JP2005211040A - Method for differentiating and proliferating endothelial cell - Google Patents
Method for differentiating and proliferating endothelial cell Download PDFInfo
- Publication number
- JP2005211040A JP2005211040A JP2004025631A JP2004025631A JP2005211040A JP 2005211040 A JP2005211040 A JP 2005211040A JP 2004025631 A JP2004025631 A JP 2004025631A JP 2004025631 A JP2004025631 A JP 2004025631A JP 2005211040 A JP2005211040 A JP 2005211040A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- substance
- adrenomedullin
- vascular
- endothelial cells
- 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
- 210000002889 endothelial cell Anatomy 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 63
- 230000002062 proliferating effect Effects 0.000 title claims abstract description 17
- ULCUCJFASIJEOE-NPECTJMMSA-N adrenomedullin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H]1C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CSSC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)[C@@H](C)O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 ULCUCJFASIJEOE-NPECTJMMSA-N 0.000 claims abstract description 56
- 230000002792 vascular Effects 0.000 claims abstract description 54
- 102000004379 Adrenomedullin Human genes 0.000 claims abstract description 50
- 101800004616 Adrenomedullin Proteins 0.000 claims abstract description 50
- 210000001671 embryonic stem cell Anatomy 0.000 claims abstract description 47
- 210000004618 arterial endothelial cell Anatomy 0.000 claims abstract description 26
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 claims abstract description 15
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 claims abstract description 15
- 230000035755 proliferation Effects 0.000 claims abstract description 15
- 230000003834 intracellular effect Effects 0.000 claims description 51
- 239000000126 substance Substances 0.000 claims description 43
- 210000000130 stem cell Anatomy 0.000 claims description 33
- 230000004069 differentiation Effects 0.000 claims description 29
- 230000001965 increasing effect Effects 0.000 claims description 27
- 239000000203 mixture Substances 0.000 claims description 26
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 claims description 20
- 102000037602 Platelet Endothelial Cell Adhesion Molecule-1 Human genes 0.000 claims description 20
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 17
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 17
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 17
- 210000004204 blood vessel Anatomy 0.000 claims description 15
- 230000033115 angiogenesis Effects 0.000 claims description 13
- 201000010099 disease Diseases 0.000 claims description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 12
- 230000001939 inductive effect Effects 0.000 claims description 12
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 claims description 10
- WGJJROVFWIXTPA-OALUTQOASA-N prostanoic acid Chemical class CCCCCCCC[C@H]1CCC[C@@H]1CCCCCCC(O)=O WGJJROVFWIXTPA-OALUTQOASA-N 0.000 claims description 10
- 102100031983 Ephrin type-B receptor 4 Human genes 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 8
- 239000002571 phosphodiesterase inhibitor Substances 0.000 claims description 8
- 230000001737 promoting effect Effects 0.000 claims description 8
- 229960002890 beraprost Drugs 0.000 claims description 7
- CTPOHARTNNSRSR-APJZLKAGSA-N beraprost Chemical compound O([C@H]1C[C@@H](O)[C@@H]([C@@H]21)/C=C/[C@@H](O)C(C)CC#CC)C1=C2C=CC=C1CCCC(O)=O CTPOHARTNNSRSR-APJZLKAGSA-N 0.000 claims description 7
- 229940082638 cardiac stimulant phosphodiesterase inhibitors Drugs 0.000 claims description 7
- 230000017531 blood circulation Effects 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 5
- 102100021243 G-protein coupled receptor 182 Human genes 0.000 claims description 5
- 108010063640 adrenomedullin receptors Proteins 0.000 claims description 5
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 5
- 208000026106 cerebrovascular disease Diseases 0.000 claims description 5
- 238000002054 transplantation Methods 0.000 claims description 5
- 102000000905 Cadherin Human genes 0.000 claims description 4
- 108050007957 Cadherin Proteins 0.000 claims description 4
- 208000031225 myocardial ischemia Diseases 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 239000006143 cell culture medium Substances 0.000 claims description 2
- 230000000414 obstructive effect Effects 0.000 claims description 2
- 239000002870 angiogenesis inducing agent Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 57
- 230000000250 revascularization Effects 0.000 abstract description 11
- 230000003511 endothelial effect Effects 0.000 abstract description 2
- 230000003028 elevating effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 24
- 230000000694 effects Effects 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 13
- 210000001519 tissue Anatomy 0.000 description 12
- 102000005962 receptors Human genes 0.000 description 10
- 108020003175 receptors Proteins 0.000 description 10
- 238000011161 development Methods 0.000 description 9
- 230000018109 developmental process Effects 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 7
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 239000012894 fetal calf serum Substances 0.000 description 6
- 102000008790 VE-cadherin Human genes 0.000 description 5
- 108010004469 allophycocyanin Proteins 0.000 description 5
- 108010018828 cadherin 5 Proteins 0.000 description 5
- 230000009762 endothelial cell differentiation Effects 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 150000003174 prostaglandin I2 derivatives Chemical class 0.000 description 5
- 210000003556 vascular endothelial cell Anatomy 0.000 description 5
- 102100024654 Calcitonin gene-related peptide type 1 receptor Human genes 0.000 description 4
- 101710118454 Calcitonin gene-related peptide type 1 receptor Proteins 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 206010012289 Dementia Diseases 0.000 description 3
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010047289 Ventricular extrasystoles Diseases 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000005129 volume perturbation calorimetry Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000030831 Peripheral arterial occlusive disease Diseases 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 102000007228 Receptor Activity-Modifying Protein 3 Human genes 0.000 description 2
- 108010033585 Receptor Activity-Modifying Protein 3 Proteins 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 210000001943 adrenal medulla Anatomy 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000002771 cell marker Substances 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 210000003038 endothelium Anatomy 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 239000007758 minimum essential medium Substances 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 2
- DVKQVRZMKBDMDH-UUOKFMHZSA-N 8-Br-cAMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1Br DVKQVRZMKBDMDH-UUOKFMHZSA-N 0.000 description 1
- 101150096544 AM gene Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 101100456543 Caenorhabditis elegans mec-4 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000609499 Palicourea Species 0.000 description 1
- 229940099471 Phosphodiesterase inhibitor Drugs 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 101500024174 Rattus norvegicus Adrenomedullin Proteins 0.000 description 1
- 108010033461 Receptor Activity-Modifying Protein 2 Proteins 0.000 description 1
- 102100030696 Receptor activity-modifying protein 2 Human genes 0.000 description 1
- 206010053648 Vascular occlusion Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- CJGYSWNGNKCJSB-YVLZZHOMSA-N bucladesine Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](OC(=O)CCC)[C@@H]2N1C(N=CN=C2NC(=O)CCC)=C2N=C1 CJGYSWNGNKCJSB-YVLZZHOMSA-N 0.000 description 1
- 229960005263 bucladesine Drugs 0.000 description 1
- 108010050923 calcitonin gene-related peptide (8-37) Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000003693 cell processing method Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229940095074 cyclic amp Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 231100001129 embryonic lethality Toxicity 0.000 description 1
- 230000009764 endothelial cell sprouting Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 150000008040 ionic compounds Chemical class 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 230000008338 local blood flow Effects 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000009094 negative regulation of endothelial cell differentiation Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000003668 pericyte Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- DMRMZQATXPQOTP-GWTDSMLYSA-M sodium;(4ar,6r,7r,7as)-6-(6-amino-8-bromopurin-9-yl)-2-oxido-2-oxo-4a,6,7,7a-tetrahydro-4h-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol Chemical compound [Na+].C([C@H]1O2)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1Br DMRMZQATXPQOTP-GWTDSMLYSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 208000023516 stroke disease Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- NDACAFBDTQIYCQ-YVQXRMNASA-N val(8)-phe(37)-cgrp Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C1=CN=CN1 NDACAFBDTQIYCQ-YVQXRMNASA-N 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 208000021331 vascular occlusion disease Diseases 0.000 description 1
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
本発明は、血管前駆細胞から内皮細胞を分化増殖させ、細胞移植に供する新規な方法に関する。詳細には、本発明は血管前駆細胞からの内皮細胞の分化増殖を有効に促進し、治療効果を期待した生体への移植に供する細胞を得るための新規な方法に関する。すなわち、移植可能な分化段階にまで増殖させた胚性幹細胞由来の細胞を十分量確保しうる方法を教示するものである。 The present invention relates to a novel method for differentiating and proliferating endothelial cells from vascular progenitor cells and subjecting them to cell transplantation. Specifically, the present invention relates to a novel method for effectively promoting the differentiation and proliferation of endothelial cells from vascular progenitor cells and obtaining cells for transplantation to a living body that is expected to have a therapeutic effect. That is, it teaches a method capable of securing a sufficient amount of embryonic stem cell-derived cells grown to a transplantable differentiation stage.
本発明者らは、血管を構成する内皮細胞および壁細胞(ペリサイトおよび血管平滑筋細胞)の双方に分化し、インビトロおよび生体への移植によりインビボで血管を構築し得る、マウス胚性幹細胞から分化増殖させた「血管前駆細胞(vascular progenitor cells; VPC)」を同定し報告した(非特許文献１)。VPCは、血管内皮増殖因子(vascular endothelial growth factor; VEGF)の受容体であるFlk-1 (VEGFR-2)陽性の細胞である。VPCは、成獣マウスに移植することで血管を形成し血流を増加させることを本発明者等は報告した(非特許文献２)。
特に本発明は、胚性幹細胞から得られる血管前駆細胞から内皮細胞への分化を有効に誘導しかつ増殖を促進することを目的とし、細胞内cAMP濃度を上昇させるため、cAMPアナログおよびアドレノメジュリン等を使用することに関する。
From mouse embryonic stem cells that can differentiate into both endothelial cells and mural cells (pericytes and vascular smooth muscle cells) that make up blood vessels and can construct blood vessels in vitro and in vivo by in vivo transplantation. Differentiated and proliferated “vascular progenitor cells (VPC)” were identified and reported (Non-patent Document 1). VPC is a positive cell for Flk-1 (VEGFR-2), which is a receptor for vascular endothelial growth factor (VEGF). The present inventors have reported that VPCs form blood vessels and increase blood flow when transplanted into adult mice (Non-patent Document 2).
In particular, the present invention aims to effectively induce differentiation from vascular progenitor cells obtained from embryonic stem cells to endothelial cells and promote proliferation, and to increase intracellular cAMP concentration, cAMP analogs and adrenomedullin And so on.
アドレノメジュリン(adrenomedullin: AM)はKitamura等により1993年に最初に報告された52アミノ酸のペプチドである(非特許文献３)。AMは正常な副腎髄質および心臓、血管(内皮細胞、血管平滑筋細胞)、肺、脳、腎臓など多くの組織に含有されている。AMはその受容体であるカルシトニン受容体様受容体（calcitonin-receptor-like receptor (CRLR)）と受容体活性改変プロテイン−２（receptor-activity-modyfying protein-2 (RAMP-2)）との複合体に結合し、細胞内cAMP濃度を上昇させることによりその生物学的活性を発揮する（非特許文献４）。AMノックアウトマウスでは、血管形成不全にて胎性致死となり、AMが発生期における血管の発生分化に重要な生理的因子であることが示されている(非特許文献５)。AMは、胎性期においても、発生心血管組織に発現していることが報告されている(非特許文献６)。また、本発明者らを含む複数の研究室の報告により、AMは培養内皮細胞の遊走増殖を促進し(非特許文献７)、さらに生体への投与により血管そのものの新生あるいはその再生を促進することが明らかにされている(非特許文献８)。
しかしながら、今日まで、胚性幹細胞へのAMの作用は報告されていない。また、VPCでの細胞内cAMP濃度上昇の効果に関する報告もない。
Adrenomedullin (AM) is a 52 amino acid peptide first reported in 1993 by Kitamura et al. AM is contained in many tissues such as normal adrenal medulla and heart, blood vessels (endothelial cells, vascular smooth muscle cells), lungs, brain and kidneys. AM is a complex of its receptor calcitonin-receptor-like receptor (CRLR) and receptor-activity-modyfying protein-2 (RAMP-2) It binds to the body and exerts its biological activity by increasing intracellular cAMP concentration (Non-patent Document 4). In AM knockout mice, embryonic lethality is caused by angiogenesis insufficiency, and AM has been shown to be an important physiological factor for the development and differentiation of blood vessels during development (Non-patent Document 5). It has been reported that AM is expressed in the developing cardiovascular tissue even in the fetal stage (Non-patent Document 6). In addition, according to reports of a plurality of laboratories including the present inventors, AM promotes the migration and proliferation of cultured endothelial cells (Non-patent Document 7), and further promotes the renewal of the blood vessel itself or its regeneration by administration to a living body. (Non-Patent Document 8).
However, to date, no effect of AM on embryonic stem cells has been reported. There is also no report on the effect of increasing intracellular cAMP concentration in VPC.
本発明者等は、胚性幹細胞由来VPCにおける細胞内cAMP濃度上昇が内皮細胞への分化を飛躍的に促進するとともに、分化した内皮細胞が動脈系内皮細胞としての性状を獲得することを発見し、さらにVPCの細胞内cAMP濃度上昇の方法として、AMの投与が有効であることを見出した。動脈系内皮細胞は、新生血管部位にその発現が認められ、血管新生時に重要な意義を有すると考えられている(非特許文献９)。しかしながら、動脈内皮マーカーであるEphrinB2の発現も含め、内皮細胞の動脈化に関して、これまでその調節機構についての報告は極めて少なく、また胚性幹細胞での検討はない。 The present inventors have discovered that an increase in intracellular cAMP concentration in an embryonic stem cell-derived VPC dramatically promotes differentiation into endothelial cells, and that the differentiated endothelial cells acquire properties as arterial endothelial cells. Furthermore, it has been found that administration of AM is effective as a method for increasing the intracellular cAMP concentration of VPC. The expression of arterial endothelial cells is recognized at a neovascular site, and is considered to have an important significance during angiogenesis (Non-patent Document 9). However, regarding the endothelialization of endothelial cells, including the expression of EphrinB2, which is an arterial endothelial marker, there have been very few reports on the regulatory mechanism, and there have been no studies on embryonic stem cells.
胚性幹細胞から持定の細胞、例えば神経細胞、骨細胞、免疫細胞および膵臓β細胞に分化するため、培地中に添加する試薬としてcAMPが記載されている(特許文献１）。しかし、ここには血管内皮細胞への分化増殖誘導に関する記載はない。胚性幹細胞から血管内皮細胞を誘導する方法として、血清および血管内皮増殖因子を含む培地を用いて培養する方法が記載されている(特許文献２)。しかし、ここにはアドレノメジュリン、プロスタン酸や細胞内cAMPに関する記載はない。 In order to differentiate from embryonic stem cells into fixed cells such as nerve cells, bone cells, immune cells and pancreatic β cells, cAMP is described as a reagent to be added to the medium (Patent Document 1). However, there is no description regarding differentiation and proliferation induction into vascular endothelial cells. As a method for inducing vascular endothelial cells from embryonic stem cells, a method of culturing using a medium containing serum and vascular endothelial growth factor has been described (Patent Document 2). However, there is no description of adrenomedullin, prostanoic acid or intracellular cAMP here.
現在日本人の3人に1人は、動脈硬化症による脳卒中や虚血性心疾患により死亡しており、また、これらの疾患は寝たきり、痴呆などの患者の生活の質(quality of life; QOL)を著しく低下させている。したがって、動脈硬化症の克服は、現代医療上最重要課題のひとつである。動脈硬化症の根本的な治療法は、血管閉塞部位において新たに血管を再生させる方法が最も望ましい。本発明は、胚性幹細胞由来のVPCおよびVPCから分化増殖させた内皮細胞の移植による新しい血管再生療法の開発のための方法を提供するものである。 Currently, 1 in 3 Japanese people have died of stroke or ischemic heart disease due to arteriosclerosis, and these diseases are bedridden, quality of life (QOL) of patients such as dementia Is significantly reduced. Therefore, overcoming arteriosclerosis is one of the most important issues in modern medicine. As a fundamental treatment method for arteriosclerosis, a method of newly regenerating a blood vessel at a vascular occlusion site is most desirable. The present invention provides a method for the development of a new revascularization therapy by transplanting an embryonic stem cell-derived VPC and endothelial cells differentiated and proliferated from the VPC.
本発明は、多くの特徴を有し、胚性幹細胞由来VPCおよびVPCから分化増殖させた内皮細胞を用いた新しい血管再生医療の開発につながる。
胚性幹細胞から内皮細胞を大量に分化増殖させるため、培地中のcAMP濃度を上昇させるための工程を包含する、血管再生医療の方法を提供する。
培地中のcAMP濃度を上昇させるために、cAMPアナログおよびホスホジエステラーゼ阻害薬、またはプロスタン酸誘導体であるプロスタグランジンI2誘導体ベラプロスト又はその塩を用いる工程を包含する、血管再生医療の方法を提供する。
また、培地中のcAMP濃度を上昇させる別の方法として、AMおよびその誘導体の投与あるいはAM遺伝子を組み込んだ発現プラスミドの投与、さらにAM受容体を活性化させる工程を包含する、血管再生医療の方法を提供する。
The present invention has many features and leads to the development of new revascularization medicine using embryonic stem cell-derived VPC and endothelial cells differentiated and proliferated from VPC.
In order to differentiate and proliferate endothelial cells in large quantities from embryonic stem cells, a method for revascularization medical treatment is provided which includes a step for increasing the cAMP concentration in a medium.
In order to increase cAMP concentration in a medium, a method of revascularization medicine comprising a step of using a cAMP analog and a phosphodiesterase inhibitor, or a prostaglandin I2 derivative beraprost or a salt thereof, which is a prostanoic acid derivative, is provided.
In addition, as another method for increasing the cAMP concentration in the culture medium, a method of revascularization medicine comprising the administration of AM and its derivatives or the administration of an expression plasmid incorporating the AM gene, and further the step of activating the AM receptor. I will provide a.
即ち、本発明は、
（１） 血管前駆細胞、具体的には胚性幹細胞由来の血管前駆細胞、さらに具体的には単離された血管前駆細胞を細胞内cAMP濃度上昇物質の存在下に分化増殖させることを特徴とする、血管前駆細胞から内皮細胞、特に動脈系内皮細胞を分化増殖させる方法；
（２） 本発明の方法により調製される単離された内皮細胞；
（３） 本発明の単離された内皮細胞を３次元様式で培養することを特徴とする、インビトロにおいて血管構造組織を調製する方法；
（４） 本発明の単離された内皮細胞を生体に移植することを特徴とする、移植部位において血管を形成させ、血流を増加させる方法；
（５） 本発明の単離された内皮細胞を生体に移植することを特徴とする、血管閉塞性疾患を処置する方法；
（６） 細胞内cAMP濃度上昇物質を含有する、血管前駆細胞から内皮細胞または動脈系内皮細胞を分化増殖させるための組成物；
（７） 細胞内cAMP濃度上昇物質を含有する、血管構造組織を形成するための組成物；
（８） 細胞内cAMP濃度上昇物質を含有する、血管閉塞性疾患を処置するための医薬組成物；
（９） 細胞内cAMP濃度上昇物質を含有する、血管形成誘導因子における分化増殖能を促進させるための組成物；に関する。
That is, the present invention
(1) It is characterized by differentiating and proliferating vascular progenitor cells, specifically vascular progenitor cells derived from embryonic stem cells, more specifically isolated vascular progenitor cells in the presence of a substance that increases intracellular cAMP concentration. A method of differentiating and proliferating endothelial cells, particularly arterial endothelial cells, from vascular progenitor cells;
(2) isolated endothelial cells prepared by the method of the present invention;
(3) A method for preparing vascular structural tissue in vitro, comprising culturing the isolated endothelial cells of the present invention in a three-dimensional manner;
(4) A method of increasing blood flow by forming blood vessels at the site of transplantation, wherein the isolated endothelial cells of the present invention are transplanted into a living body;
(5) A method for treating a vascular occlusive disease, which comprises transplanting the isolated endothelial cell of the present invention into a living body;
(6) A composition for differentiating and proliferating endothelial cells or arterial endothelial cells from vascular progenitor cells, comprising an intracellular cAMP concentration increasing substance;
(7) A composition for forming a vascular structural tissue, comprising a substance that increases intracellular cAMP concentration;
(8) A pharmaceutical composition for treating a vascular occlusive disease, comprising a substance that increases intracellular cAMP concentration;
(9) A composition for promoting differentiation and proliferation ability of an angiogenesis-inducing factor, comprising a substance that increases intracellular cAMP concentration.
本発明の利点は、本発明の以下の説明により明らかにされる。
細胞内cAMP濃度を上昇させることにより、従来の内皮細胞誘導方法にくらべてはるかに大量に内皮細胞を誘導できる。
細胞内cAMP濃度を上昇させることにより分化増殖される内皮細胞は、EphrinB2などのマーカーを有した動脈系内皮細胞が主体であり、これは本発明の方法により胚性幹細胞から分化増殖される内皮細胞が動脈系内皮細胞としての性質を持つことを意味する。
細胞内cAMP濃度を上昇させることにより分化増殖される内皮細胞により、インビトロで有効に３次元様式で培養することにより血管構造を構築できること、および生体に移植することで、血管を形成し移植部の血流を増加し得ること。
本発明により、血管再生が可能となり、血管閉塞性疾患すなわち、閉塞性動脈硬化症(Aterosclerosis Obliterans; ASO)、虚血性心疾患、脳血管障害(脳梗塞、痴呆を含む)などに対して、血管を再生し、血流を再開、増加させるという根本的な治療が可能となり、血管再生医療の開発にもつながる。
The advantages of the present invention will become apparent from the following description of the invention.
By increasing the intracellular cAMP concentration, endothelial cells can be induced in a much larger amount than conventional endothelial cell induction methods.
Endothelial cells differentiated and proliferated by increasing intracellular cAMP concentration are mainly arterial endothelial cells having markers such as EphrinB2, which are differentiated and proliferated from embryonic stem cells by the method of the present invention. Means that it has properties as an arterial endothelial cell.
Endothelial cells that are differentiated and proliferated by increasing the intracellular cAMP concentration can be constructed in vitro in a three-dimensional manner, and the vascular structure can be constructed. Can increase blood flow.
According to the present invention, revascularization is possible, and vascular obstructive diseases, that is, atherosclerosis obliterans (ASO), ischemic heart disease, cerebrovascular disorders (including cerebral infarction, dementia), etc. , And the fundamental treatment of resuming and increasing blood flow is possible, leading to the development of revascularization medicine.
（１） 血管前駆細胞を細胞内cAMP濃度上昇物質の存在下に分化増殖させることを特徴とする、血管前駆細胞から内皮細胞を分化増殖させる方法
出発原料としての「血管前駆細胞」は例えば、Yamasita J.等、Nature. 2000, 408(6808): 92-96に記載のようにして、胚性幹細胞から分化させ、入手することができる。胚性幹細胞（embryonic stem cell）は胚幹細胞あるいはＥＳ細胞とも呼ばれ、胚盤胞の内部に存在する未分化幹細胞である内部細胞塊を構成している細胞を培養に移し、頻繁に細胞塊の解離と継代を繰り返すことで樹立できる。この細胞は正常核型を維持しながらほぼ無限に増殖と継代を繰り返すことができ、内部細胞塊と同じようにあらゆる種類の細胞に分化することができる多分化能を保つことが知られている。
また、血管前駆細胞は、胚性幹細胞のほか、胎仔および骨髄由来細胞などの自己複製可能な他の多能性幹細胞からも調製できる。本発明においては、ヒト、サル、マウスなどの多様な動物に由来する多能性幹細胞を使用できる。
(1) A method for differentiating and proliferating endothelial cells from vascular progenitor cells, characterized by differentiating and proliferating vascular progenitor cells in the presence of an intracellular cAMP concentration-increasing substance. J. et al., Nature. 2000, 408 (6808): 92-96, can be differentiated from embryonic stem cells and obtained. Embryonic stem cells, also called embryonic stem cells or ES cells, transfer cells that constitute the inner cell mass, which is an undifferentiated stem cell present in the blastocyst, to the culture, and frequently It can be established by repeating dissociation and passage. These cells are known to maintain normal karyotypes and to proliferate and pass through indefinitely, maintaining the pluripotency that can differentiate into all types of cells, just like the inner cell mass. Yes.
In addition to embryonic stem cells, vascular progenitor cells can be prepared from other pluripotent stem cells capable of self-replication, such as fetal and bone marrow-derived cells. In the present invention, pluripotent stem cells derived from various animals such as humans, monkeys and mice can be used.
本発明では「単離された血管前駆細胞」を用いることができる。「単離された」とは、生体から物理的に分離された状態、または他の種類の細胞の混入がない状態を表す。血管前駆細胞はセルソーターなどを用いて、血管内皮増殖因子(VEGF)の受容体であるFlk-1 (VEGFR-2)陽性を指標として分離できる。 In the present invention, “isolated vascular progenitor cells” can be used. “Isolated” refers to a state of being physically separated from a living body or being free from contamination with other types of cells. Vascular progenitor cells can be isolated using a cell sorter or the like as a marker of positive for Flk-1 (VEGFR-2), which is a receptor for vascular endothelial growth factor (VEGF).
本発明において、「細胞内cAMP濃度上昇物質」とは、血管前駆細胞内のcAMP濃度を上昇させるあらゆる物質を意味し、これにはcAMPアナログ、アドレノメジュリンおよびアドレノメジュリン誘導体、ホスホジエステラーゼ阻害薬、およびプロスタン酸誘導体が含まれる。
「cAMPアナログ」とは、細胞に接触させると細胞内cAMP濃度を上昇させる、cAMPに類似した構造を有する化合物であり、これには例えば8-ブロモアデノシン-3': 5'-サイクリック一リン酸ナトリウム塩(8-br-cAMPナトリウム塩)、ジブチリル-cAMPが含まれる。
「アドレノメジュリン」または「AM」は、正常な副腎髄質および心臓、血管(内皮細胞、血管平滑筋細胞)、肺、脳、腎臓など多くの組織に含有されている52アミノ酸のペプチドである(Kitamura K.等,Biochem. Biophys. Res. Commun (192: 553-560 (1993))。AMは特開平7-196693に記載の方法により容易に調製できる。
AMはその受容体であるカルシトニン受容体様受容体（calcitonin-receptor-like receptor (CRLR)）と受容体活性改変プロテイン−２（receptor-activity-modifying protein-2 (RAMP-2)）あるいは受容体活性改変プロテイン−３（receptor-activity-modifying protein-3 (RAMP-3)）との複合体に結合し、細胞内cAMP濃度を上昇させることによりその生物学的活性を発揮することが知られている。よって、「アドレノメジュリン誘導体」とは、胚性幹細胞上に局在するアドレノメジュリン受容体を活性化させるあらゆる物質を意味し、例えば、アドレノメジュリンを構成する５２アミノ酸残基の１または数個のアミノ酸を欠失、置換もしくは付加したアミノ酸配列を有し、かつアドレノメジュリンが有する活性を保持したペプチドが挙げられ、例えばAM(22-52)やCGRP(8-37)が例示される。
In the present invention, “intracellular cAMP concentration increasing substance” means any substance that increases the cAMP concentration in vascular progenitor cells, including cAMP analogs, adrenomedullin and adrenomedullin derivatives, phosphodiesterase inhibitors, And prostanoic acid derivatives.
A “cAMP analog” is a compound having a structure similar to cAMP that increases intracellular cAMP concentration when contacted with a cell, such as 8-bromoadenosine-3 ′: 5′-cyclic monolin. Acid sodium salt (8-br-cAMP sodium salt), dibutyryl-cAMP.
`` Adrenomedullin '' or `` AM '' is a 52 amino acid peptide contained in many tissues such as normal adrenal medulla and heart, blood vessels (endothelial cells, vascular smooth muscle cells), lungs, brain, kidneys, etc. Kitamura K. et al., Biochem. Biophys. Res. Commun (192: 553-560 (1993)) AM can be easily prepared by the method described in JP-A-7-196693.
AM is its receptor calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein-2 (RAMP-2) or receptor It is known to bind to a complex with receptor-activity-modifying protein-3 (RAMP-3) and exert its biological activity by increasing intracellular cAMP concentration. Yes. Therefore, the term “adrenomedullin derivative” means any substance that activates the adrenomedullin receptor localized on embryonic stem cells. For example, one or a number of 52 amino acid residues constituting adrenomedullin Examples include peptides having an amino acid sequence in which one amino acid is deleted, substituted or added, and retaining the activity of adrenomedullin, such as AM (22-52) and CGRP (8-37). .
「ホスホジエステラーゼ阻害薬」には例えば、3-イソブチルメチルキサンチン(IBMX)が含まれる。
本発明において使用される「プロスタン酸誘導体」とはプロスタン酸を基本骨格とし、これに二重結合やヒドロキシ基等が加わった数多くの不飽和脂肪酸を意味し、これにはプロスタグランジン類、トロンボキサン類、ロイコトリエン類が含まれる。本発明においては特に、プロスタグランジンI２誘導体が好ましく、ベラプロストまたはその塩が特に好ましい。ここに、「塩」とはイオン化合物を意味する。
“Phosphodiesterase inhibitors” include, for example, 3-isobutylmethylxanthine (IBMX).
The “prostanoic acid derivative” used in the present invention means a number of unsaturated fatty acids having prostanoic acid as a basic skeleton and having a double bond, a hydroxy group or the like added thereto. Xanes and leukotrienes are included. In the present invention, a prostaglandin I 2 derivative is particularly preferable, and beraprost or a salt thereof is particularly preferable. Here, “salt” means an ionic compound.
血管前駆細胞を細胞内cAMP濃度上昇物質の存在下に「分化増殖」させるとは、血管前駆細胞を細胞内cAMP濃度上昇物質とともに培養することを意味する。培養は、細胞の維持または分化の目的に適する組成の培地を使用し、それに本発明の細胞内cAMP濃度上昇物質を添加し、当業者に周知の温度等の環境下にて行う。例えば、用いる培地としては、通常、細胞培養用の最小必須培地に5x10-5M2-メルカプトエタノール、10%FCS等を添加したものを使用する。各培地には、抗生物質などの培養に有用な他の物質を添加することができ、各成分に代えて、同等の機能を有する代替物を使用してもよい。また、培地の各成分は、各々適する方法で滅菌して使用する。本発明においては、血管形成誘導因子、例えば血管内皮増殖因子（VEGF）を培地に添加して培養を行う。 “Differentiating and proliferating” vascular progenitor cells in the presence of an intracellular cAMP concentration-increasing substance means culturing vascular progenitor cells together with the intracellular cAMP concentration-increasing substance. The culture is carried out using a medium having a composition suitable for the purpose of cell maintenance or differentiation, adding the substance for increasing intracellular cAMP concentration of the present invention thereto, and under an environment known to those skilled in the art such as temperature. For example, as a medium to be used, a medium obtained by adding 5 × 10 −5 M2-mercaptoethanol, 10% FCS or the like to a minimum essential medium for cell culture is usually used. Other substances useful for culture such as antibiotics can be added to each medium, and substitutes having equivalent functions may be used in place of the respective components. In addition, each component of the medium is used after being sterilized by a suitable method. In the present invention, angiogenesis-inducing factor, for example, vascular endothelial growth factor (VEGF) is added to the medium for culturing.
本発明のある態様では、血管前駆細胞をコラーゲンIVでコートした培養容器内、10％FCSおよびVEGFの存在下に培養する。このような条件で培養すると、通常３日目に本発明の内皮細胞が最適に得られる。あるいは、無血清状態でもある程度、内皮細胞に分化させることができる。 In one embodiment of the invention, vascular progenitor cells are cultured in the presence of 10% FCS and VEGF in a culture vessel coated with collagen IV. When cultured under such conditions, the endothelial cells of the present invention are optimally obtained usually on the third day. Alternatively, it can be differentiated into endothelial cells to some extent even in a serum-free state.
内皮細胞の単離は、適当な標識物、アロフィコシアニン（allophycocyanin(APC)）を結合したFlk-1に対する抗体、および適当な標識物、フルオレッセインイソチオシアネート（FITC）を結合したE-カドヘリンに対する抗体を用い、セルソーター等により、Flk-1陽性かつE-カドヘリン陰性細胞分画を採取することにより行うことができる。また、APC結合VE-カドヘリン抗体、FITC結合PECAM-1抗体、FITC結合CD34抗体を用いて、FACSセルソーターにて、内皮細胞の分化および分化した内皮細胞の性状を検討できる。本発明において、細胞の単離・精製に用いる抗体は、ポリクローナル抗体またはモノクローナル抗体のいずれでもよい。ＦＡＣＳを使用する場合、モノクローナル抗体が好ましい。そのような抗体は、当業者であれば容易に作成でき、または市販のものを使用してもよい。 Endothelial cell isolation is directed against an appropriate label, an antibody to Flk-1 conjugated with allophycocyanin (APC), and an E-cadherin conjugated with an appropriate label, fluorescein isothiocyanate (FITC). The antibody can be used to collect a Flk-1-positive and E-cadherin-negative cell fraction using a cell sorter or the like. In addition, using an APC-conjugated VE-cadherin antibody, FITC-conjugated PECAM-1 antibody, and FITC-conjugated CD34 antibody, differentiation of endothelial cells and the properties of the differentiated endothelial cells can be examined using a FACS cell sorter. In the present invention, the antibody used for cell isolation / purification may be either a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are preferred when using FACS. Such antibodies can be easily prepared by those skilled in the art, or commercially available antibodies may be used.
本発明において単離された内皮細胞の多くは、Flk-1陽性、PECAM-1陽性、EphB4結合分子陽性、かつE-カドヘリン陰性である場合、それは動脈系内皮細胞である。動脈系内皮細胞とは、動脈血管を構成する内皮細胞を意味する。細胞内cAMP濃度上昇物質の存在下に増殖させた内皮細胞は１００％ではないが、EphB4結合分子（EphrinB2）発現が上昇している動脈内皮細胞の性質を有するものが多く、他方、細胞内cAMP濃度上昇物質の不存在下に増殖させた内皮細胞はEphB4結合分子発現のない静脈内皮細胞の性質を主に持っている（図６）。 Many of the endothelial cells isolated in the present invention are arterial endothelial cells when they are Flk-1 positive, PECAM-1 positive, EphB4 binding molecule positive, and E-cadherin negative. Arterial endothelial cells mean endothelial cells that constitute arterial blood vessels. Endothelial cells grown in the presence of substances that increase intracellular cAMP concentration are not 100%, but many have the properties of arterial endothelial cells with increased expression of EphB4 binding molecule (EphrinB2), while intracellular cAMP Endothelial cells grown in the absence of a concentration-enhancing substance mainly have the properties of venous endothelial cells without EphB4 binding molecule expression (FIG. 6).
本明細書において、ある分子について「陽性」とは細胞が当該分子を発現していることを意味し、「陰性」とは、発現していないことを意味する。細胞がある分子を発現しているか否かは、ＦＡＣＳ等により判定できる。ＦＡＣＳは、通常、フローサイトメーター、レーザー発生装置、光学系、データ処理装置および細胞分取装置を備えている。ＦＡＣＳにより、特定物質で蛍光標識した細胞にについて、散乱光（前方散乱光や側方散乱光）や蛍光のシグナル情報を測定し、特定のシグナル情報を発する細胞を分取することができる。 In the present specification, “positive” for a certain molecule means that the cell expresses the molecule, and “negative” means not expressing the molecule. Whether a cell expresses a certain molecule can be determined by FACS or the like. The FACS usually includes a flow cytometer, a laser generator, an optical system, a data processing device, and a cell sorting device. By FACS, scattered light (forward scattered light and side scattered light) and fluorescent signal information can be measured on cells fluorescently labeled with a specific substance, and cells emitting specific signal information can be collected.
（２） 本発明の方法により調製される単離された内皮細胞
本発明は別の態様として、本発明の方法により調製される単離された内皮細胞を提供する。
本発明において、「単離された」とは、生体から物理的に分離された状態、または他の種類の細胞の混入がない状態を表す。
本発明において単離される内皮細胞の多くは上記のとおり、Flk-1陽性、PECAM-1陽性、EphB4結合分子陽性かつE-カドヘリン陰性であり、それは動脈系内皮細胞である。
(2) Isolated endothelial cell prepared by the method of the present invention As another aspect, the present invention provides an isolated endothelial cell prepared by the method of the present invention.
In the present invention, “isolated” means a state in which the substance is physically separated from a living body or a state in which there is no contamination of other types of cells.
Many of the endothelial cells isolated in the present invention are Flk-1 positive, PECAM-1 positive, EphB4 binding molecule positive and E-cadherin negative, as described above, and are arterial endothelial cells.
本発明の細胞は、アドレノメジュリン受容体をさらに発現しているものが好ましい。アドレノメジュリン受容体は、例えばマウスの場合、マウス抗AMモノクローナル抗体(Michibata H等、Kidney Int 53: 979-985 (1998))を用いて確認できる。 The cells of the present invention preferably further express an adrenomedullin receptor. The adrenomedullin receptor can be confirmed using, for example, a mouse anti-AM monoclonal antibody (Michibata H et al., Kidney Int 53: 979-985 (1998)) in the case of a mouse.
（３） 本発明の単離された動脈系内皮細胞または内皮細胞を３次元様式で培養することを特徴とする、インビトロにおいて血管構造組織を調製する方法
「３次元様式での培養」とは、例えばゲルの中に細胞を埋め込んで培養する立体的培養を意味し、本発明ではこれにより血管構造組織の構築を促すことができる。「血管構造組織」とは、動脈、静脈、リンパ管等の脈管を構成する組織を意味する。
(3) A method for preparing a vascular structure tissue in vitro, characterized by culturing the isolated arterial endothelial cells or endothelial cells of the present invention in a three-dimensional manner. For example, it means three-dimensional culture in which cells are embedded in a gel and cultured, and in the present invention, this can promote the construction of a vascular structure tissue. “Vascular structure tissue” means a tissue that constitutes a blood vessel such as an artery, vein, or lymphatic vessel.
（４） 本発明の単離された動脈系内皮細胞または内皮細胞を生体に移植することを特徴とする、移植部位において血管を形成させ、血流を増加させる方法
移植の方法としては、局所注入、血管内注入（動脈内、静脈内）を挙げることができる。
(4) A method for forming blood vessels at a transplant site and increasing blood flow, characterized by transplanting the isolated arterial endothelial cells or endothelial cells of the present invention into a living body. And intravascular injection (intraarterial, intravenous).
（５） 本発明の単離された動脈系内皮細胞または内皮細胞を生体に移植することを特徴とする、血管閉塞性疾患を処置する方法
本発明において、「血管閉塞性疾患」とは閉塞性動脈硬化症、虚血性心疾患、脳血管障害、例えば脳梗塞、痴呆である。
この態様において、本発明は、本発明の単離された動脈系内皮細胞または内皮細胞における血管閉塞性疾患を処置する使用に関する。
(5) A method for treating a vascular occlusive disease, characterized by transplanting the isolated arterial endothelial cell or endothelial cell of the present invention into a living body. Arteriosclerosis, ischemic heart disease, cerebrovascular disorder such as cerebral infarction, dementia.
In this aspect, the present invention relates to the use of the isolated arterial endothelial cells or endothelial cells of the present invention for treating vascular occlusive disease.
（６） 細胞内cAMP濃度上昇物質を含有する、血管前駆細胞から内皮細胞または動脈系内皮細胞を分化増殖させるための組成物
本発明は別の態様として、血管前駆細胞から内皮細胞または動脈系内皮細胞を分化増殖させるために用いられる、細胞内cAMP濃度上昇物質を含有する組成物に関する。
「細胞内cAMP濃度上昇物質」は上記のとおりである。かかる組成物は通常、胚性幹細胞由来血管前駆細胞に対し、ex vivoで細胞内cAMP濃度を上昇させる目的で細胞内cAMP濃度上昇物質を添加するのに用いられる。
この態様において、本発明は、血管前駆細胞から内皮細胞または動脈系内皮細胞を分化増殖するための組成物を調製するための、細胞内cAMP濃度上昇物質の使用に関する。
この態様における組成物は典型的には、細胞内cAMP濃度上昇物質を含有する、細胞培養培地である。
(6) A composition for differentiating and proliferating endothelial cells or arterial endothelial cells from vascular progenitor cells, which contains a substance that increases intracellular cAMP concentration. As another aspect, the present invention is directed to endothelial cells or arterial endothelium from vascular progenitor cells. The present invention relates to a composition containing a substance that increases intracellular cAMP concentration, which is used to differentiate and proliferate cells.
The “intracellular cAMP concentration increasing substance” is as described above. Such a composition is usually used for adding an intracellular cAMP concentration increasing substance to an embryonic stem cell-derived vascular progenitor cell for the purpose of increasing the intracellular cAMP concentration ex vivo.
In this aspect, the present invention relates to the use of an intracellular cAMP concentration increasing substance for preparing a composition for differentiating and proliferating endothelial cells or arterial endothelial cells from vascular progenitor cells.
The composition in this embodiment is typically a cell culture medium containing a substance that increases intracellular cAMP concentration.
（７） 細胞内cAMP濃度上昇物質を含有する、血管構造組織を形成するための組成物
この態様の発明は、血管構造組織を形成するための試薬として利用される。
(7) Composition for Forming Vascular Structure Tissue Containing Substance for Increasing Intracellular cAMP Concentration The invention of this aspect is used as a reagent for forming vascular structure tissue.
（８） 細胞内cAMP濃度上昇物質を含有する、血管閉塞性疾患を処置するための医薬組成物
この態様の発明は通常、注射により生体に投与される。投与部位は目的とする疾患により異なり、通常、静脈内投与である。用量は適宜医師により決定されるが、通常、０．０５μg/分/kg体重である。
(8) A pharmaceutical composition for treating a vascular occlusive disease, comprising a substance that increases intracellular cAMP concentration. The invention of this embodiment is usually administered to a living body by injection. The administration site varies depending on the target disease and is usually intravenous administration. The dose is appropriately determined by a doctor and is usually 0.05 μg / min / kg body weight.
（９）細胞内cAMP濃度上昇物質を含有する、血管形成誘導因子における分化増殖能を促進させるための組成物
本発明は別の態様として、血管形成誘導因子が有する血管形成を促進、増大、増進させるために用いられる、細胞内cAMP濃度上昇物質を含有する組成物に関する。
本発明において、血管形成誘導因子とは、破壊局所からの血管内皮細胞の発芽、血管外への遊走・増殖、管腔形成の諸過程を促進し、新たな毛細血管の形成を促す活性を有する因子を意味する。これには例えば、血管内皮増殖因子（VEGF）、繊維芽細胞増殖因子−２、塩基性繊維芽細胞増殖因子（bFGF）、血小板由来増殖因子（PDGF）等が挙げられる。本発明に用いられる血管形成誘導因子は天然由来のものあるいは遺伝子操作により調製されたものいずれも使用することができる。
(9) A composition for promoting the differentiation and proliferation ability of an angiogenesis inducing factor, which contains a substance that increases intracellular cAMP concentration. As another aspect, the present invention promotes, increases and enhances angiogenesis of an angiogenesis inducing factor. The present invention relates to a composition containing a substance that increases intracellular cAMP concentration.
In the present invention, the angiogenesis-inducing factor has an activity of promoting the formation of new capillaries by accelerating various processes of vascular endothelial cell sprouting from the destruction site, migration / proliferation outside the blood vessel, and lumen formation. Means a factor. This includes, for example, vascular endothelial growth factor (VEGF), fibroblast growth factor-2, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and the like. The angiogenesis-inducing factor used in the present invention can be either naturally derived or prepared by genetic manipulation.
（１０）
本発明は別の態様として、細胞内cAMP濃度上昇物質を含有する、血管構造組織を形成する、または血管閉塞性疾患を処置する組成物を調製するための、本発明の方法による胚性幹細胞から内皮細胞ないし動脈系内皮細胞にいたるインビトロの分化培養系の使用を提供する。また、本発明の方法による胚性幹細胞から内皮細胞ないし動脈系内皮細胞にいたるインビトロの分化培養系における分化過程の細胞での遺伝子発現を網羅的に解析する方法（例えば、PNAマイクロアレイ法など）を提供する。さらに、遺伝子発現のデータベース開発により、そのデータベースから新しい血管形成誘導遺伝子を見つけることが可能となる。
(10)
In another aspect, the present invention provides an embryonic stem cell according to the method of the present invention for preparing a composition containing an intracellular cAMP-concentrating substance, forming a vascular structural tissue, or treating a vascular occlusive disease. The use of an in vitro differentiation culture system from endothelial cells to arterial endothelial cells is provided. In addition, a method (for example, PNA microarray method) for comprehensively analyzing gene expression in cells undergoing differentiation in an in vitro differentiation culture system from embryonic stem cells to endothelial cells or arterial endothelial cells according to the method of the present invention. provide. Furthermore, the development of a gene expression database makes it possible to find new angiogenesis-inducing genes from the database.
以下に本発明を実施例によりさらに詳細に説明するが、本発明はこれら実施例に制限されるものではない。
参考例
胚性幹細胞からのFlk-1陽性細胞への分化増殖
本発明者らが以前に報告した方法(Yamashita J等、Nature 408: 92-96 (2000)により、Flk-1陽性細胞を分化増殖させた。すなわち、マウス胚性幹細胞（Department of Molecular Embryology, Max-Planck-Institute fur Immunobiology, Freiburg, GermanyのBM I Evans博士より供与）を10％胎児ウシ血清(FCS)および5×10-5M 2-メルカプトエタノール含有アルファー最小必須培地（分化メデイウム）にて、タイプIVコラーゲンにてコートした培養皿(ベクトンデイキンソン社)上で、５％CO2および９５％空気の環境下３７℃で４日間培養した。
分化増殖させたFlk-1陽性細胞を、既報の方法に従い(Nishikawa SI 等、Development 125: 1747-1457 (1998))、FACSヴァンテージ(ベクトンデイキンソン社)にて97%以上の純度で単離した。
EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.
Reference Example Differentiation and Proliferation from Embryonic Stem Cells to Flk-1 Positive Cells Differentiation and proliferation of Flk-1 positive cells by the method previously reported by the present inventors (Yamashita J et al., Nature 408: 92-96 (2000)) That is, mouse embryonic stem cells (provided by Dr. BM I Evans of Department of Molecular Embryology, Max-Planck-Institute fur Immunobiology, Freiburg, Germany) with 10% fetal calf serum (FCS) and 5 × 10 −5 M 4-Mercaptoethanol-containing alpha minimum essential medium (differentiation medium) on a culture dish (Becton Dakinson) coated with type IV collagen for 4 days at 37 ° C. in an environment of 5% CO 2 and 95% air Cultured.
Differentiated and proliferated Flk-1-positive cells were isolated with a purity of 97% or more by FACS Vantage (Becton Deakinson) according to a previously reported method (Nishikawa SI et al., Development 125: 1747-1457 (1998)). .
実施例１
Flk-1陽性細胞からの内皮細胞への分化増殖に対する種々の添加剤の効果
内皮細胞の単離では、APC結合抗-Flk-1モノクローナル抗体(AVAIS12 (Dev.Growth Differ. 39, 729-740 (1997)))およびFITC結合抗-E-カドヘリンモノクローナル抗体(ECCD2 (Cell Struct.Funct. 11, 245-252(1986)))を用い、Flk-1陽性かつE-カドヘリン陰性細胞分画を採取した。
内皮細胞の分化および分化した内皮細胞の性状の検討は、APC結合VE-カドヘリン抗体(VECD1 (Proc.Assoc.Am.Physicians 109, 362-371 (1997)))、FITC結合PECAM-1抗体(Mec13.3 (Pharmigen))、およびFITC結合CD34抗体(RAM34 (Pharmigen))を用いて、FACSセルソーターにて行った。
Example 1
Effect of various additives on differentiation and proliferation from Flk-1-positive cells to endothelial cells APC-conjugated anti-Flk-1 monoclonal antibody (AVAIS12 (Dev.Growth Differ. 39, 729-740 ( 1997))) and FITC-conjugated anti-E-cadherin monoclonal antibody (ECCD2 (Cell Struct. Funct. 11, 245-252 (1986))) was used to collect Flk-1-positive and E-cadherin-negative cell fractions. .
Endothelial cell differentiation and the properties of differentiated endothelial cells were investigated using APC-conjugated VE-cadherin antibody (VECD1 (Proc. Assoc. Am. Physicians 109, 362-371 (1997))), FITC-conjugated PECAM-1 antibody (Mec13 .3 (Pharmigen)), and FITC-conjugated CD34 antibody (RAM34 (Pharmigen)) using a FACS cell sorter.
参考例にて単離した純化Flk-1陽性細胞であるマウス胚性幹細胞由来VPCは、コラーゲンIVコート培養皿上にて10％FCSおよびVEGF (10ng/ml、RアンドDシステム)存在下にて４日間培養すると、PECAM-1およびVE-カドヘリン陽性の内皮細胞およびSMA陽性の壁細胞の二種類の細胞に分化した。この条件において、VPCから内皮細胞への分化率はFACS解析より全体の細胞の約３０％であることが判明した。
10％FCSおよびVEGF (50ng/ml)含有分化メデイウム(この培養メデイウムを以下メデイウムAと称する)に添加剤として、ラットAMおよびAM誘導体であるヒトAM-(22-52)-アミド(ともにペプチド研究所)、8-ブロモアデノシン-3': 5'-サイクリック一リン酸ナトリウム塩(8-br-cyclic AMP)、IBMX、およびプロスタグランジンI2誘導体であるベラプロストを加える以外は、同様に培養し、Flk-1陽性細胞からの内皮細胞への分化増殖に対する種々の添加剤の効果を調べた。
メデイウムAに8-br-cAMPを添加し、胚性幹細胞由来VPCをコラーゲンIVコート培養皿上にて４日間培養し内皮細胞マーカ一であるPECAM-1および壁細胞マーカーであるSMAによる免疫学的二重染色をおこなった。得られた結果を図１に示す(PECAM-1(一部を矢印にて図中に示す)、SMA(一部を矢印にて図中に示す)、40倍)。図１は、8-br-cAMP 0.1mM〜0.5mMの濃度において濃度依存性に内皮細胞への分化を促進していることを示している。また、0.5mMの8-br-cAMP添加にて内皮細胞に分化した細胞は全体の約７０％に達していることが判明した。なお、VEGF非存在下では、8-brc-AMPの内皮細胞分化促進作用は認められなかった。
Mouse embryonic stem cell-derived VPC, which is a purified Flk-1-positive cell isolated in the Reference Example, is present on a collagen IV-coated culture dish in the presence of 10% FCS and VEGF (10 ng / ml, R and D system). When cultured for 4 days, it differentiated into two types of cells: PECAM-1 and VE-cadherin positive endothelial cells and SMA positive wall cells. Under these conditions, the differentiation rate from VPC to endothelial cells was found to be about 30% of the total cells by FACS analysis.
Rat AM and AM derivative human AM- (22-52) -amide (both peptide studies) as an additive to differentiation medium containing 10% FCS and VEGF (50 ng / ml) (this culture medium is hereinafter referred to as medium A) ), 8-bromoadenosine-3 ': 5'-cyclic monophosphate sodium salt (8-br-cyclic AMP), IBMX, and except that beraprost, a prostaglandin I2 derivative, was added and cultured in the same manner. The effect of various additives on the differentiation and proliferation from Flk-1 positive cells to endothelial cells was examined.
8-Br-cAMP is added to medium A, and embryonic stem cell-derived VPCs are cultured on a collagen IV-coated culture dish for 4 days, and then the immunology by PECAM-1 which is an endothelial cell marker and SMA which is a wall cell marker Double staining was performed. The obtained results are shown in FIG. 1 (PECAM-1 (partially indicated by arrows), SMA (partly indicated by arrows), 40 times). FIG. 1 shows that the differentiation into endothelial cells is promoted in a concentration-dependent manner at a concentration of 8-br-cAMP of 0.1 mM to 0.5 mM. In addition, it was found that about 70% of the cells differentiated into endothelial cells by addition of 0.5 mM 8-br-cAMP. In the absence of VEGF, endothelial cell differentiation promoting action of 8-brc-AMP was not observed.
メデイウムAにIBMX(10-4M)を添加し、同様に培養した結果を図２に示す。メディウムA単独の培養と比較し、IBMX10-4M添加条件では胚性幹細胞由来VPCからのPECAM-1陽性内皮細胞(青)の分化を促進した(図２、PECAM-1(一部を矢印にて図中に示す)、SMA(一部を矢印にて図中に示す)、100倍)。
メデイウムAにアドレノメジュリン（AM）を添加し、同様に培養した結果を図３に示す。AM(10-9〜10-6M)の添加は、メディウムA単独の培養と比較し、濃度依存的に胚性幹細胞由来VPCからPECAM-1陽性内皮細胞の分化増殖を促進した（内皮細胞への分化を示す細胞数が増加）(図３、PECAM-1(一部を矢印にて図中に示す)、SMA(一部を矢印にて図中に示す)、200倍)。AM10-6M添加により内皮細胞に分化した細胞は全体の細胞の50%に達した。AM(10-7M)添加時に認められる胚性幹細胞由来VPCからのPECAM-1陽性内皮細胞(青)分化の促進効果は、AM誘導体であるAM(２２−５２)(10-4M)の添加によりメディウムA単独培養条件と同程度に阻害された（図４参照、PECAM-1(一部を矢印にて図中に示す)、SMA(一部を矢印にて図中に示す)、100倍)）。このことから、AMの分化促進効果はAM受容体を介した作用であると考えられた。同様の作用は、プロスタグランジンI2誘導体であるベラプロスト(10nM)添加においても認められた。
FIG. 2 shows the results obtained by adding IBMX (10 −4 M) to medium A and culturing in the same manner. Compared with medium A alone, the addition of IBMX10 -4 M promoted the differentiation of PECAM-1-positive endothelial cells (blue) from embryonic stem cell-derived VPC (Fig. 2, PECAM-1 (partly with arrows) And SMA (partially indicated by arrows), 100 times).
The result of adding adrenomedullin (AM) to medium A and culturing in the same manner is shown in FIG. Addition of AM (10 -9 to 10 -6 M) promoted the differentiation and proliferation of PECAM-1-positive endothelial cells from embryonic stem cell-derived VPC in a concentration-dependent manner compared to culture with medium A alone (to endothelial cells) (FIG. 3, PECAM-1 (partly indicated by arrows), SMA (partly indicated by arrows), 200 times). Cells differentiated into endothelial cells by AM10 -6 M addition reached 50% of the total cells. The effect of promoting differentiation of PECAM-1-positive endothelial cells (blue) from VPCs derived from embryonic stem cells observed when AM (10 −7 M) is added is the effect of AM (22-52) (10 −4 M), which is an AM derivative. It was inhibited to the same extent as the medium A culture condition by addition (see FIG. 4, PECAM-1 (partly indicated by arrows), SMA (partly indicated by arrows), 100 Times)). From this, it was considered that the differentiation promoting effect of AM is an action through the AM receptor. A similar effect was observed when beraprost (10 nM), a prostaglandin I2 derivative, was added.
さらに、AMおよびcAMPによるVE-カドヘリン/PECAM-1陽性内皮細胞の誘導効果をFACS解析により確認した。結果を図５に示す。図５により、メディウムA単独においてはVE-cad/CD31陽性細胞の誘導が20%であるのに対し、AM10-6M添加時には37.9%、8-br-cAMP添加時には51.9%の高い誘導効果を認めた。 Furthermore, the effect of inducing VE-cadherin / PECAM-1-positive endothelial cells by AM and cAMP was confirmed by FACS analysis. The results are shown in FIG. According to FIG. 5, the induction of VE-cad / CD31-positive cells is 20% with Medium A alone, while the high induction effect is 37.9% when AM10 -6 M is added and 51.9% when 8-br-cAMP is added. Admitted.
実施例２
胚性幹細胞より分化した内皮細胞の性状検討
EphrinB2は動脈系内皮細胞に発現し、その受容体であるEphB4は静脈系内皮細胞に発現することが知られている(Adams, R et al Genes Dev. 13: 295-306 (1999))。
そこで、既に報告している免疫組織化学的手法 (Hirashima等、Blood 93: 1253-1263 (1999))により、実施例１にて分化増殖した細胞をメタノール固定後、Mec 13.3や抗アルファ平滑筋アクチン(SMA)抗体(1A4 (SIGMA社))にて処理し、アルカリフォスファターゼ結合抗ラットIgG抗体(EphB4-Feキメラ (ZYMED社))、あるいは西洋ワサビペルオキシダーゼ(HRP)結合抗マウスIgG抗体(ZYMED社)等を用いて染色した。
さらに、EphrinB2発現の検討には、EphrinB2が特異的に結合するEphB4とヒトIgGのキメラタンパクとペルオシダーゼ結合抗ヒトIgGFcヤギIgG（ICN/CAPPEL社）を用いた。この際、EphB4-ヒトIgGキメラタンパクとEphrinB2の結合の検出感度を上げるため、Tyramid Signal Amplication Biotinシステム(Perkin Elmerライフサイエンス社)を用いた。
Example 2
Characterization of endothelial cells differentiated from embryonic stem cells
It is known that EphrinB2 is expressed in arterial endothelial cells and its receptor EphB4 is expressed in venous endothelial cells (Adams, R et al Genes Dev. 13: 295-306 (1999)).
Therefore, after the cells that had been differentiated and proliferated in Example 1 were fixed with methanol by the immunohistochemical method (Hirashima et al., Blood 93: 1253-1263 (1999)) already reported, Mec 13.3 or anti-alpha smooth muscle actin (SMA) antibody (1A4 (SIGMA)) treated with alkaline phosphatase-conjugated anti-rat IgG antibody (EphB4-Fe chimera (ZYMED)) or horseradish peroxidase (HRP) -conjugated anti-mouse IgG antibody (ZYMED) Etc. were used for staining.
Furthermore, EphinB2 expression was examined by using EphB4 and human IgG chimeric protein to which EphrinB2 specifically binds, and perosidase-conjugated anti-human IgGFc goat IgG (ICN / CAPPEL). At this time, a Tyramid Signal Amplication Biotin system (Perkin Elmer Life Sciences) was used in order to increase the detection sensitivity of the binding between EphB4-human IgG chimeric protein and EphrinB2.
メデイウムAに8-brcAMP(0.5mM)添加し、上記のように胚性幹細胞より分化した内皮細胞の性状を検討した。得られた結果を図６に示す。メディウムA単独の培養条件でVPCから分化したPECAM-1陽性内皮細胞(図６a左、右)はEphB4-Fcキメラと結合しないEphrinB2陰性の内皮細胞であった(図６a)。AM10-6Mおよび8-br-cAMP0.5mM添加時にはPECAM-1陽性内皮細胞の多くはEphB4-Fcキメラとの結合性を有する(図６b, c中央、右)EphrinB2陽性内皮細胞であった(図６、PECAM-1(左)、EphB4-Fcキメラ(中央、右)、200倍)。図６は、メデイウムAに8-brcAMP(0.5mM)添加すると、メデイウムAのみに比べ、EphB4結合分子 (EphrinB2と考えられる)の有意の発現増加が認められることを示している(P<0.05)。AM10-6M添加にても同様の効果が認められた(P<0.05)。
これは、AMあるいは8-br-cAMPが胚性幹細胞由来の血管前駆細胞から動脈系内皮細胞を分化させたことを示している。
8-brcAMP (0.5 mM) was added to medium A, and the properties of endothelial cells differentiated from embryonic stem cells as described above were examined. The obtained result is shown in FIG. PECAM-1-positive endothelial cells differentiated from VPC under the culture conditions of medium A alone (FIG. 6a left, right) were EphrinB2-negative endothelial cells that did not bind to EphB4-Fc chimera (FIG. 6a). When AM10 -6 M and 8-br-cAMP 0.5 mM were added, most of the PECAM-1 positive endothelial cells had EphB4-Fc chimera binding properties (Fig. 6b, c middle, right) EphrinB2 positive endothelial cells ( FIG. 6, PECAM-1 (left), EphB4-Fc chimera (center, right), 200 times). FIG. 6 shows that when 8-brcAMP (0.5 mM) is added to medium A, a significant increase in the expression of an EphB4 binding molecule (considered EphrinB2) is observed compared to medium A alone (P <0.05). . A similar effect was observed when AM10 -6 M was added (P <0.05).
This indicates that AM or 8-br-cAMP differentiated arterial endothelial cells from vascular progenitor cells derived from embryonic stem cells.
実施例３
マウス胚性幹細胞におけるAMの発現はすでに本発明者らが開発したマウス抗AMモノクローナル抗体(Michibata H等、Kidney Int 53: 979-985 (1998))を用いて、本発明者らの既報の方法に準じて(Naruko T等、Circulation 94: 3103-3108 (1996))行った。
胚性幹細胞由来VPCより分化した血管細胞におけるAMおよびその受容体の発現を検討したところ、胚性幹細胞由来VPCより分化した壁細胞においてAMの発現を認め、一方、内皮細胞においてAM受容体の発現を認めた。
これは、胚性幹細胞由来の内皮細胞にAMが作用できること、および同時に分化する壁細胞から分泌されるAMが内皮細胞に作用する可能性を示している。
Example 3
The expression of AM in mouse embryonic stem cells was performed using the mouse anti-AM monoclonal antibody (Michibata H et al., Kidney Int 53: 979-985 (1998)) already developed by the present inventors. (Naruko T et al., Circulation 94: 3103-3108 (1996)).
We examined the expression of AM and its receptor in vascular cells differentiated from embryonic stem cell-derived VPC, and we found AM expression in mural cells differentiated from embryonic stem cell-derived VPC, while AM receptor expression in endothelial cells Admitted.
This indicates that AM can act on endothelial cells derived from embryonic stem cells, and that AM secreted from simultaneously differentiated mural cells can act on endothelial cells.
上記結果は明らかに、胚性幹細胞由来VPCにcAMPアナログおよびIBMX、またはブロスタン酸誘導体であるプロスタグランジンI2誘導体ベラプロスト又はその塩あるいは、AMを投与し、細胞内cAMP濃度を上昇させることで、内皮細胞の分化が飛躍的に進むことを示している。
結果はさらに上記添加剤により、血管新生において重要な動脈系の内皮細胞への分化増殖を促進されたことを示している。
これらのAMの胚性幹細胞への作用は、AM受容体の媒介を通して発現すると考えられる。従って、本発明は、胚性幹細胞を用いた血管再生医療の開発に新しい方法を提供する。つまり、胚性幹細胞を虚血部に移植することで生体内で血管を形成し局所の血流を回復させる際、胚性幹細胞由来移植内皮細胞の量的確保および質的な変化調整をめざす新規のセルプロセシング方法を提供する。
また、胚性幹細胞から内皮細胞ないし動脈系内皮細胞にいたるインビトロの分化培養系における分化過程の細胞群の遺伝子発現をDNAマイクロアレイ法などを用いて、網羅的に解析する方法も新規の血管再生誘導遺伝子の探索、血管再生医薬の開発に有用であると考えられる。
The above results clearly show that by administering cAMP analog and IBMX, or prostaglandin I2 derivative beraprost or its salt or AM, or AM to embryonic stem cell-derived VPC, and increasing intracellular cAMP concentration, endothelium It shows that the differentiation of cells progresses dramatically.
The results further indicate that the above-mentioned additive promoted the differentiation and proliferation into endothelial cells of arterial system important in angiogenesis.
These effects of AM on embryonic stem cells are thought to be expressed through AM receptor mediation. Therefore, the present invention provides a new method for the development of revascularization medicine using embryonic stem cells. In other words, when embryonic stem cells are transplanted into the ischemic region to form blood vessels in the body and restore local blood flow, a new aim is to secure quantitative and adjust qualitative changes in embryonic stem cell-derived transplanted endothelial cells A cell processing method is provided.
In addition, a novel method for inducing revascularization is also a method for exhaustively analyzing gene expression of cells in the differentiation process in in vitro differentiation culture system from embryonic stem cells to endothelial cells or arterial endothelial cells using DNA microarray method etc. It is thought that it is useful for the search of genes and the development of revascularization drugs.
Claims (30)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004025631A JP4895479B2 (en) | 2004-02-02 | 2004-02-02 | Endothelial cell differentiation and proliferation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004025631A JP4895479B2 (en) | 2004-02-02 | 2004-02-02 | Endothelial cell differentiation and proliferation method |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2009010927A Division JP5081172B2 (en) | 2009-01-21 | 2009-01-21 | Endothelial cell differentiation and proliferation method |
| JP2009010932A Division JP2009148273A (en) | 2009-01-21 | 2009-01-21 | Method for differentiating and proliferating endothelial cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2005211040A true JP2005211040A (en) | 2005-08-11 |
| JP4895479B2 JP4895479B2 (en) | 2012-03-14 |
Family
ID=34907958
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2004025631A Expired - Lifetime JP4895479B2 (en) | 2004-02-02 | 2004-02-02 | Endothelial cell differentiation and proliferation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4895479B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497874B (en) * | 2009-03-04 | 2012-05-23 | 中山大学中山眼科中心 | Method for promoting somatic cell proliferation |
| WO2014192925A1 (en) * | 2013-05-31 | 2014-12-04 | ｉＨｅａｒｔ Ｊａｐａｎ株式会社 | Efficient method of deriving endothelial cells |
-
2004
- 2004-02-02 JP JP2004025631A patent/JP4895479B2/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497874B (en) * | 2009-03-04 | 2012-05-23 | 中山大学中山眼科中心 | Method for promoting somatic cell proliferation |
| WO2014192925A1 (en) * | 2013-05-31 | 2014-12-04 | ｉＨｅａｒｔ Ｊａｐａｎ株式会社 | Efficient method of deriving endothelial cells |
| JP5842289B2 (en) * | 2013-05-31 | 2016-01-13 | ｉＨｅａｒｔ Ｊａｐａｎ株式会社 | Efficient method for inducing endothelial cells |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4895479B2 (en) | 2012-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nicosia | The aortic ring model of angiogenesis: a quarter century of search and discovery | |
| KR101730052B1 (en) | Pluripotent stem cell that induces repair and regeneration after myocardial infarction | |
| JP6691921B2 (en) | Generation of arterial endothelial cell population | |
| JP7097814B2 (en) | Genetic markers for engraftment of human ventricular progenitor cells | |
| JP5588587B2 (en) | Human liver progenitor cells and methods of use thereof | |
| US20050003529A1 (en) | Stem cells and method of separating the same | |
| JP7275109B2 (en) | Use of Neuropilin-1 (NRP1) as a Cell Surface Marker to Isolate Human Ventricular Progenitor Cells | |
| EP3183337B1 (en) | Use of jagged 1/frizzled 4 as a cell surface marker for isolating human cardiac ventricular progenitor cells | |
| CN108350423A (en) | Method for obtaining mankind's brown/cream-coloured adipocyte | |
| JPH08510451A (en) | Prevention and treatment of pathogenesis associated with hyperproliferative smooth muscle cells | |
| KR20050000398A (en) | Methods for identification of modulators of angiogenesis, compounds discovered thereby, and methods of treatment using the compounds | |
| US10596200B2 (en) | Use of LIFR or FGFR3 as a cell surface marker for isolating human cardiac ventricular progenitor cells | |
| JP2007143554A (en) | Method and reagent for cell transplantation | |
| BRPI0620523A2 (en) | method of increasing implant potential and cell attraction, method of preparing cells for transplantation in a subject, cell population and pharmaceutical composition | |
| KR20140137444A (en) | Isolation and use of human lymphoid organ-derived suppressive stromal cells | |
| JP4895479B2 (en) | Endothelial cell differentiation and proliferation method | |
| JP5081172B2 (en) | Endothelial cell differentiation and proliferation method | |
| WO2019144971A1 (en) | Icam-1 marker and application thereof | |
| JP2009148273A (en) | Method for differentiating and proliferating endothelial cell | |
| JPWO2005116194A1 (en) | Myoblast or myoblast-like cell culture method for in vitro myofiber formation | |
| CN113846052A (en) | Application of endothelial cells and precursor cells thereof in treating demyelinating diseases | |
| JP2006034199A (en) | Method for separation of myocardial precursor cell and separation device | |
| Steinthal | Exploring the role of CD248/endosialin/TEM-1 on lymphoid stromal cells in secondary lymphoid organs | |
| Gupte | Pluripotent Hematopoietic Stem Cells Augment α-Adrenergic Receptor-Mediated 1 Contraction of Pulmonary Artery and Contribute to the Pathogenesis of Pulmonary 2 Hypertension 3 | |
| Cunningham | Focal adhesions as mechanotransducers: Differential localization of focal adhesion components in response to mechanical strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20070111 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080624 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20080822 |
|
| RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20080911 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20081007 |
|
| AA91 | Notification that invitation to amend document was cancelled |
Free format text: JAPANESE INTERMEDIATE CODE: A971091 Effective date: 20081111 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20081125 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20090121 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20090204 |
|
| A912 | Re-examination (zenchi) completed and case transferred to appeal board |
Free format text: JAPANESE INTERMEDIATE CODE: A912 Effective date: 20090717 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20111115 |
|
| 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: 20111220 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 4895479 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20150106 Year of fee payment: 3 |
|
| 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 |
|
| 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 |
|
| EXPY | Cancellation because of completion of term |