03 | ||
JP2004212255 | 2004-07-20 | ||
JP2004212255 | 2004-07-20 | ||
JP2005514483A JP4684108B2 (ja) | 2003-10-03 | 2004-10-04 | 幹細胞から心筋細胞を分化誘導する方法 |
PCT/JP2004/014598 WO2005033298A1 (ja) | 2003-10-03 | 2004-10-04 | 幹細胞から心筋細胞を分化誘導する方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2005033298A1 true JPWO2005033298A1 (ja) | 2006-12-14 |
JP4684108B2 JP4684108B2 (ja) | 2011-05-18 |
Family
ID=34425354
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2005514483A Active JP4684108B2 (ja) | 2003-10-03 | 2004-10-04 | 幹細胞から心筋細胞を分化誘導する方法 |
Country Status (11)
Country | Link |
---|---|
US (1) | US7727762B2 (ja) |
EP (1) | EP1674562A4 (ja) |
JP (1) | JP4684108B2 (ja) |
KR (1) | KR101164104B1 (ja) |
CN (2) | CN1863904B (ja) |
AU (1) | AU2004278634B2 (ja) |
BR (1) | BRPI0414961A (ja) |
CA (1) | CA2540135C (ja) |
IL (1) | IL174606A (ja) |
RU (1) | RU2392315C2 (ja) |
WO (1) | WO2005033298A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10918671B2 (en) * | 2007-07-31 | 2021-02-16 | Daiichi Sankyo Company, Limited | Method of constructing masses of myocardial cells and use of the myocardial cell mass |
Families Citing this family (55)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7410798B2 (en) | 2001-01-10 | 2008-08-12 | Geron Corporation | Culture system for rapid expansion of human embryonic stem cells |
EP1444326A4 (en) * | 2001-08-24 | 2006-06-28 | Advanced Cell Tech Inc | SCREENING ASSAYS FOR IDENTIFICATION OF AGENTS INDUCING DIFFERENTIATION, AND PRODUCTION OF CELLS DIFFERENTIATED FOR CELL THERAPY |
GB2441488C (en) | 2005-06-22 | 2011-10-05 | Geron Corp | Suspension culture of human embryonic stem cells |
EP1739179A1 (en) * | 2005-06-30 | 2007-01-03 | Octapharma AG | Serum-free stable transfection and production of recombinant human proteins in human cell lines |
JP5149791B2 (ja) | 2006-04-28 | 2013-02-20 | 第一三共株式会社 | 多能性幹細胞から心筋細胞を分化誘導する方法 |
US20100291042A1 (en) | 2007-05-03 | 2010-11-18 | The Brigham And Women's Hospital, Inc. | Multipotent stem cells and uses thereof |
US8574567B2 (en) | 2007-05-03 | 2013-11-05 | The Brigham And Women's Hospital, Inc. | Multipotent stem cells and uses thereof |
JP5174019B2 (ja) * | 2007-06-07 | 2013-04-03 | 恵一 福田 | G−csfを用いた心筋細胞への分化誘導方法 |
US8799249B2 (en) * | 2007-06-08 | 2014-08-05 | Yahoo! Inc. | Method and system for rendering a collection of media items |
KR100937457B1 (ko) * | 2007-08-10 | 2010-01-19 | 한국생명공학연구원 | 인간배아줄기세포와 인간배아종양세포를 심근세포 직계열로분화시키는 방법 |
US9962409B2 (en) | 2007-10-01 | 2018-05-08 | Vestion, Inc. | Therapy using cardiac stem cells and mesenchymal stem cells |
JP5453612B2 (ja) * | 2007-12-06 | 2014-03-26 | 公益財団法人ヒューマンサイエンス振興財団 | 心筋細胞分化誘導促進剤及びその使用方法 |
AU2009225665B9 (en) | 2008-03-17 | 2015-01-15 | The Scripps Research Institute | Combined chemical and genetic approaches for generation of induced pluripotent stem cells |
WO2009154770A2 (en) * | 2008-06-18 | 2009-12-23 | The Texas A & M University System | Mesenchymal stem cells, compositions, and methods for treatment of cardiac tissue damage |
CN102317442B (zh) | 2008-12-17 | 2014-08-13 | 斯克里普斯研究所 | 干细胞的产生和保持 |
EP2433125B1 (en) * | 2009-05-20 | 2018-11-28 | Mayo Foundation For Medical Education And Research | Method for determining the cardio-generative potential of mammalian cells |
CA2767825A1 (en) * | 2009-07-09 | 2011-01-13 | Janssen Biotech, Inc. | Cardiac tissue-derived cells |
WO2011047300A1 (en) | 2009-10-16 | 2011-04-21 | The Scripps Research Institute | Induction of pluripotent cells |
EP4364797A3 (en) | 2009-10-19 | 2024-07-10 | FUJIFILM Cellular Dynamics, Inc. | Cardiomyocyte production |
KR101168053B1 (ko) * | 2009-11-06 | 2012-07-24 | 연세대학교 산학협력단 | 효율적이고 보편적인 전분화능 줄기세포의 신경세포 분화 유도방법 |
JPWO2011071118A1 (ja) * | 2009-12-09 | 2013-04-22 | 国立大学法人京都大学 | ニトロビンを含む多能性幹細胞の心筋細胞への分化促進剤 |
AU2011235212B2 (en) | 2010-03-31 | 2014-07-31 | The Scripps Research Institute | Reprogramming cells |
CN103228782B (zh) | 2010-06-13 | 2016-03-30 | 中国科学院生物物理研究所 | 由干细胞制备心肌细胞的方法和组合物及其用途 |
EP2580320B1 (en) | 2010-06-14 | 2018-08-01 | The Scripps Research Institute | Reprogramming of cells to a new fate |
US9499790B2 (en) | 2010-08-26 | 2016-11-22 | Kyoto University | Method for promoting differentiation of pluripotent stem cells into cardiac muscle cells |
JP5930205B2 (ja) * | 2010-08-26 | 2016-06-08 | 国立大学法人京都大学 | 多能性幹細胞の心筋分化促進剤 |
BR112013004514A2 (pt) * | 2010-08-31 | 2016-06-07 | Janssen Biotech Inc | diferenciação das células-tronco embrionárias humanas |
US9181529B2 (en) | 2010-10-19 | 2015-11-10 | Cellular Dynamics International, Inc. | Titration of differentiation medium components |
CN103380212B (zh) | 2010-12-22 | 2017-04-05 | 菲特治疗公司 | 用于单细胞分选与增强ipsc重新编程的细胞培养平台 |
WO2012135621A2 (en) | 2011-03-30 | 2012-10-04 | Cellular Dynamics International. Inc | Priming of pluripotent stem cells for neural differentiation |
JP5840855B2 (ja) | 2011-03-30 | 2016-01-06 | 学校法人東京女子医科大学 | 胚性幹細胞から心筋シートを製造する方法 |
WO2013111875A1 (ja) | 2012-01-27 | 2013-08-01 | 国立大学法人京都大学 | 多能性幹細胞の心筋分化誘導法 |
JP5920741B2 (ja) * | 2012-03-15 | 2016-05-18 | iHeart Japan株式会社 | 人工多能性幹細胞から心筋および血管系混合細胞群を製造する方法 |
EP2891712A4 (en) | 2012-07-23 | 2016-04-06 | Inst Biophysics Cn Acad Sci | METHOD OF INDUCING THE DIFFERENTIATION OF PLURIPOTENT STEM CELLS IN VITRICULAR MYOCYTES IN VITRO |
EP2925863B1 (en) | 2012-11-30 | 2020-07-15 | Vestion Inc. | Cardiac stem cells and methods of identifying and using the same |
EP2966166B1 (en) | 2013-03-08 | 2019-04-03 | Kyoto University | Promoter of differentiation of pluripotent stem cell into myocardium, which comprises egf receptor inhibitor |
US9493742B2 (en) | 2013-03-15 | 2016-11-15 | Emory University | Purification of cell mixtures using molecular beacons targeting cell specific RNA |
EP3006559B1 (en) | 2013-05-31 | 2019-11-06 | iHeart Japan Corporation | Layered cell sheet incorporating hydrogel |
WO2015066197A2 (en) | 2013-10-29 | 2015-05-07 | Vestion, Inc. | Cardiac neural crest cells and methods of use thereof |
KR102460549B1 (ko) | 2014-03-04 | 2022-10-28 | 페이트 세러퓨틱스, 인코포레이티드 | 개선된 재프로그래밍 방법 및 세포 배양 플랫폼 |
CN106661553B (zh) * | 2014-05-06 | 2020-09-01 | 豪夫迈·罗氏有限公司 | 用于使多能干细胞分化为心肌细胞的方法 |
US10233426B2 (en) | 2014-05-30 | 2019-03-19 | Kyoto University | Method for inducing cardiac differentiation of pluripotent stem cell with low-molecular compounds |
KR20160142587A (ko) * | 2015-06-03 | 2016-12-13 | 서울대학교산학협력단 | 전기·기계적 자극을 가하여 줄기세포를 심근세포로 분화시키는 방법 |
SG11201802957PA (en) | 2015-10-16 | 2018-05-30 | Fate Therapeutics Inc | Platform for the induction & maintenance of ground state pluripotency |
SG11201903254PA (en) * | 2016-10-17 | 2019-05-30 | Univ Keio | Undifferentiated stem cell-removing agent, and method for removing undifferentiated stem cells |
JP2018093823A (ja) | 2016-12-15 | 2018-06-21 | Heartseed株式会社 | 未分化幹細胞除去剤及び未分化幹細胞除去方法 |
JP6909454B2 (ja) * | 2017-01-26 | 2021-07-28 | 日本メナード化粧品株式会社 | 幹細胞の品質を評価する方法及び幹細胞の品質評価用キット |
JP6978045B2 (ja) * | 2017-10-03 | 2021-12-08 | Heartseed株式会社 | 未分化幹細胞を培養し心筋細胞を分化誘導するシステム及び心筋細胞の製造方法 |
ES2966207T3 (es) | 2018-03-30 | 2024-04-18 | Orizuru Therapeutics Inc | Compuesto heterocíclico |
JP7344486B2 (ja) | 2018-03-30 | 2023-09-14 | 国立大学法人京都大学 | 心筋細胞成熟促進剤 |
US20210054406A1 (en) | 2018-03-30 | 2021-02-25 | Kyoto University | Cell production method |
CN112639111B (zh) | 2018-08-10 | 2023-07-21 | 国立大学法人京都大学 | 使用阳离子性脂质的转染心肌细胞的方法 |
CN110907644B (zh) * | 2019-12-11 | 2023-01-06 | 深圳市达科为生物工程有限公司 | 多种细胞鉴定试剂盒及操作方法 |
GB202308408D0 (en) | 2023-06-06 | 2023-07-19 | Cambridge Entpr Ltd | Stem cells based three-dimensional embryo model |
CN117398427B (zh) * | 2023-12-14 | 2024-02-27 | 再少年(北京)生物科技有限公司 | 包含iPS诱导定向分化成心肌细胞的药物组合在用于治疗心力衰竭中的应用 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1397646A (zh) * | 2001-07-13 | 2003-02-19 | 上海杰隆生物工程股份有限公司 | 一种克隆心肌细胞、心脏组织及心脏的方法 |
EP1456374A4 (en) * | 2001-11-26 | 2005-08-17 | Advanced Cell Tech Inc | METHODS FOR THE PRODUCTION AND USE OF REPROGRAMME HUMAN SOMATIC CELL CORES AND AUTOLOGOUS AND ISOGENIC HUMAN STEM CELLS |
CN1164741C (zh) * | 2002-12-30 | 2004-09-01 | 浙江大学 | 淫羊藿苷在诱导胚胎干细胞体外定向分化方面的用途 |
-
2004
- 2004-10-04 BR BRPI0414961-0A patent/BRPI0414961A/pt not_active Application Discontinuation
- 2004-10-04 KR KR1020067006275A patent/KR101164104B1/ko not_active IP Right Cessation
- 2004-10-04 CN CN200480028917.7A patent/CN1863904B/zh not_active Expired - Fee Related
- 2004-10-04 AU AU2004278634A patent/AU2004278634B2/en not_active Ceased
- 2004-10-04 WO PCT/JP2004/014598 patent/WO2005033298A1/ja active Application Filing
- 2004-10-04 US US10/574,530 patent/US7727762B2/en not_active Expired - Fee Related
- 2004-10-04 RU RU2006114832/13A patent/RU2392315C2/ru not_active IP Right Cessation
- 2004-10-04 CA CA2540135A patent/CA2540135C/en not_active Expired - Fee Related
- 2004-10-04 CN CN201310492188.1A patent/CN103602633A/zh active Pending
- 2004-10-04 JP JP2005514483A patent/JP4684108B2/ja active Active
- 2004-10-04 EP EP04792009A patent/EP1674562A4/en not_active Withdrawn
-
2006
- 2006-03-27 IL IL174606A patent/IL174606A/en not_active IP Right Cessation
Non-Patent Citations (4)
Title |
---|
JPN6010050264, Mol. Cell. Biol., 1999, 19(10), p.7096−7105 * |
JPN6010050265, Nat. Biotechnol., 2005, 23(5), p.607−611 * |
JPN6010050266, J. Cell Sci., 200403, 117(7), p.1269−1280 * |
JPN6010050267, Circ J., 200406, 68, p.691−702 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10918671B2 (en) * | 2007-07-31 | 2021-02-16 | Daiichi Sankyo Company, Limited | Method of constructing masses of myocardial cells and use of the myocardial cell mass |
Also Published As
Publication number | Publication date |
---|---|
IL174606A (en) | 2011-07-31 |
BRPI0414961A (pt) | 2006-11-07 |
EP1674562A4 (en) | 2007-04-11 |
EP1674562A1 (en) | 2006-06-28 |
CA2540135A1 (en) | 2005-04-14 |
CN103602633A (zh) | 2014-02-26 |
AU2004278634A1 (en) | 2005-04-14 |
US20070134215A1 (en) | 2007-06-14 |
US7727762B2 (en) | 2010-06-01 |
CA2540135C (en) | 2012-12-04 |
CN1863904B (zh) | 2014-05-28 |
KR20060080934A (ko) | 2006-07-11 |
KR101164104B1 (ko) | 2012-07-12 |
RU2392315C2 (ru) | 2010-06-20 |
CN1863904A (zh) | 2006-11-15 |
WO2005033298A1 (ja) | 2005-04-14 |
IL174606A0 (en) | 2006-08-20 |
AU2004278634B2 (en) | 2009-10-01 |
RU2006114832A (ru) | 2007-11-10 |
JP4684108B2 (ja) | 2011-05-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4684108B2 (ja) | 幹細胞から心筋細胞を分化誘導する方法 | |
JP5149791B2 (ja) | 多能性幹細胞から心筋細胞を分化誘導する方法 | |
US20100166714A1 (en) | Cardiovascular stem cells, methods for stem cell isolation, and uses thereof | |
US20160194608A1 (en) | Methods for Inducing Cardiomyogenesis | |
JP5174019B2 (ja) | G−csfを用いた心筋細胞への分化誘導方法 | |
JP2007252220A (ja) | 心筋細胞の製造方法および分化誘導剤 | |
JP4122365B2 (ja) | 移植用細胞の製造方法 | |
JP2007267658A (ja) | 心筋細胞の製造方法及び心筋細胞への分化誘導促進剤 | |
Misfeldt | Endocardial cells are a distinct endothelial lineage derived from multipotent cardiovascular progenitors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20070911 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20070911 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20091005 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20100830 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20101029 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20101029 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20101118 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20101213 |
|
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: 20110111 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110208 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140218 Year of fee payment: 3 |
|
R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 Ref document number: 4684108 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |