WO2024071382A1 - Pluripotential stem cell and cell differentiated and induced from stem cell having glp-1 secretion function - Google Patents
Pluripotential stem cell and cell differentiated and induced from stem cell having glp-1 secretion function Download PDFInfo
- Publication number
- WO2024071382A1 WO2024071382A1 PCT/JP2023/035640 JP2023035640W WO2024071382A1 WO 2024071382 A1 WO2024071382 A1 WO 2024071382A1 JP 2023035640 W JP2023035640 W JP 2023035640W WO 2024071382 A1 WO2024071382 A1 WO 2024071382A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- glp
- cell
- induced
- stem cells
- Prior art date
Links
- 210000004027 cell Anatomy 0.000 title claims abstract description 267
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 31
- 230000028327 secretion Effects 0.000 title abstract description 30
- 101100337060 Caenorhabditis elegans glp-1 gene Proteins 0.000 title description 2
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims abstract description 106
- 210000001778 pluripotent stem cell Anatomy 0.000 claims abstract description 43
- 210000002889 endothelial cell Anatomy 0.000 claims abstract description 25
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 7
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 claims abstract 9
- 102100025101 GATA-type zinc finger protein 1 Human genes 0.000 claims abstract 9
- 238000000034 method Methods 0.000 claims description 47
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 150000007523 nucleic acids Chemical class 0.000 claims description 29
- 239000013604 expression vector Substances 0.000 claims description 28
- 108020004707 nucleic acids Proteins 0.000 claims description 28
- 102000039446 nucleic acids Human genes 0.000 claims description 28
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 239000008194 pharmaceutical composition Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 10
- 210000000399 corneal endothelial cell Anatomy 0.000 claims description 9
- 238000010362 genome editing Methods 0.000 claims description 8
- 206010019280 Heart failures Diseases 0.000 claims description 3
- 208000008589 Obesity Diseases 0.000 claims description 3
- 206010035664 Pneumonia Diseases 0.000 claims description 3
- 208000025371 Taste disease Diseases 0.000 claims description 3
- 208000015114 central nervous system disease Diseases 0.000 claims description 3
- 208000019423 liver disease Diseases 0.000 claims description 3
- 230000005976 liver dysfunction Effects 0.000 claims description 3
- 208000031225 myocardial ischemia Diseases 0.000 claims description 3
- 235000020824 obesity Nutrition 0.000 claims description 3
- 208000027232 peripheral nervous system disease Diseases 0.000 claims description 3
- 230000008085 renal dysfunction Effects 0.000 claims description 3
- 235000019669 taste disorders Nutrition 0.000 claims description 3
- 238000002659 cell therapy Methods 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 210000000871 endothelium corneal Anatomy 0.000 abstract 1
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 98
- 102100040918 Pro-glucagon Human genes 0.000 description 98
- 239000005090 green fluorescent protein Substances 0.000 description 49
- 239000002609 medium Substances 0.000 description 37
- 230000014509 gene expression Effects 0.000 description 35
- 239000013598 vector Substances 0.000 description 35
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 32
- 239000012228 culture supernatant Substances 0.000 description 27
- 239000013612 plasmid Substances 0.000 description 26
- 239000013603 viral vector Substances 0.000 description 24
- 230000003511 endothelial effect Effects 0.000 description 22
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 17
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 17
- 230000004069 differentiation Effects 0.000 description 16
- 230000003248 secreting effect Effects 0.000 description 15
- 241000702421 Dependoparvovirus Species 0.000 description 12
- 230000004927 fusion Effects 0.000 description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 239000008103 glucose Substances 0.000 description 11
- 230000006698 induction Effects 0.000 description 11
- 208000015181 infectious disease Diseases 0.000 description 11
- 238000012546 transfer Methods 0.000 description 10
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 238000004113 cell culture Methods 0.000 description 9
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 102000000905 Cadherin Human genes 0.000 description 6
- 108050007957 Cadherin Proteins 0.000 description 6
- 101000595669 Homo sapiens Pituitary homeobox 2 Proteins 0.000 description 6
- 108050000637 N-cadherin Proteins 0.000 description 6
- 102100036090 Pituitary homeobox 2 Human genes 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000012744 immunostaining Methods 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 210000004379 membrane Anatomy 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 239000013600 plasmid vector Substances 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 210000001082 somatic cell Anatomy 0.000 description 6
- 238000011579 SCID mouse model Methods 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 102000034287 fluorescent proteins Human genes 0.000 description 5
- 108091006047 fluorescent proteins Proteins 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 230000009385 viral infection Effects 0.000 description 5
- 101800004266 Glucagon-like peptide 1(7-37) Proteins 0.000 description 4
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000288906 Primates Species 0.000 description 4
- 102000040739 Secretory proteins Human genes 0.000 description 4
- 108091058545 Secretory proteins Proteins 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000003501 co-culture Methods 0.000 description 4
- 238000012790 confirmation Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 230000003914 insulin secretion Effects 0.000 description 4
- 238000001638 lipofection Methods 0.000 description 4
- 239000007758 minimum essential medium Substances 0.000 description 4
- 230000008672 reprogramming Effects 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000012096 transfection reagent Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 3
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- -1 i.e. Proteins 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000001578 tight junction Anatomy 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 2
- 101100239628 Danio rerio myca gene Proteins 0.000 description 2
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 2
- 102100030801 Elongation factor 1-alpha 1 Human genes 0.000 description 2
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 102000007446 Glucagon-Like Peptide-1 Receptor Human genes 0.000 description 2
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 2
- 102400000324 Glucagon-like peptide 1(7-37) Human genes 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 101000920078 Homo sapiens Elongation factor 1-alpha 1 Proteins 0.000 description 2
- 101000615613 Homo sapiens Mineralocorticoid receptor Proteins 0.000 description 2
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 2
- 241000283953 Lagomorpha Species 0.000 description 2
- 101150039798 MYC gene Proteins 0.000 description 2
- 102100021316 Mineralocorticoid receptor Human genes 0.000 description 2
- 101000716728 Mus musculus Kit ligand Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 2
- 101150086694 SLC22A3 gene Proteins 0.000 description 2
- 108010008125 Tenascin Proteins 0.000 description 2
- 102100038126 Tenascin Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 101100459258 Xenopus laevis myc-a gene Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000017455 cell-cell adhesion Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 210000001368 germline stem cell Anatomy 0.000 description 2
- 230000002641 glycemic effect Effects 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000007410 oral glucose tolerance test Methods 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 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
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 206010010996 Corneal degeneration Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 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 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 101150099612 Esrrb gene Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 241000282323 Felidae Species 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 229940089838 Glucagon-like peptide 1 receptor agonist Drugs 0.000 description 1
- 241000175212 Herpesvirales Species 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101500028774 Homo sapiens Glucagon-like peptide 1 Proteins 0.000 description 1
- 101000701334 Homo sapiens Sodium/potassium-transporting ATPase subunit alpha-1 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010021518 Impaired gastric emptying Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 241000288903 Lemuridae Species 0.000 description 1
- 241000288986 Lorisidae Species 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 description 1
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 241000288754 Scandentia Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102100030458 Sodium/potassium-transporting ATPase subunit alpha-1 Human genes 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000168914 Strepsirrhini Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000004781 bullous keratopathy Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000030414 genetic transfer Effects 0.000 description 1
- 101150102822 glp-1 gene Proteins 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000003897 hepatic stem cell Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 210000004966 intestinal stem cell Anatomy 0.000 description 1
- 101150108076 lin28a gene Proteins 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 235000011475 lollipops Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 210000001665 muscle stem cell Anatomy 0.000 description 1
- 229940028444 muse Drugs 0.000 description 1
- 230000003880 negative regulation of appetite Effects 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108010056030 retronectin Proteins 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the present invention relates to pluripotent stem cells having the function of secreting GLP-1 (Glucagon-like peptide-1) and cells induced to differentiate from the stem cells.
- the present invention also relates to endothelial cells induced to differentiate and have the function of secreting GLP-1.
- CECSi cells Corneal endothelial cell substitute from iPS cells
- Patent Documents 1 to 3 Corneal endothelial cell substitute from iPS cells
- CECSi cells have the advantage that they can be used to mass-produce differentiated endothelial cells with uniform quality and high adhesiveness to cells and extracellular matrix from iPS cells in a short period of time (approximately two weeks). If it is possible to give additional functions to CECSi cells by gene editing or gene introduction while taking advantage of this characteristic, it is possible to aim to expand the application to other disease areas as well as the treatment of the cornea region.
- Gene introduction techniques are broadly divided into non-viral introduction methods and introduction methods using viruses, and in gene therapy, gene introduction techniques using viruses are used, and many viral vectors have been developed.
- Patent Document 4 describes a transfected cell that expresses glucagon-like peptide-1 (GLP-1).
- GLP-1 is a hormone secreted from L cells in the small intestine, which acts on the GLP-1 receptor present in the ⁇ cells of the pancreas to promote insulin secretion.
- GLP-1 receptor agonists are actually used to treat diabetes.
- the objective of the present invention is to provide pluripotent stem cells to which new functions have been imparted, and cells induced to differentiate from stem cells.
- the objective of the present invention is to provide corneal endothelial replacement cells to which new functions have been imparted, which are induced from pluripotent stem cells, particularly iPS cells.
- the inventors attempted to impart GLP-1 secretion function to the corneal endothelial replacement cells that they had been developing. They induced the differentiation of iPS cells into corneal endothelial replacement cells, introduced the GLP-1 gene into the resulting corneal endothelial replacement cells, expressed the gene, and confirmed that the GLP-1 protein was secreted, thus completing the present invention.
- a pluripotent stem cell or a cell induced to differentiate from a stem cell, which has a GLP-1 secretion function [2] The cell described in [1] above, wherein the cell induced to differentiate from a stem cell is an endothelial cell. [3] The cell described in [1] or [2] above, wherein the pluripotent stem cell or stem cell is an iPS cell. [4] The cell described in [2] above, wherein the endothelial cell is a corneal endothelial cell. [5] The cell according to any one of [1] to [4] above, which expresses GLP-1. [A] CECSi cells having GLP-1 secretion function.
- a method for producing a cell expressing GLP-1 comprising the step of introducing a nucleic acid encoding GLP-1 into a cell, wherein the cell is a pluripotent stem cell or a cell induced to differentiate from a stem cell.
- [11] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid; (2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
- a method for producing a cell expressing GLP-1 comprising: [12] The method according to [11] above, wherein the cells induced to differentiate from stem cells are endothelial cells. [13] The method according to [11] or [12] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
- a method for producing a CECSi cell expressing GLP-1 comprising: [15] A pharmaceutical composition comprising the cell according to any one of [1] to [5] and [A] above. [16] The pharmaceutical composition according to [15], which is for treating diabetes, obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction, taste disorders, and pneumonia.
- the present invention has made it possible to endow CECSi cells with the ability to secrete GLP-1.
- CECSi cells that have the ability to secrete GLP-1 for a long period of time will enable the development of new cell therapies for diabetes and other conditions.
- FIG. 1 shows that iPS cells were induced to differentiate into CECSi cells by immunostaining for PITX2, N-Cadherin, and ZO-1.
- This figure shows the results of confirming GLP-1 expression in CECSi cells 48 hours after transfection with each plasmid (sig-peptide, sig-GLP-1), showing the image observed by a fluorescent microscope (A) and the amount of EGFP (GFP) in the cell culture supernatant (B).
- This figure shows the results of confirming GLP-1 expression in CECSi cells 65 hours after transfection with each plasmid (sig-peptide, sig-GLP-1), showing the images observed by a fluorescence microscope (A) and the amount of EGFP (GFP) in the cell culture supernatant (B).
- A fluorescence microscope
- GFP EGFP
- B cell culture supernatant
- n 2, mean ⁇ standard error
- This is a diagram showing the results of confirming GLP-1 expression in CECSi cells 5 days after transfection with each plasmid (sig-peptide, sig-GLP-1). Fluorescence microscopy images (A) and intracellular mRNA expression levels (B) are shown. n 2, mean ⁇ standard error
- the construction diagram of various plasmids (sig-peptide (a), sig-GLP-1 (b), sig-GFP (c), sig-GLP-1-GFP (d)) is shown.
- IRES was used instead of T2A.
- This figure shows the results of confirming GLP-1 expression in CECSi cells 19 hours after transfection with each plasmid (sig-peptide (a), sig-GLP-1 (b), sig-GFP (c), sig-GLP-1-GFP (d)). Detection was performed by Western blotting using an anti-GFP antibody. Western blot images are shown. 1 shows the results of confirming GLP-1 expression in CECSi cells 13 days after infection with AAV containing each plasmid (sig-GFP, sig-GLP-1-GFP), with (A) images observed by a fluorescence microscope and (B) amounts of EGFP (GFP) in the cell culture supernatant.
- This figure shows the results of confirming GLP-1 expression in CECSi cells 48 hours after infection with AAV containing each plasmid (sig-peptide, sig-GLP-1), showing the image observed by a fluorescence microscope (A) and the amount of EGFP (GFP) in the cell culture supernatant (B).
- A fluorescence microscope
- GFP EGFP
- B cell culture supernatant
- 1 shows the results of confirming GLP-1 expression in CECSi cells 32 days after infection with AAV containing each plasmid (sig-peptide, sig-GLP-1).
- 1 shows the results of co-culturing sig-GLP-1-GFP (fusion type)-secreting CECSi cells and human iPS-derived pancreatic ⁇ cells to examine whether or not c-peptide secretion is promoted.
- FIG. 1 shows the results of co-culturing sig-GLP-1-GFP (fusion type)-secreting CECSi cells with human iPS-derived pancreatic ⁇ cells to verify the presence or absence of c-peptide secretion promotion.
- concentration (ng/ml) of c-peptide secreted from pancreatic ⁇ cells in the medium is shown.
- n 2, mean ⁇ standard error
- This figure shows the results of examining the effect on blood glucose control when sig-GLP-1-GFP (fusion type)-introduced CECSi cells were transplanted intraperitoneally into healthy SCID mice.
- 1 is a graph showing the results of examining the effect on blood glucose control when sig-GLP-1-GFP (fusion type)-introduced CECSi cells are intraperitoneally transplanted into healthy SCID mice.
- 2 is a graph showing the results of measuring the amount of sig-GLP-1-GFP in the blood at the start of OGTT and 0.5 hours after the start.
- GLP-1 secretion function refers to a function capable of producing GLP-1 in cells and then secreting it outside the cells.
- GLP-1 refers to active human glucagon-like peptide-1, i.e., GLP-1 having the amino acid sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 1], GLP-1 (7-37) having the amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 2], which is its active form, or a variant thereof.
- GLP-1 refers to an amino acid sequence in which one or more amino acids have been changed from the native sequence by substitution, deletion or insertion of one or more amino acid residues without changing the activity of GLP-1.
- a "GLP-1 variant” may have one or more deleted amino acid residues, or may be a fragment of GLP-1 shorter than GLP-1(7-37) that has the same activity as the native GLP-1 sequence, for example, the activity of promoting insulin secretion by pancreatic ⁇ cells, by having an amino acid residue important for GLP-1 activity or a residue that can be substituted for one or more of the important amino acid residues.
- GLP-1 is a concept that includes all of these.
- one or more amino acid residues may be 2, 3, 4, or 5 amino acid residues.
- One or more amino acid residues is preferably 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid residue.
- the amount is not limited and differs depending on the type of biological action serving as an indicator of GLP-1 secretion. It is sufficient if there is a significant difference between the case where the function is exerted and the case where it is not exerted.
- a preferred indicator of GLP-1 secretory function is its insulin secretion promoting effect.
- the present invention provides cells having a GLP-1 secretion function (hereinafter, also simply referred to as the cells of the present invention).
- the cells include pluripotent stem cells and cells obtained by inducing differentiation from the pluripotent stem cells or stem cells (hereinafter, also simply referred to as differentiated cells), in particular endothelial cells such as corneal endothelial replacement cells.
- Stem cells are cells that have the ability to replicate themselves and differentiate into multiple other cell lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and skin stem cells.
- ES cells embryonic stem cells
- iPS cells induced pluripotent stem cells
- neural stem cells hematopoietic stem cells
- mesenchymal stem cells mesenchymal stem cells
- pancreatic stem cells pancreatic stem cells
- muscle stem cells germline stem cells
- intestinal stem cells intestinal stem cells
- cancer stem cells hair follicle stem cells
- hair follicle stem cells and skin stem cells.
- Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc.
- pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells).
- Muse cells Multi-lineage differentiating Stress Enduring cells obtained from mesenchymal stem cells (MSCs), and GS cells produced from germ cells (e.g. testes) are also included in pluripotent stem cells. When simply referring to stem cells, it is intended to include pluripotent stem cells.
- ES cells can be produced by culturing the inner cell population on feeder cells or in medium containing leukemia inhibitory factor (LIF). They are also available from designated institutions and can be purchased commercially.
- LIF leukemia inhibitory factor
- ntES cells nuclear transfer ES cells
- a type of ES cell can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.
- EG cells can be produced by culturing primordial germ cells in a medium containing mouse stem cell factor (mSCF), LIF, and basic fibroblast growth factor (bFGF) (Cell, 70:841-847, 1992).
- mSCF mouse stem cell factor
- LIF mouse stem cell factor
- bFGF basic fibroblast growth factor
- iPS cells are cells in which pluripotency has been induced by reprogramming somatic cells using known methods.
- Specific examples of iPS cells include cells in which pluripotency has been induced by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., through the expression of multiple genes selected from a group of reprogramming genes including Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc.
- Yamanaka et al. established induced pluripotent stem cells from mouse cells (Cell, 2006, 126(4) pp.663-676).
- induced pluripotent stem cells were established from human fibroblasts, and like embryonic stem cells, they have pluripotency and the ability to self-replicate (Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106).
- induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp.651-654).
- Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells, T cells, etc.), liver cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.
- tissue-derived fibroblasts e.g., peripheral blood mononuclear cells, T cells, etc.
- blood cells e.g., peripheral blood mononuclear cells, T cells, etc.
- liver cells e.g., pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.
- the means for expressing the genes is not particularly limited.
- means for expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, and electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, and electroporation method), and direct protein injection methods.
- viral vectors e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors
- gene transfer methods using plasmid vectors e.g., plasmid vectors, episomal vectors
- RNA vectors e
- the iPS cells include 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 836B3, FF-I14s03, FF-I01s04, MH09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs09, QHJI14s03, QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs 14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all from iPS Academia Japan, Inc., or Kyoto University iPS Research Foundation), Tic (JCRB1331 strain), Dot
- “Mammals” includes rodents, ungulates, felines, lagomorphs, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Felidae include dogs and cats. Lagomorphs include rabbits, etc. "Primates” refers to mammals belonging to the order Primates, and includes prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans.
- the pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, and most preferably human pluripotent stem cells.
- rodent e.g., mouse, rat
- primate e.g., human, monkey
- the differentiated cells used in the present invention include those induced to differentiate from the above stem cells, preferably the above pluripotent stem cells.
- endothelial cells preferably corneal endothelial cells, more preferably corneal endothelial-like cells developed by the present inventors, so-called corneal endothelial substitute cells
- they are corneal endothelial cell substitute from iPS cells (CECSi cells), which have corneal endothelial cell-like properties and functions and are characterized by enhanced expression of the NR3C2 (nuclear receptor subfamily 3, group C, member 2) gene (Patent Document 3).
- a method for producing corneal endothelial replacement cells is as follows.
- the iPS cells are cultured for one week in a culture dish coated with iMatrix-511 (0.6 ⁇ g/cm 2 ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells are seeded again on a culture dish coated with iMatrix-511 (0.3 ⁇ g/cm 2 ), and differentiation induction culture of the iPS cells into corneal endothelial substitute cells is performed for 8 to 14 days using the differentiation induction medium described below (Table 1). Note that when using cryopreserved iPS cells, differentiation induction is performed after two passages and culture.
- corneal endothelial cell-like properties and functions possessed by the corneal endothelial substitute cells include the following characteristics (i) to (iv), and among these characteristics, the cells have at least one, preferably two, more preferably three, and even more preferably all four.
- Cell-cell adhesion is composed of N-cadherin.
- Tight junctions are formed between the cells.
- Expression of the transcription factor PITX2 is observed in the cell nucleus. Whether or not the cell-cell adhesion is composed of N-Cadherin can be confirmed by immunostaining for N-Cadherin.
- Whether or not tight junctions are formed between cells can be confirmed by observing the presence of ZO-1, a protein that constitutes tight junctions, using immunostaining for ZO-1, or by directly observing the structure using an electron microscope. Whether or not Na,K-ATPase ⁇ 1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining of ZO-1 and Na,K-ATPase ⁇ 1 subunit by immunostaining. Whether or not the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining for PITX2.
- the cells of the present invention are the above-mentioned pluripotent stem cells or their differentiated cells, particularly endothelial cells, to which a GLP-1 secretion function has been imparted.
- the process of imparting a GLP-1 secretion function to cells will be described in detail in "2. Cell manufacturing method” below, but specifically, this is carried out by introducing a nucleic acid encoding GLP-1 into the above-mentioned pluripotent stem cells or their differentiated cells, particularly endothelial cells. Therefore, the cells of the present invention are cells that express GLP-1.
- a signal peptide is attached to the GLP-1 expressed in the cells. Therefore, it is preferable that the nucleic acid encoding GLP-1 introduced into the cells in the present invention is linked to a signal sequence.
- the present invention provides a method for producing cells having a GLP-1 secretion function (hereinafter, also simply referred to as the method for producing the cells of the present invention).
- the method for producing cells of the present invention is characterized in that it imparts a GLP-1 secretion function to cells.
- cells include pluripotent stem cells and their differentiated cells, particularly endothelial cells such as corneal endothelial replacement cells.
- the GLP-1 secretion function may be imparted to cells at any stage.
- the GLP-1 secretion function may be imparted to pluripotent stem cells, or to differentiated cells, particularly endothelial cells, obtained by inducing differentiation of pluripotent stem cells.
- Examples of endothelial cells include CECSi cells, which are corneal endothelial replacement cells developed by the present inventors and described above in the section "1. Cells”.
- a nucleic acid encoding GLP-1 preferably a nucleic acid linked to a signal sequence
- an appropriate expression vector two types of expression vectors may be used if necessary.
- this expression vector is introduced into cells to express GLP-1.
- One embodiment of the method for producing the cells of the present invention is the following method. (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid; (2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
- a method for producing a cell expressing GLP-1 comprising:
- the vector can be a viral vector or a non-viral vector.
- viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors.
- retroviral vectors, lentiviral vectors, and adeno-associated viral vectors the target gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected.
- Each viral vector can be prepared according to a conventional method or using a commercially available dedicated kit.
- non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, and artificial chromosome vectors. These vectors can also be used in gene therapy, preferably using adeno-associated virus vectors.
- the expression vector is introduced into a cell by infection with a virus.
- a conventional method such as electroporation, lipofection, calcium phosphate, or nucleofection can be used for introduction into a cell, and the lipofection method is preferred.
- GLP-1 The ability to secrete GLP-1 may be imparted to cells by genome editing.
- Gene editing is a technique for intentionally modifying a target gene or genome region by site-specific cleavage of a genomic DNA strand using a nuclease, or by chemical conversion of bases.
- site-specific nucleases include zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas9.
- ZFNs zinc finger nucleases
- TALENs TALENs
- CRISPR/Cas9 CRISPR/Cas9.
- the nucleic acid encoding GLP-1 that is introduced to impart GLP-1 secretion function to cells is not particularly limited as long as it can cause the cells to express the desired protein (i.e., GLP-1).
- Examples of the base sequence of the nucleic acid to be introduced include the following.
- GLP-1(7-37) CATGCTGAAGGGACCTTTACCAGTGATGGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 3)
- GLP-1 (7-37) (linked to a sequence encoding a signal peptide on the 5' side) TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCGCATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 4)
- the cell culture in the subsequent expansion culture is preferably carried out in a medium for culturing pluripotent stem cells.
- a medium for pluripotent stem cells a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of pluripotent stem cells.
- DMEM, DMEMHG, EMEM, IMDM Iscove's Modified Dulbecco's Medium
- GMEM Gasgow's MEM
- RPMI-1640 ⁇ -MEM
- the medium include BMOC-3 Medium, E8 medium (Nature Methods, 2011, 8, 424-429), ReproFF2 medium (ReproCell), StemFit (registered trademark) AK medium (Ajinomoto), and a
- the thus obtained pluripotent stem cells having the function of secreting GLP-1 can be induced to differentiate into desired differentiated cells. Differentiation induction is appropriately determined depending on the type of differentiated cells to be obtained, and is carried out using known materials and methods.
- corneal endothelial replacement cells which are corneal endothelial-like cells, can be obtained by inducing differentiation of pluripotent stem cells having the function of secreting GLP-1 according to the descriptions in Patent Documents 1 to 3.
- the obtained corneal endothelial replacement cells have the function of secreting GLP-1.
- the cell culture in the subsequent expansion culture is preferably performed in a medium for culturing differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells.
- a medium a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of differentiated cells.
- DMEM, DMEMHG, EMEM, IMDM Iscove's Modified Dulbecco's Medium
- GMEM Gasgow's MEM
- RPMI-1640 ⁇ -MEM
- the medium include medium and mixed mediums thereof.
- the differentiation of pluripotent stem cells into differentiated cells can be carried out by known methods.
- the differentiation of pluripotent stem cells into corneal endothelial replacement cells can be carried
- composition of the present invention provides a pharmaceutical composition comprising cells capable of secreting GLP-1 as an active ingredient (hereinafter, also simply referred to as the pharmaceutical composition of the present invention).
- the cells capable of secreting GLP-1 contained as an active ingredient in the pharmaceutical composition of the present invention are the cells described above in “1. Cells” and are cells produced by the method described above in “2. Method for producing cells”.
- compositions of the present invention can be prepared by mixing the cells of the present invention, which are the active ingredient, with a pharma- ceutical acceptable carrier.
- pharmaceutical-ceutical acceptable carrier includes diluents, adjuvants, excipients, stabilizers, vehicles, or supports (such as cell fiber (alginate hydrogel)) that are non-toxic to cells exposed thereto at the doses and concentrations used.
- the carrier is an aqueous pH buffer solution, an antioxidant, a low molecular weight (less than about 10 residues) polypeptide, a hydrophilic polymer, an amino acid, a monosaccharide, a disaccharide, a chelating agent such as EDTA, a salt-forming counterion such as sodium; and a non-ionic surfactant such as TWEEN (registered trademark), polyethylene glycol (PEG), and PLURONICS (registered trademark).
- a preferred carrier is a saline solution.
- the pharmaceutical composition of the present invention contains the cells of the present invention as an active ingredient, and the cells of the present invention are capable of secreting a therapeutically effective amount of GLP-1.
- a therapeutically effective amount is an amount that, when the pharmaceutical composition of the present invention is administered to a subject, can provide a therapeutic effect against the above-mentioned diseases, compared to a subject not administered the pharmaceutical composition.
- a specific therapeutically effective amount is appropriately determined depending on the administration method, the purpose of use, and the age, weight, symptoms, etc. of the subject.
- GLP-1 can be secreted in the human body using cells having GLP-1 secretion function.
- the secreted GLP-1 acts on the GLP-1 receptor present in the ⁇ cells of the pancreas, promoting the secretion of insulin.
- Diseases to which the pharmaceutical composition of the present invention can be applied include various diseases for which GLP-1 is known to be useful for the prevention and treatment of, and specific examples include, but are not limited to, diabetes (type 1 diabetes, type 2 diabetes), obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction (non-alcoholic liver disease (NASH), etc.), taste disorders, pneumonia, etc.
- Experimental Example 1 Construction of an expression vector for GLP-1 A vector for mammals, pRP, was constructed using the EF1A promoter, and was added with a signal sequence (TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCG) (SEQ ID NO: 5) derived from IL2 with the start codon ATG added, and a sequence of GLP-1 (CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA) (SEQ ID NO: 6).
- EGFP Enhanced Green Fluorescent Protein
- T2A linker By linking to EGFP (Enhanced Green Fluorescent Protein) with a T2A linker, EGFP remains in the cell during translation, and the target protein (GLP-1) is secreted into the culture supernatant (sig-GLP-1, FIG. 6A(b)).
- GLP-1 target protein
- the expression of EGFP is considered to be equivalent to the state in which GLP-1 is expressed.
- the cleavage efficiency of the T2A linker is not 100%, some of the fusion protein is secreted into the culture supernatant. Therefore, it is possible to confirm that GLP-1 is expressed in the cells by detecting GFP in the culture supernatant.
- a structure in which only the signal peptide is secreted was prepared (sig-peptide, FIG.
- an EGFP-fused signal sequence-added GLP-1 was also constructed using 3xGGGGS (SEQ ID NO: 11) instead of the T2A linker (sig-GLP-1-GFP, FIG. 6A(d)).
- sig-GLP-1-GFP T2A linker
- FIG. 6A(c) a sequence in which the signal sequence and EGFP were directly linked was used as a control.
- the EF1A promoter was used as in the plasmid vector, and the target vector was constructed using IRES instead of the T2A linker (sig-GLP-1, FIG. 6A(b)).
- the fusion protein type was constructed using the same sequence as in the plasmid vector (EGFP fusion signal sequence-added GLP-1 using 3xGGGGS instead of the T2A linker) in the pAAV viral vector (sig-GLP-1-GFP, FIG. 6A(d)).
- Experimental Example 2 Genetic transfer of GLP-1 expression plasmid into iPS cells [0223] 2 ⁇ g of each of the plasmids prepared in Experimental Example 1 was transfected into iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) using Lipofectamine TM Stem Transfection Reagent (Thermo Fisher STEM00001), and the EGFP fluorescence after 24 hours was observed using a fluorescence microscope (KEYENCE BZ-X810, magnification x40, exposure time 6 sec). By introducing the plasmid into iPS cells, expression of EGFP was confirmed in the cells.
- ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026) Lipofectamine TM Stem Transfection Reagent (Thermo
- the cells were seeded again on a culture dish coated with iMatrix-511 (0.3 ⁇ g/cm 2 ) and differentiation induction culture of the iPS cells into corneal endothelial substitute cells was performed for 14 days using the differentiation induction medium in Table 1, and the cells were then frozen.
- the frozen cells were thawed on the 14th day after the induction of differentiation, and the state was observed under a fluorescent microscope on the 3rd day after the start of adhesion culture to confirm that the cells had been induced to differentiate into corneal endothelial replacement cells (CECSi cells), and the cells were then subjected to the subsequent experiments.
- the CECSi cells obtained by induction from the iPS cells purchased from ATCC may be referred to as ATCC CECSI cells.
- the confirmation of induction of differentiation into CECSi cells was carried out by fluorescent immunostaining of ZO-1, N-Cadherin, and PITX2.
- the culture supernatant was removed, and the wells were washed twice with 500 ⁇ l of 1xDPBS(-).
- the 1xDPBS(-) was removed, 500 ⁇ l of 4% PFA was added, and the wells were left to stand at room temperature for 15 minutes.
- the 4% PFA was removed from the wells, and the wells were washed twice with fresh 1xDPBS(-), and blocked with antibody diluent (0.1% Triton/1% donkey serum/1xDPBS(-)) for 30 minutes or more.
- the blocked cells were reacted with primary antibodies at room temperature for 12 hours. Each primary antibody was diluted with antibody diluent; ZO-1 (500-fold dilution), N-Cadherin (100-fold dilution), and PITX2 (200-fold dilution). After the reaction, the cells were rinsed once with 1xDPBS(-), and then washed twice by adding 1xDPBS(-) and leaving the wells to stand for 5 minutes.
- Each secondary antibody was diluted with antibody diluent, and DAPI (1000-fold dilution) was added to prepare secondary antibody solutions; Alexa488-Rabbit IgG (150-fold dilution), Cy3-mouse IgG (200-fold dilution).
- the secondary antibody solutions were reacted with the cells and left to stand for 1.5 hours. After rinsing once with 1xDPBS(-), 1xDPBS(-) was added and left to stand for 5 minutes, this washing was repeated twice, and after removing 1xDPBS(-), the cell surface was covered with a mounting agent and observed under a fluorescent microscope ( Figure 1).
- Figure 2A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 2B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 48 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.
- Figure 3A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 3B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 65 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.
- 3-3 Confirmation of gene expression 24 hours or 5 days after plasmid introduction
- ATCC CECSi cells were used, and 2 ⁇ g of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6(b))) prepared in Experimental Example 1 was introduced using Lipofectamine TM 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 24 hours or 5 days was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 9 sec).
- mRNA was extracted from the cells using RNeasy Plus Mini kit (Qiagen 74134), and 0.5 ⁇ g of mRNA was reverse transcribed with RevaTraAce reverse transcriptase (Takara TRT-101) to prepare cDNA.
- the following qPCR primers were used.
- Experimental Example (Example) 4 Detection of GLP-1 in cell culture supernatant
- 3-1 ATCC CECSi cells were used, and 2 ⁇ g of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b)), sig-GFP (FIG. 6A(c)), sig-GLP-1-GFP (FIG. 6A(d))) prepared in Experimental Example 1 was introduced, and after 19 hours, the GLP-1 protein (GFP fusion type) secreted into the culture supernatant was detected by Western blotting.
- GLP-1 protein GLP-1 protein
- the membrane after transfer was subjected to blocking treatment with Blocking One (Nacalai Tesque 03953-66) at room temperature for 2 hours, and then the primary antibody Anti GFP (Green Fluorescent Protein) pAb (MBL 598) was diluted 1000 times and reacted overnight at 4 ° C. After washing the membrane with 1x TBS / 0.05% Tween buffer, the membrane was reacted with the secondary antibody HRP-linked IgG (CST # 7074S) diluted 2000 times for 1 hour. After the reaction, the membrane was washed with 1x TBS/0.05% Tween buffer and subjected to ELC reaction using Nacalai Chemi-lumi One Ultra (Nacalai Tesque 11644).
- the chemiluminescence of the membrane was detected using iBright FL1000 Imaging Systems. The results are shown in Figure 6B.
- the upper panel shows schematic diagrams of each peptide and the plasmids encoding them.
- the lower panel shows the results of Western blotting using an anti-GFP antibody. Since the protein assumed to be sig-GLP-1-GFP was confirmed in the culture supernatant by GFP, it is assumed that sig-GLP-1 is also present in the culture supernatant.
- iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were induced to differentiate according to the method of Experimental Example 3.
- the cells were frozen 14 days after differentiation induction (frozen ATCC CECSi cells).
- Frozen ATCC CECSi cells were seeded at 5.5x104 cells in 24 wells and infected at approximately MOI: 100,000.
- the virus was removed the next day, and the medium was replaced with a medium (medium containing only DMEM/F12, ITS, and IGF-1 in the medium composition in Table 1), and then fluorescence observation was performed 24 hours later using a fluorescent microscope KEYENCE BZ-X810.
- GFP expression in the cells 13 days after virus infection is shown in Figure 7A (fluorescence, bright field).
- the secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 ⁇ l of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in Figure 7B. Even on day 13 after infection, intracellular expression of EGFP was confirmed, and EGFP was also detected in the culture supernatant. Similar results were obtained on day 18.
- Frozen ATCC CECSi cells frozen 10 days after differentiation induction, were placed in a 24-well plate at 1.1 x 105 cells/well and then infected with AAV2-sigGLP-1-GFP (fusion type) viral vector and control GFP viral vector at MOI: 100,000 on the fourth day, and the viral vector was removed the next day.
- the medium was replaced every two days, and co-culture was performed on the fourth day after infection.
- the medium was replaced with Krebs buffer (KRB), and after 24 hours, the supernatant of pancreatic ⁇ cells was collected, KRB was added again, and the amount of c-peptide contained in the supernatant after 24 hours was measured using a c-peptide ELISA kit (AB Clone RKO3588).
- the AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the plasmid sig-GLP-1-GFP ( Figure 6A(d)) or a plasmid containing only the EGFP sequence, respectively.
- Example 7 Effect on glycemic control when CECSi cells expressing and secreting GLP-1 are intraperitoneally transplanted into healthy SCID mice
- AAV2-sig-GLP-1-GFP fusion type virus vector
- control GFP virus vector 100000 for 24 hours. After 2 days of virus removal, the cells were collected and suspended in 100 ⁇ l of physiological saline. SCID mice were fasted for 4 hours, and the prepared cells were transplanted into the abdominal cavity.
- the AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the sig-GLP-1 plasmid ( Figure 6A(b)) or a plasmid carrying only the EGFP sequence, respectively.
- Corneal endothelial substitute cells having a GLP-1 secretion function make it possible to develop a new cell therapy for diseases involving GLP-1, particularly diseases in which GLP-1 secretion is useful (diabetes, etc.).
- This application is based on Patent Application No. 2022-158838 filed in Japan (filing date: September 30, 2022), the contents of which are incorporated in their entirety herein.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Diabetes (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Reproductive Health (AREA)
- Immunology (AREA)
- Virology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Endocrinology (AREA)
- Epidemiology (AREA)
- Vascular Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Emergency Medicine (AREA)
Abstract
The present invention addresses the problem of providing a pluripotent stem cell and a cell (especially, an endothelial cell) differentiated and induced from a stem cell, to which a new function is imparted. The pluripotent stem cell having a GLP-1 secretion function or the corneal endothelium replacement cell that is induced from a pluripotent stem cell and that has a GLP-1 secretion function according to the present invention has a function to secrete GLP-1 for a long period and thus enables development of a novel cellular therapy for diabetes or the like.
Description
本発明は、GLP-1(Glucagon-like peptide-1;グルカゴン様ペプチド-1)分泌機能を有する多能性幹細胞及び幹細胞から分化誘導された細胞に関する。本発明は、また、GLP-1分泌機能を有する、分化誘導された内皮細胞に関する。
The present invention relates to pluripotent stem cells having the function of secreting GLP-1 (Glucagon-like peptide-1) and cells induced to differentiate from the stem cells. The present invention also relates to endothelial cells induced to differentiate and have the function of secreting GLP-1.
iPS細胞由来角膜内皮代替細胞(CECSi cells; Corneal Endothelial Cell Substitute from iPS cells;CECSi細胞)は従来、水疱性角膜症への治療目的で開発された細胞である(特許文献1~3)。一方、CECSi細胞には、iPS細胞から短期間(約2週間)で、均一な品質で、細胞や細胞外基質等へ高接着性の、分化した内皮細胞を大量生産できるという特徴がある。
この特徴を生かしつつ、CECSi細胞に遺伝子編集あるいは遺伝子導入により追加の機能を付与することができれば、角膜領域の治療だけでなく、他の疾患領域への適応拡大を目指すことができる。遺伝子編集技術としては、例えば、ジンクフィンガーヌクレアーゼ、TALEN(転写活性化様エフェクターヌクレアーゼ)、CRISPR(Clustered Regularly Interspaced Short Palindromic Repeat)-Casシステム等のエンドヌクレアーゼを用いる技術が開発されている。遺伝子導入技術としては、非ウイルス的な導入法と、ウイルスを用いた導入法に大別されるが、遺伝子治療においては、ウイルスを用いた遺伝子導入技術が使用され、多くのウイルスベクターが開発されている。 Corneal endothelial cell substitute from iPS cells (CECSi cells) have been developed for the treatment of bullous keratopathy (Patent Documents 1 to 3). On the other hand, CECSi cells have the advantage that they can be used to mass-produce differentiated endothelial cells with uniform quality and high adhesiveness to cells and extracellular matrix from iPS cells in a short period of time (approximately two weeks).
If it is possible to give additional functions to CECSi cells by gene editing or gene introduction while taking advantage of this characteristic, it is possible to aim to expand the application to other disease areas as well as the treatment of the cornea region. As gene editing techniques, for example, techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems have been developed. Gene introduction techniques are broadly divided into non-viral introduction methods and introduction methods using viruses, and in gene therapy, gene introduction techniques using viruses are used, and many viral vectors have been developed.
この特徴を生かしつつ、CECSi細胞に遺伝子編集あるいは遺伝子導入により追加の機能を付与することができれば、角膜領域の治療だけでなく、他の疾患領域への適応拡大を目指すことができる。遺伝子編集技術としては、例えば、ジンクフィンガーヌクレアーゼ、TALEN(転写活性化様エフェクターヌクレアーゼ)、CRISPR(Clustered Regularly Interspaced Short Palindromic Repeat)-Casシステム等のエンドヌクレアーゼを用いる技術が開発されている。遺伝子導入技術としては、非ウイルス的な導入法と、ウイルスを用いた導入法に大別されるが、遺伝子治療においては、ウイルスを用いた遺伝子導入技術が使用され、多くのウイルスベクターが開発されている。 Corneal endothelial cell substitute from iPS cells (CECSi cells) have been developed for the treatment of bullous keratopathy (Patent Documents 1 to 3). On the other hand, CECSi cells have the advantage that they can be used to mass-produce differentiated endothelial cells with uniform quality and high adhesiveness to cells and extracellular matrix from iPS cells in a short period of time (approximately two weeks).
If it is possible to give additional functions to CECSi cells by gene editing or gene introduction while taking advantage of this characteristic, it is possible to aim to expand the application to other disease areas as well as the treatment of the cornea region. As gene editing techniques, for example, techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems have been developed. Gene introduction techniques are broadly divided into non-viral introduction methods and introduction methods using viruses, and in gene therapy, gene introduction techniques using viruses are used, and many viral vectors have been developed.
導入する遺伝子もヒトの遺伝子治療に有益な種々のものが試みられている。例えば特許文献4には、グルカゴン様ペプチド-1(GLP-1)を発現するトランスフェクトされた細胞が記載されている。
GLP-1は、小腸内のL細胞から分泌されるホルモンで、膵臓のβ細胞に存在するGLP-1受容体に作用し、インスリンの分泌を促す因子である。実際にGLP-1受容体作動薬は糖尿病の治療に用いられている。 Regarding the genes to be introduced, various types of genes that are useful for human gene therapy have been attempted. For example, Patent Document 4 describes a transfected cell that expresses glucagon-like peptide-1 (GLP-1).
GLP-1 is a hormone secreted from L cells in the small intestine, which acts on the GLP-1 receptor present in the β cells of the pancreas to promote insulin secretion. GLP-1 receptor agonists are actually used to treat diabetes.
GLP-1は、小腸内のL細胞から分泌されるホルモンで、膵臓のβ細胞に存在するGLP-1受容体に作用し、インスリンの分泌を促す因子である。実際にGLP-1受容体作動薬は糖尿病の治療に用いられている。 Regarding the genes to be introduced, various types of genes that are useful for human gene therapy have been attempted. For example, Patent Document 4 describes a transfected cell that expresses glucagon-like peptide-1 (GLP-1).
GLP-1 is a hormone secreted from L cells in the small intestine, which acts on the GLP-1 receptor present in the β cells of the pancreas to promote insulin secretion. GLP-1 receptor agonists are actually used to treat diabetes.
本発明の課題は、新たな機能が付与された、多能性幹細胞、及び幹細胞から分化誘導された細胞を提供することにある。特に、本発明は、多能性幹細胞、特にiPS細胞から誘導された、新たな機能が付与された角膜内皮代替細胞を提供することを課題とする。
The objective of the present invention is to provide pluripotent stem cells to which new functions have been imparted, and cells induced to differentiate from stem cells. In particular, the objective of the present invention is to provide corneal endothelial replacement cells to which new functions have been imparted, which are induced from pluripotent stem cells, particularly iPS cells.
上記課題に鑑み、本発明者らは、本発明者らがこれまで開発を進めてきた角膜内皮代替細胞にGLP-1分泌機能を付与することを試みた。iPS細胞を角膜内皮代替細胞へと分化誘導し、得られた角膜内皮代替細胞にGLP-1遺伝子を導入、該遺伝子を発現させ、GLP-1タンパク質を分泌することを確認して本発明を完成するに至った。
In light of the above problems, the inventors attempted to impart GLP-1 secretion function to the corneal endothelial replacement cells that they had been developing. They induced the differentiation of iPS cells into corneal endothelial replacement cells, introduced the GLP-1 gene into the resulting corneal endothelial replacement cells, expressed the gene, and confirmed that the GLP-1 protein was secreted, thus completing the present invention.
即ち、本発明は、以下を提供する。
[1]GLP-1分泌機能を有する、多能性幹細胞、又は幹細胞から分化誘導された細胞。
[2]幹細胞から分化誘導された細胞が内皮細胞である、上記[1]記載の細胞。
[3]多能性幹細胞又は幹細胞がiPS細胞である、上記[1]又は[2]記載の細胞。
[4]内皮細胞が角膜内皮細胞である、上記[2]記載の細胞。
[5]GLP-1を発現する上記[1]~[4]のいずれかに記載の細胞。
[A]GLP-1分泌機能を有する、CECSi細胞。
[6]GLP-1をコードする核酸を細胞に導入する工程を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞、又は幹細胞から分化誘導された細胞である、方法。 That is, the present invention provides the following.
[1] A pluripotent stem cell or a cell induced to differentiate from a stem cell, which has a GLP-1 secretion function.
[2] The cell described in [1] above, wherein the cell induced to differentiate from a stem cell is an endothelial cell.
[3] The cell described in [1] or [2] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[4] The cell described in [2] above, wherein the endothelial cell is a corneal endothelial cell.
[5] The cell according to any one of [1] to [4] above, which expresses GLP-1.
[A] CECSi cells having GLP-1 secretion function.
[6] A method for producing a cell expressing GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into a cell, wherein the cell is a pluripotent stem cell or a cell induced to differentiate from a stem cell.
[1]GLP-1分泌機能を有する、多能性幹細胞、又は幹細胞から分化誘導された細胞。
[2]幹細胞から分化誘導された細胞が内皮細胞である、上記[1]記載の細胞。
[3]多能性幹細胞又は幹細胞がiPS細胞である、上記[1]又は[2]記載の細胞。
[4]内皮細胞が角膜内皮細胞である、上記[2]記載の細胞。
[5]GLP-1を発現する上記[1]~[4]のいずれかに記載の細胞。
[A]GLP-1分泌機能を有する、CECSi細胞。
[6]GLP-1をコードする核酸を細胞に導入する工程を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞、又は幹細胞から分化誘導された細胞である、方法。 That is, the present invention provides the following.
[1] A pluripotent stem cell or a cell induced to differentiate from a stem cell, which has a GLP-1 secretion function.
[2] The cell described in [1] above, wherein the cell induced to differentiate from a stem cell is an endothelial cell.
[3] The cell described in [1] or [2] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[4] The cell described in [2] above, wherein the endothelial cell is a corneal endothelial cell.
[5] The cell according to any one of [1] to [4] above, which expresses GLP-1.
[A] CECSi cells having GLP-1 secretion function.
[6] A method for producing a cell expressing GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into a cell, wherein the cell is a pluripotent stem cell or a cell induced to differentiate from a stem cell.
[7]核酸の細胞への導入が遺伝子導入又はゲノム編集によるものである、上記[6]記載の方法。
[8]幹細胞から分化誘導された細胞が内皮細胞である、上記[6]又は[7]に記載の方法。
[9]多能性幹細胞又は幹細胞がiPS細胞である、上記[6]~[8]のいずれかに記載の方法。
[10]内皮細胞が角膜内皮細胞である、上記[8]記載の方法。
[B]GLP-1をコードする核酸をCECSi細胞に導入する工程を含む、GLP-1を発現するCECSi細胞の製造方法。
[11](1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いて細胞に該核酸を導入し、発現ベクターを含む細胞を作製する工程、及び
(3)前記発現ベクターを含む細胞を培養する工程、
を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞、又は幹細胞から分化誘導された細胞である、方法。
[12]幹細胞から分化誘導された細胞が内皮細胞である、上記[11]に記載の方法。
[13]多能性幹細胞又は幹細胞がiPS細胞である、上記[11]又は[12]に記載の方法。
[14]内皮細胞が角膜内皮細胞である、上記[12]記載の方法。
[C](1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いてCECSi細胞に該核酸を導入し、発現ベクターを含むCECSi細胞を作製する工程、及び
(3)前記発現ベクターを含むCECSi細胞を培養する工程、
を含む、GLP-1を発現するCECSi細胞の製造方法。
[15]上記[1]~[5]及び[A]のいずれかに記載の細胞を含む医薬組成物。
[16]糖尿病、肥満、中枢神経障害、末梢神経障害、腎機能障害、心不全、虚血性心疾患、肝機能障害、味覚障害、肺炎の治療用である、[15]に記載の医薬組成物。 [7] The method described in [6] above, wherein the introduction of the nucleic acid into the cell is by gene introduction or genome editing.
[8] The method according to [6] or [7] above, wherein the cells induced to differentiate from stem cells are endothelial cells.
[9] The method according to any one of [6] to [8] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[10] The method described in [8] above, wherein the endothelial cells are corneal endothelial cells.
[B] A method for producing CECSi cells expressing GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into CECSi cells.
[11] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:
[12] The method according to [11] above, wherein the cells induced to differentiate from stem cells are endothelial cells.
[13] The method according to [11] or [12] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[14] The method described in [12] above, wherein the endothelial cells are corneal endothelial cells.
[C] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a CECSi cell using an expression vector containing the nucleic acid to prepare a CECSi cell containing the expression vector; and (3) culturing the CECSi cell containing the expression vector.
A method for producing a CECSi cell expressing GLP-1, comprising:
[15] A pharmaceutical composition comprising the cell according to any one of [1] to [5] and [A] above.
[16] The pharmaceutical composition according to [15], which is for treating diabetes, obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction, taste disorders, and pneumonia.
[8]幹細胞から分化誘導された細胞が内皮細胞である、上記[6]又は[7]に記載の方法。
[9]多能性幹細胞又は幹細胞がiPS細胞である、上記[6]~[8]のいずれかに記載の方法。
[10]内皮細胞が角膜内皮細胞である、上記[8]記載の方法。
[B]GLP-1をコードする核酸をCECSi細胞に導入する工程を含む、GLP-1を発現するCECSi細胞の製造方法。
[11](1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いて細胞に該核酸を導入し、発現ベクターを含む細胞を作製する工程、及び
(3)前記発現ベクターを含む細胞を培養する工程、
を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞、又は幹細胞から分化誘導された細胞である、方法。
[12]幹細胞から分化誘導された細胞が内皮細胞である、上記[11]に記載の方法。
[13]多能性幹細胞又は幹細胞がiPS細胞である、上記[11]又は[12]に記載の方法。
[14]内皮細胞が角膜内皮細胞である、上記[12]記載の方法。
[C](1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いてCECSi細胞に該核酸を導入し、発現ベクターを含むCECSi細胞を作製する工程、及び
(3)前記発現ベクターを含むCECSi細胞を培養する工程、
を含む、GLP-1を発現するCECSi細胞の製造方法。
[15]上記[1]~[5]及び[A]のいずれかに記載の細胞を含む医薬組成物。
[16]糖尿病、肥満、中枢神経障害、末梢神経障害、腎機能障害、心不全、虚血性心疾患、肝機能障害、味覚障害、肺炎の治療用である、[15]に記載の医薬組成物。 [7] The method described in [6] above, wherein the introduction of the nucleic acid into the cell is by gene introduction or genome editing.
[8] The method according to [6] or [7] above, wherein the cells induced to differentiate from stem cells are endothelial cells.
[9] The method according to any one of [6] to [8] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[10] The method described in [8] above, wherein the endothelial cells are corneal endothelial cells.
[B] A method for producing CECSi cells expressing GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into CECSi cells.
[11] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:
[12] The method according to [11] above, wherein the cells induced to differentiate from stem cells are endothelial cells.
[13] The method according to [11] or [12] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[14] The method described in [12] above, wherein the endothelial cells are corneal endothelial cells.
[C] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a CECSi cell using an expression vector containing the nucleic acid to prepare a CECSi cell containing the expression vector; and (3) culturing the CECSi cell containing the expression vector.
A method for producing a CECSi cell expressing GLP-1, comprising:
[15] A pharmaceutical composition comprising the cell according to any one of [1] to [5] and [A] above.
[16] The pharmaceutical composition according to [15], which is for treating diabetes, obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction, taste disorders, and pneumonia.
本発明によりCECSi細胞にGLP-1分泌機能を持たせることができた。GLP-1を長期間分泌する機能を有するCECSi細胞により糖尿病等への新規細胞治療の開発が可能となる。
The present invention has made it possible to endow CECSi cells with the ability to secrete GLP-1. CECSi cells that have the ability to secrete GLP-1 for a long period of time will enable the development of new cell therapies for diabetes and other conditions.
以下、本発明を説明する。本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味を有する。
The present invention is described below. Terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified.
本発明において「GLP-1分泌機能」とは、細胞内でGLP-1を産生し、次いで細胞外に分泌し得る機能を意味する。本明細書において、GLP-1は、活性ヒトグルカゴン様ペプチド-1、すなわちアミノ酸配列がHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG[配列番号1]であるGLP-1、その活性型であるアミノ酸配列がHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG[配列番号2]であるGLP-1(7-37)またはそれらのバリアントを意味する。本明細書において、GLP-1の「バリアント」は、GLP-1の活性を変化させることなく、アミノ酸残基の1以上の置換、欠失または挿入により、天然配列から1以上のアミノ酸が変えられたアミノ酸配列を意味する。「GLP-1バリアント」は、1以上のアミノ酸残基の欠失を有していてもよく、GLP-1活性に重要なアミノ酸残基または該重要なアミノ酸残基の1以上と置換できる残基を有することにより、天然GLP-1配列と同じ活性、例えば膵臓β細胞によるインスリン分泌を促進させる活性を有する、GLP-1(7-37)より短いGLP-1の断片であり得る。本明細書において、特に明記しない場合には、「GLP-1」はこれら全てを包含する概念である。
ここで、「アミノ酸残基の1以上」は、2つ、3つ、4つ又は5つのアミノ酸残基であってもよい。「アミノ酸残基の1以上」は好ましくは1~5、1~4、1~3、1若しくは2、又は1つのアミノ酸残基である。
GLP-1分泌機能において、GLP-1が分泌されることによって期待される生物学的作用(例、プレインスリン(c-peptide)分泌促進作用、インスリン分泌促進作用、グルカゴン分泌抑制作用、膵臓β細胞増殖作用、体重減少、グルコース取り込み作用、腎臓保護作用、心臓保護作用、神経保護、胃排泄遅延、食欲抑制、利尿作用)が認められる限り、その量は限定されず、GLP-1分泌の指標となる生物学的作用の種類によっても異なる。当該機能が発揮された場合と発揮されない場合との間に有意な差があればよい。 In the present invention, the term "GLP-1 secretion function" refers to a function capable of producing GLP-1 in cells and then secreting it outside the cells. In the present specification, GLP-1 refers to active human glucagon-like peptide-1, i.e., GLP-1 having the amino acid sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 1], GLP-1 (7-37) having the amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 2], which is its active form, or a variant thereof. In the present specification, the term "variant" of GLP-1 refers to an amino acid sequence in which one or more amino acids have been changed from the native sequence by substitution, deletion or insertion of one or more amino acid residues without changing the activity of GLP-1. A "GLP-1 variant" may have one or more deleted amino acid residues, or may be a fragment of GLP-1 shorter than GLP-1(7-37) that has the same activity as the native GLP-1 sequence, for example, the activity of promoting insulin secretion by pancreatic β cells, by having an amino acid residue important for GLP-1 activity or a residue that can be substituted for one or more of the important amino acid residues. In this specification, unless otherwise specified, "GLP-1" is a concept that includes all of these.
Here, "one or more amino acid residues" may be 2, 3, 4, or 5 amino acid residues. "One or more amino acid residues" is preferably 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid residue.
In the GLP-1 secretion function, as long as the biological action expected from the secretion of GLP-1 (e.g., preinsulin (c-peptide) secretion promoting effect, insulin secretion promoting effect, glucagon secretion suppressing effect, pancreatic β cell proliferation effect, weight loss, glucose uptake effect, renal protective effect, cardiac protective effect, neuroprotection, delayed gastric emptying, appetite suppression, diuretic effect) is observed, the amount is not limited and differs depending on the type of biological action serving as an indicator of GLP-1 secretion. It is sufficient if there is a significant difference between the case where the function is exerted and the case where it is not exerted.
ここで、「アミノ酸残基の1以上」は、2つ、3つ、4つ又は5つのアミノ酸残基であってもよい。「アミノ酸残基の1以上」は好ましくは1~5、1~4、1~3、1若しくは2、又は1つのアミノ酸残基である。
GLP-1分泌機能において、GLP-1が分泌されることによって期待される生物学的作用(例、プレインスリン(c-peptide)分泌促進作用、インスリン分泌促進作用、グルカゴン分泌抑制作用、膵臓β細胞増殖作用、体重減少、グルコース取り込み作用、腎臓保護作用、心臓保護作用、神経保護、胃排泄遅延、食欲抑制、利尿作用)が認められる限り、その量は限定されず、GLP-1分泌の指標となる生物学的作用の種類によっても異なる。当該機能が発揮された場合と発揮されない場合との間に有意な差があればよい。 In the present invention, the term "GLP-1 secretion function" refers to a function capable of producing GLP-1 in cells and then secreting it outside the cells. In the present specification, GLP-1 refers to active human glucagon-like peptide-1, i.e., GLP-1 having the amino acid sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 1], GLP-1 (7-37) having the amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 2], which is its active form, or a variant thereof. In the present specification, the term "variant" of GLP-1 refers to an amino acid sequence in which one or more amino acids have been changed from the native sequence by substitution, deletion or insertion of one or more amino acid residues without changing the activity of GLP-1. A "GLP-1 variant" may have one or more deleted amino acid residues, or may be a fragment of GLP-1 shorter than GLP-1(7-37) that has the same activity as the native GLP-1 sequence, for example, the activity of promoting insulin secretion by pancreatic β cells, by having an amino acid residue important for GLP-1 activity or a residue that can be substituted for one or more of the important amino acid residues. In this specification, unless otherwise specified, "GLP-1" is a concept that includes all of these.
Here, "one or more amino acid residues" may be 2, 3, 4, or 5 amino acid residues. "One or more amino acid residues" is preferably 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid residue.
In the GLP-1 secretion function, as long as the biological action expected from the secretion of GLP-1 (e.g., preinsulin (c-peptide) secretion promoting effect, insulin secretion promoting effect, glucagon secretion suppressing effect, pancreatic β cell proliferation effect, weight loss, glucose uptake effect, renal protective effect, cardiac protective effect, neuroprotection, delayed gastric emptying, appetite suppression, diuretic effect) is observed, the amount is not limited and differs depending on the type of biological action serving as an indicator of GLP-1 secretion. It is sufficient if there is a significant difference between the case where the function is exerted and the case where it is not exerted.
GLP-1分泌機能の指標として好ましくはインスリン分泌促進作用が挙げられる。
A preferred indicator of GLP-1 secretory function is its insulin secretion promoting effect.
1.細胞
本発明は、GLP-1分泌機能を有する細胞(以下、単に本発明の細胞とも称する)を提供する。ここで、細胞としては、多能性幹細胞や該多能性幹細胞又は幹細胞から分化誘導して得られる細胞(以下、単に分化細胞とも称する)、特に角膜内皮代替細胞のような内皮細胞が挙げられる。 1. Cells The present invention provides cells having a GLP-1 secretion function (hereinafter, also simply referred to as the cells of the present invention). Examples of the cells include pluripotent stem cells and cells obtained by inducing differentiation from the pluripotent stem cells or stem cells (hereinafter, also simply referred to as differentiated cells), in particular endothelial cells such as corneal endothelial replacement cells.
本発明は、GLP-1分泌機能を有する細胞(以下、単に本発明の細胞とも称する)を提供する。ここで、細胞としては、多能性幹細胞や該多能性幹細胞又は幹細胞から分化誘導して得られる細胞(以下、単に分化細胞とも称する)、特に角膜内皮代替細胞のような内皮細胞が挙げられる。 1. Cells The present invention provides cells having a GLP-1 secretion function (hereinafter, also simply referred to as the cells of the present invention). Examples of the cells include pluripotent stem cells and cells obtained by inducing differentiation from the pluripotent stem cells or stem cells (hereinafter, also simply referred to as differentiated cells), in particular endothelial cells such as corneal endothelial replacement cells.
幹細胞とは、自分自身を複製する能力と他の複数系統の細胞に分化する能力を兼ね備えた細胞であり、その例としては、以下に限定されるものではないが、胚性幹細胞(ES細胞)、胚性腫瘍細胞、胚性生殖幹細胞、人工多能性幹細胞(iPS細胞)、神経幹細胞、造血幹細胞、間葉系幹細胞、肝幹細胞、膵幹細胞、筋幹細胞、生殖幹細胞、腸幹細胞、がん幹細胞、毛包幹細胞、皮膚幹細胞などが挙げられる。
Stem cells are cells that have the ability to replicate themselves and differentiate into multiple other cell lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and skin stem cells.
多能性幹細胞は、受精卵、クローン胚、生殖幹細胞、組織内幹細胞、体細胞等から誘導することができる。多能性幹細胞としては、胚性幹細胞(ES細胞:Embryonic stem cell)、EG細胞(Embryonic germ cell)、人工多能性幹細胞(iPS細胞:induced pluripotent stem cell)等を挙げることができる。間葉系幹細胞(mesenchymal stem cell:MSC)から得られるMuse細胞(Multi-lineage differentiating Stress Enduring cell)、及び生殖細胞(例えば精巣)から作製されるGS細胞も多能性幹細胞に包含される。単に幹細胞と称する場合は、多能性幹細胞も包含する意図である。
Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating Stress Enduring cells) obtained from mesenchymal stem cells (MSCs), and GS cells produced from germ cells (e.g. testes) are also included in pluripotent stem cells. When simply referring to stem cells, it is intended to include pluripotent stem cells.
ES細胞は、内部細胞集団をフィーダー細胞上又はleukemia inhibitory factor(LIF)を含む培地中で培養することにより製造することができる。また、所定の機関より入手でき、市販品を購入することもできる。ES細胞の1つである核移植ES細胞(ntES細胞)は、細胞核を取り除いた卵子に体細胞の細胞核を移植して作ったクローン胚から樹立することができる。
ES cells can be produced by culturing the inner cell population on feeder cells or in medium containing leukemia inhibitory factor (LIF). They are also available from designated institutions and can be purchased commercially. Nuclear transfer ES cells (ntES cells), a type of ES cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.
EG細胞は、始原生殖細胞をマウス幹細胞因子(mSCF)、LIF及び塩基性線維芽細胞増殖因子(bFGF)を含む培地中で培養することにより製造することができる(Cell, 70:841-847, 1992)。
EG cells can be produced by culturing primordial germ cells in a medium containing mouse stem cell factor (mSCF), LIF, and basic fibroblast growth factor (bFGF) (Cell, 70:841-847, 1992).
iPS細胞とは、体細胞を公知の方法等により初期化(reprogramming)することにより、多能性を誘導した細胞である。iPS細胞としては、具体的には線維芽細胞、末梢血単核球等に分化した体細胞をOct3/4、Sox2、Klf4、Myc(c-Myc、N-Myc、L-Myc)、Glis1、Nanog、Sall4、lin28、Esrrb等を含む初期化遺伝子群から選ばれる複数の遺伝子の発現により初期化して多分化能を誘導した細胞が挙げられる。2006年、山中らによりマウス細胞で人工多能性幹細胞が樹立された(Cell, 2006, 126(4) pp.663-676)。人工多能性幹細胞は、2007年にヒト線維芽細胞でも樹立され、胚性幹細胞と同様に多能性と自己複製能を有する(Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106)。人工多能性幹細胞として、遺伝子発現による直接初期化で製造する方法以外に、化合物の添加などにより体細胞より人工多能性幹細胞を誘導することもできる(Science, 2013, 341, pp.651-654)。
iPS cells are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specific examples of iPS cells include cells in which pluripotency has been induced by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., through the expression of multiple genes selected from a group of reprogramming genes including Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. In 2006, Yamanaka et al. established induced pluripotent stem cells from mouse cells (Cell, 2006, 126(4) pp.663-676). In 2007, induced pluripotent stem cells were established from human fibroblasts, and like embryonic stem cells, they have pluripotency and the ability to self-replicate (Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106). In addition to producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp.651-654).
人工多能性幹細胞を製造する際に用いられる体細胞としては、特に限定は無いが、組織由来の線維芽細胞、血球系細胞(例えば末梢血単核球、T細胞等)、肝細胞、膵臓細胞、腸上皮細胞、平滑筋細胞等が挙げられる。
Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells, T cells, etc.), liver cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.
人工多能性幹細胞を製造する際に、数種類の遺伝子(例えばOct3/4、Sox2、Klf4及びMycの4因子)の発現により初期化する場合、遺伝子を発現させるための手段は特に限定されない。遺伝子を発現させるための手段としては、例えばウイルスベクター(例えばレトロウイルスベクター、レンチウイルスベクター、センダイウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター)を用いた感染法、プラスミドベクター(例えばプラスミドベクター、エピソーマルベクター)を用いた遺伝子導入法(例えばリン酸カルシウム法、リポフェクション法、レトロネクチン法、エレクトロポレーション法)、RNAベクターを用いた遺伝子導入法(例えばリン酸カルシウム法、リポフェクション法、エレクトロポレーション法)、タンパク質の直接注入法等が挙げられる。
When producing artificial pluripotent stem cells, if initialization is performed by expression of several types of genes (e.g., four factors: Oct3/4, Sox2, Klf4, and Myc), the means for expressing the genes is not particularly limited. Examples of means for expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, and electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, and electroporation method), and direct protein injection methods.
また、株化された人工多能性幹細胞を入手する事も可能である。具体的には、iPS細胞としては、201B7、201B7-Ff、253G1、253G4、1201C1、1205D1、1210B2、836B3、FF-I14s03、FF-I01s04、MH09s01、Ff-XT18s02、Ff-WIs03、Ff-WJs513、Ff-CLs14、Ff-KVs09、QHJI14s03、QHJI01s04、RWMH09s01、DRXT18s02、RJWIs03、YZWJs513、ILCLs14、GLKVs09、 Ff-XT28s05-ABo_To,Ff-I01s04-ABII-KO,Ff-I14s04-ABII-KO(いずれもiPSアカデミアジャパン社、又は京都大学iPS研究財団)、Tic(JCRB1331株)、Dotcom(JCRB1327株)、Squeaky(JCRB1329株)、及びToe(JCRB1338株)、Lollipop(JCRB1336株)(以上成育医療センター、医薬基盤研究所難病・疾患資源研究部・JCRB細胞バンク)、UTA-1株及びUTA-1-SF-2-2株(いずれも東京大学)、21526、21528、21530、21531、31536、31538株(いずれもフジフイルム・セルラー・ダイナミクス社)、ATCC-DYP0730、ATCC-DYP0250、ATCC-HYR0103、ATCC-DYR0100、ATCC-DYR0530、ATCC-DYS0530、ATCC-DYP0530、ATCC-DYS0100、ATCC-HYS0103、ATCC-CYS0105、KYOU-DXR0109B、ATCC-BYS0110、ATCC-BYS0111、ATCC-BYS0112、ATCC-BYS0113、ATCC-BXS0114、ATCC-BXS0115、ATCC-BXS0116、ATCC-BXS0117(いずれも非営利法人American Type Culture Collection)等を用いることができる。
It is also possible to obtain established induced pluripotent stem cells. Specifically, the iPS cells include 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 836B3, FF-I14s03, FF-I01s04, MH09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs09, QHJI14s03, QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs 14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all from iPS Academia Japan, Inc., or Kyoto University iPS Research Foundation), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), Toe (JCRB1338 strain), and Lollipop (JCRB1336 strain) (all from the National Center for Child Health and Development, National Institute of Biomedical Innovation, Department of Intractable Diseases) ATCC-DYP0730, ATCC-DYP0250, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYP0530, ATCC-DYP0530, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYP0530, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-HYR0103 ... C-DYS0100, ATCC-HYS0103, ATCC-CYS0105, KYOU-DXR0109B, ATCC-BYS0110, ATCC-BYS0111, ATCC-BYS0112, ATCC-BYS0113, ATCC-BXS0114, ATCC-BXS0115, ATCC-BXS0116, ATCC-BXS0117 (all from the non-profit American Type Culture Collection) can be used.
「哺乳動物」には、げっ歯類、有蹄類、ネコ目、ウサギ目、霊長類等が包含される。げっ歯類には、マウス、ラット、ハムスター、モルモット等が包含される。有蹄類には、ブタ、ウシ、ヤギ、ウマ、ヒツジ等が包含される。ネコ目には、イヌ、ネコ等が包含される。ウサギ目には、ウサギ等が含包される。「霊長類」とは、霊長目に属する哺乳類動物をいい、霊長類としては、キツネザル、ロリス、ツバイ等の原猿亜目、及びサル、類人猿、ヒト等の真猿亜目が含まれる。
"Mammals" includes rodents, ungulates, felines, lagomorphs, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Felidae include dogs and cats. Lagomorphs include rabbits, etc. "Primates" refers to mammals belonging to the order Primates, and includes prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans.
本発明に用いる多能性幹細胞は、哺乳動物の多能性幹細胞であり、好ましくはげっ歯類(例えばマウス、ラット)又は霊長類(例えばヒト、サル)の多能性幹細胞であり、最も好ましくはヒトの多能性幹細胞である。
The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, and most preferably human pluripotent stem cells.
本発明において使用する分化細胞としては、上記幹細胞、好ましくは上記多能性幹細胞から分化誘導したものが挙げられる。例えば内皮細胞、好ましくは角膜内皮細胞、より好ましくは本発明者らによって開発された角膜内皮様の細胞、所謂角膜内皮代替細胞であり、特許文献1~3に記載の方法によって製造、調製することができる。好ましくは、角膜内皮細胞様の性状及び機能を有し、且つ、NR3C2(nuclear receptor subfamily 3, group C, member 2)の遺伝子発現量が増強されていることを特徴とする、iPS細胞由来の角膜内皮代替細胞(Corneal Endothelial Cell Substitute from iPS cells;CECSi細胞)である(特許文献3)。
角膜内皮代替細胞を製造する方法の一例としては以下の方法が挙げられる。
iPS細胞をiMatrix-511(0.6μg/cm2)をコートした培養皿で、StemFit(登録商標)AK03N培地(味の素)を用いて1週間培養する。その後、iMatrix-511(0.3μg/cm2)をコートした培養皿に播種しなおして、下記の分化誘導培地(表1)を用いて、iPS細胞から角膜内皮代替細胞への分化誘導培養を8~14日間行う。なお、凍結保存したiPS細胞を用いる場合には2回継代後、培養したのちに分化誘導を行う。 The differentiated cells used in the present invention include those induced to differentiate from the above stem cells, preferably the above pluripotent stem cells. For example, endothelial cells, preferably corneal endothelial cells, more preferably corneal endothelial-like cells developed by the present inventors, so-called corneal endothelial substitute cells, can be produced and prepared by the methods described in Patent Documents 1 to 3. Preferably, they are corneal endothelial cell substitute from iPS cells (CECSi cells), which have corneal endothelial cell-like properties and functions and are characterized by enhanced expression of the NR3C2 (nuclear receptor subfamily 3, group C, member 2) gene (Patent Document 3).
An example of a method for producing corneal endothelial replacement cells is as follows.
The iPS cells are cultured for one week in a culture dish coated with iMatrix-511 (0.6 μg/cm 2 ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells are seeded again on a culture dish coated with iMatrix-511 (0.3 μg/cm 2 ), and differentiation induction culture of the iPS cells into corneal endothelial substitute cells is performed for 8 to 14 days using the differentiation induction medium described below (Table 1). Note that when using cryopreserved iPS cells, differentiation induction is performed after two passages and culture.
角膜内皮代替細胞を製造する方法の一例としては以下の方法が挙げられる。
iPS細胞をiMatrix-511(0.6μg/cm2)をコートした培養皿で、StemFit(登録商標)AK03N培地(味の素)を用いて1週間培養する。その後、iMatrix-511(0.3μg/cm2)をコートした培養皿に播種しなおして、下記の分化誘導培地(表1)を用いて、iPS細胞から角膜内皮代替細胞への分化誘導培養を8~14日間行う。なお、凍結保存したiPS細胞を用いる場合には2回継代後、培養したのちに分化誘導を行う。 The differentiated cells used in the present invention include those induced to differentiate from the above stem cells, preferably the above pluripotent stem cells. For example, endothelial cells, preferably corneal endothelial cells, more preferably corneal endothelial-like cells developed by the present inventors, so-called corneal endothelial substitute cells, can be produced and prepared by the methods described in Patent Documents 1 to 3. Preferably, they are corneal endothelial cell substitute from iPS cells (CECSi cells), which have corneal endothelial cell-like properties and functions and are characterized by enhanced expression of the NR3C2 (nuclear receptor subfamily 3, group C, member 2) gene (Patent Document 3).
An example of a method for producing corneal endothelial replacement cells is as follows.
The iPS cells are cultured for one week in a culture dish coated with iMatrix-511 (0.6 μg/cm 2 ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells are seeded again on a culture dish coated with iMatrix-511 (0.3 μg/cm 2 ), and differentiation induction culture of the iPS cells into corneal endothelial substitute cells is performed for 8 to 14 days using the differentiation induction medium described below (Table 1). Note that when using cryopreserved iPS cells, differentiation induction is performed after two passages and culture.
角膜内皮代替細胞が有する角膜内皮細胞様の性状及び機能としては、具体的には以下の特徴(i)~(iv)が挙げられ、これらの特徴のうち少なくとも1つ、好ましくは2つ、より好ましくは3つ、いっそう好ましくは4つ全ての特徴を有する。
(i)細胞間接着がN-cadherinで構成されている。
(ii)細胞間にtight junctionが形成されている。
(iii)細胞膜上にNa,K-ATPase α1 subunitを発現する。
(iv)細胞核に転写因子PITX2の発現が観察される。
細胞間接着がN-Cadherinで構成されているか否かは、N-Cadherinに対する免疫染色で確認することができる。
細胞間にtight junctionが形成されているか否かは、tight junctionを構成するタンパク質であるZO-1の存在を、ZO-1に対する免疫染色で観察することにより確認することができる。また、電子顕微鏡により直接構造を観察することによって確認することもできる。
細胞膜上にNa,K-ATPase α1 subunit(ATP1A1)を発現しているか否かは、ZO-1とNa,K-ATPase α1 subunitに対する免疫染色により、両者が共染色されることで確認することができる。
細胞核に転写因子PITX2が発現しているか否かは、PITX2に対する免疫染色で確認することができる。 Specific examples of the corneal endothelial cell-like properties and functions possessed by the corneal endothelial substitute cells include the following characteristics (i) to (iv), and among these characteristics, the cells have at least one, preferably two, more preferably three, and even more preferably all four.
(i) Cell-cell adhesion is composed of N-cadherin.
(ii) Tight junctions are formed between the cells.
(iii) Express the Na,K-ATPase α1 subunit on the cell membrane.
(iv) Expression of the transcription factor PITX2 is observed in the cell nucleus.
Whether or not the cell-cell adhesion is composed of N-Cadherin can be confirmed by immunostaining for N-Cadherin.
Whether or not tight junctions are formed between cells can be confirmed by observing the presence of ZO-1, a protein that constitutes tight junctions, using immunostaining for ZO-1, or by directly observing the structure using an electron microscope.
Whether or not Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining of ZO-1 and Na,K-ATPase α1 subunit by immunostaining.
Whether or not the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining for PITX2.
(i)細胞間接着がN-cadherinで構成されている。
(ii)細胞間にtight junctionが形成されている。
(iii)細胞膜上にNa,K-ATPase α1 subunitを発現する。
(iv)細胞核に転写因子PITX2の発現が観察される。
細胞間接着がN-Cadherinで構成されているか否かは、N-Cadherinに対する免疫染色で確認することができる。
細胞間にtight junctionが形成されているか否かは、tight junctionを構成するタンパク質であるZO-1の存在を、ZO-1に対する免疫染色で観察することにより確認することができる。また、電子顕微鏡により直接構造を観察することによって確認することもできる。
細胞膜上にNa,K-ATPase α1 subunit(ATP1A1)を発現しているか否かは、ZO-1とNa,K-ATPase α1 subunitに対する免疫染色により、両者が共染色されることで確認することができる。
細胞核に転写因子PITX2が発現しているか否かは、PITX2に対する免疫染色で確認することができる。 Specific examples of the corneal endothelial cell-like properties and functions possessed by the corneal endothelial substitute cells include the following characteristics (i) to (iv), and among these characteristics, the cells have at least one, preferably two, more preferably three, and even more preferably all four.
(i) Cell-cell adhesion is composed of N-cadherin.
(ii) Tight junctions are formed between the cells.
(iii) Express the Na,K-ATPase α1 subunit on the cell membrane.
(iv) Expression of the transcription factor PITX2 is observed in the cell nucleus.
Whether or not the cell-cell adhesion is composed of N-Cadherin can be confirmed by immunostaining for N-Cadherin.
Whether or not tight junctions are formed between cells can be confirmed by observing the presence of ZO-1, a protein that constitutes tight junctions, using immunostaining for ZO-1, or by directly observing the structure using an electron microscope.
Whether or not Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining of ZO-1 and Na,K-ATPase α1 subunit by immunostaining.
Whether or not the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining for PITX2.
本発明の細胞は、上記多能性幹細胞やその分化細胞、特に内皮細胞にGLP-1分泌機能が付与されたものである。細胞にGLP-1分泌機能を付与する工程については後述の「2.細胞の製造方法」にて詳述されるが、具体的には、上記多能性幹細胞やその分化細胞、特に内皮細胞に、GLP-1をコードする核酸を導入することで実施される。従って、本発明の細胞は、GLP-1を発現する細胞である。GLP-1を細胞外に分泌させるためには、細胞内で発現するGLP-1にシグナルペプチドが付されていることが好ましい。従って、本発明において細胞内に導入されるGLP-1をコードする核酸はシグナル配列が連結していることが好ましい。
The cells of the present invention are the above-mentioned pluripotent stem cells or their differentiated cells, particularly endothelial cells, to which a GLP-1 secretion function has been imparted. The process of imparting a GLP-1 secretion function to cells will be described in detail in "2. Cell manufacturing method" below, but specifically, this is carried out by introducing a nucleic acid encoding GLP-1 into the above-mentioned pluripotent stem cells or their differentiated cells, particularly endothelial cells. Therefore, the cells of the present invention are cells that express GLP-1. In order to secrete GLP-1 outside the cells, it is preferable that a signal peptide is attached to the GLP-1 expressed in the cells. Therefore, it is preferable that the nucleic acid encoding GLP-1 introduced into the cells in the present invention is linked to a signal sequence.
2.細胞の製造方法
本発明は、GLP-1分泌機能を有する細胞の製造方法(以下、単に本発明の細胞の製造方法とも称する)を提供する。
本発明の細胞の製造方法は、細胞にGLP-1分泌機能を付与することを特徴とする。ここで、細胞としては、多能性幹細胞やその分化細胞、特に角膜内皮代替細胞のような内皮細胞が挙げられる。
GLP-1分泌機能の細胞への付与は、どの段階で行われてもよい。例えば、多能性幹細胞にGLP-1分泌機能を付与しても、多能性幹細胞を分化誘導して得られた分化細胞、特に内皮細胞にGLP-1分泌機能を付与してもよい。内皮細胞としては、上記「1.細胞」の項で述べた本発明者らが開発した角膜内皮代替細胞であるCECSi細胞等が挙げられる。 2. Method for Producing Cells The present invention provides a method for producing cells having a GLP-1 secretion function (hereinafter, also simply referred to as the method for producing the cells of the present invention).
The method for producing cells of the present invention is characterized in that it imparts a GLP-1 secretion function to cells. Examples of cells include pluripotent stem cells and their differentiated cells, particularly endothelial cells such as corneal endothelial replacement cells.
The GLP-1 secretion function may be imparted to cells at any stage. For example, the GLP-1 secretion function may be imparted to pluripotent stem cells, or to differentiated cells, particularly endothelial cells, obtained by inducing differentiation of pluripotent stem cells. Examples of endothelial cells include CECSi cells, which are corneal endothelial replacement cells developed by the present inventors and described above in the section "1. Cells".
本発明は、GLP-1分泌機能を有する細胞の製造方法(以下、単に本発明の細胞の製造方法とも称する)を提供する。
本発明の細胞の製造方法は、細胞にGLP-1分泌機能を付与することを特徴とする。ここで、細胞としては、多能性幹細胞やその分化細胞、特に角膜内皮代替細胞のような内皮細胞が挙げられる。
GLP-1分泌機能の細胞への付与は、どの段階で行われてもよい。例えば、多能性幹細胞にGLP-1分泌機能を付与しても、多能性幹細胞を分化誘導して得られた分化細胞、特に内皮細胞にGLP-1分泌機能を付与してもよい。内皮細胞としては、上記「1.細胞」の項で述べた本発明者らが開発した角膜内皮代替細胞であるCECSi細胞等が挙げられる。 2. Method for Producing Cells The present invention provides a method for producing cells having a GLP-1 secretion function (hereinafter, also simply referred to as the method for producing the cells of the present invention).
The method for producing cells of the present invention is characterized in that it imparts a GLP-1 secretion function to cells. Examples of cells include pluripotent stem cells and their differentiated cells, particularly endothelial cells such as corneal endothelial replacement cells.
The GLP-1 secretion function may be imparted to cells at any stage. For example, the GLP-1 secretion function may be imparted to pluripotent stem cells, or to differentiated cells, particularly endothelial cells, obtained by inducing differentiation of pluripotent stem cells. Examples of endothelial cells include CECSi cells, which are corneal endothelial replacement cells developed by the present inventors and described above in the section "1. Cells".
具体的にはGLP-1をコードする核酸、好ましくはシグナル配列が連結した核酸を適当な発現ベクターに挿入する(必要に応じて2種類の発現ベクターを用いてもよい)。その際、発現制御領域、例えば、エンハンサー、プロモーターの制御のもとで発現するよう発現ベクターに組み込む。次に、この発現ベクターを細胞に導入し、GLP-1を発現させる。
Specifically, a nucleic acid encoding GLP-1, preferably a nucleic acid linked to a signal sequence, is inserted into an appropriate expression vector (two types of expression vectors may be used if necessary). At that time, it is incorporated into the expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or promoter. Next, this expression vector is introduced into cells to express GLP-1.
本発明の細胞の製造方法の一実施態様として下記の方法が挙げられる。
(1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いて細胞に該核酸を導入し、発現ベクターを含む細胞を作製する工程、及び
(3)前記発現ベクターを含む細胞を培養する工程、
を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞又は幹細胞から分化誘導された細胞である、方法。 One embodiment of the method for producing the cells of the present invention is the following method.
(1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:
(1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いて細胞に該核酸を導入し、発現ベクターを含む細胞を作製する工程、及び
(3)前記発現ベクターを含む細胞を培養する工程、
を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞又は幹細胞から分化誘導された細胞である、方法。 One embodiment of the method for producing the cells of the present invention is the following method.
(1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:
用いることのできる発現ベクターとしては、挿入した遺伝子を安定に保持するものであれば種類に特に制限はなく、様々な種類のベクターが利用可能である。ベクターは、ウイルスベクターまたは非ウイルスベクターでありうる。ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、ヘルペスウイルスベクター、センダイウイルスベクター、ワクシニアウイルスベクター等が挙げられる。この中でもレトロウイルスベクター、レンチウイルスベクター、およびアデノ随伴ウイルスベクターでは、ベクターに組み込んだ目的遺伝子が宿主染色体へと組み込まれ、安定かつ長期的な発現が期待できる。各ウイルスベクターは、常法に従い、または市販される専用のキットを用いて、作製することができる。非ウイルスベクターとしては、プラスミドベクター、リポソームベクター、正電荷型リポソームベクター(Felgner, P.L., Gadek, T.R., Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987)、YACベクター、BACベクター、人工染色体ベクター等が挙げられる。
これらのベクターは遺伝子治療にも使用することができ、好ましくはアデノ随伴ウイルスベクターが用いられる。
細胞への発現ベクターの導入は、ウイルスベクターの場合、ウイルスの感染により細胞に導入される。プラスミドなどの非ウイルスベクターの場合、細胞への導入のため、エレクトロポレーション法、リポフェクション法、リン酸カルシウム法、ヌクレオフェクション法等の常法を用いることができ、好ましくはリポフェクション法により導入される。 There is no particular limitation on the type of expression vector that can be used as long as it stably retains the inserted gene, and various types of vectors can be used. The vector can be a viral vector or a non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors. Among these, in retroviral vectors, lentiviral vectors, and adeno-associated viral vectors, the target gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be prepared according to a conventional method or using a commercially available dedicated kit. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, and artificial chromosome vectors.
These vectors can also be used in gene therapy, preferably using adeno-associated virus vectors.
In the case of a viral vector, the expression vector is introduced into a cell by infection with a virus. In the case of a non-viral vector such as a plasmid, a conventional method such as electroporation, lipofection, calcium phosphate, or nucleofection can be used for introduction into a cell, and the lipofection method is preferred.
これらのベクターは遺伝子治療にも使用することができ、好ましくはアデノ随伴ウイルスベクターが用いられる。
細胞への発現ベクターの導入は、ウイルスベクターの場合、ウイルスの感染により細胞に導入される。プラスミドなどの非ウイルスベクターの場合、細胞への導入のため、エレクトロポレーション法、リポフェクション法、リン酸カルシウム法、ヌクレオフェクション法等の常法を用いることができ、好ましくはリポフェクション法により導入される。 There is no particular limitation on the type of expression vector that can be used as long as it stably retains the inserted gene, and various types of vectors can be used. The vector can be a viral vector or a non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors. Among these, in retroviral vectors, lentiviral vectors, and adeno-associated viral vectors, the target gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be prepared according to a conventional method or using a commercially available dedicated kit. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, and artificial chromosome vectors.
These vectors can also be used in gene therapy, preferably using adeno-associated virus vectors.
In the case of a viral vector, the expression vector is introduced into a cell by infection with a virus. In the case of a non-viral vector such as a plasmid, a conventional method such as electroporation, lipofection, calcium phosphate, or nucleofection can be used for introduction into a cell, and the lipofection method is preferred.
GLP-1分泌機能の細胞への付与は、ゲノム編集によって行われてもよい。「ゲノム編集」とは、ヌクレアーゼを用いた部位特異的なゲノムDNA鎖の切断、又は塩基の化学的変換等の原理により標的遺伝子もしくはゲノム領域を意図的に改変する技術である。部位特異的ヌクレアーゼとしては、ジンクフィンガーヌクレアーゼ(ZFN)、TALEN、CRISPR/Cas9等が挙げられる。ゲノム編集技術を用いることにより、特定の遺伝子を欠失したノックアウト細胞株、特定の遺伝子座に人工的に別の配列を挿入したノックイン細胞株等を作製することができる。本発明ではゲノム編集の技術を用いて、多能性幹細胞又はその分化細胞、特に内皮細胞にGLP-1をコードする核酸を導入する。
The ability to secrete GLP-1 may be imparted to cells by genome editing. "Genome editing" is a technique for intentionally modifying a target gene or genome region by site-specific cleavage of a genomic DNA strand using a nuclease, or by chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas9. By using genome editing techniques, it is possible to create knockout cell lines in which a specific gene has been deleted, knock-in cell lines in which a different sequence has been artificially inserted into a specific gene locus, and the like. In the present invention, a nucleic acid encoding GLP-1 is introduced into pluripotent stem cells or their differentiated cells, particularly endothelial cells, using genome editing techniques.
GLP-1分泌機能を細胞に付与する為に導入されるGLP-1をコードする核酸は、細胞に所望するタンパク質(即ちGLP-1)を発現させ得る限り特に限定されない。導入される核酸の塩基配列の例として以下のものが挙げられる。
The nucleic acid encoding GLP-1 that is introduced to impart GLP-1 secretion function to cells is not particularly limited as long as it can cause the cells to express the desired protein (i.e., GLP-1). Examples of the base sequence of the nucleic acid to be introduced include the following.
・GLP-1(7-37)をコードする遺伝子
CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA(配列番号3) The gene encoding GLP-1(7-37)
CATGCTGAAGGGACCTTTACCAGTGATGGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 3)
CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA(配列番号3) The gene encoding GLP-1(7-37)
CATGCTGAAGGGACCTTTACCAGTGATGGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 3)
・GLP-1(7-37)をコードする遺伝子(5’側にシグナルペプチドをコードする配列が連結している)
TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCGCATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA(配列番号4) A gene encoding GLP-1 (7-37) (linked to a sequence encoding a signal peptide on the 5' side)
TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCGCATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 4)
TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCGCATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA(配列番号4) A gene encoding GLP-1 (7-37) (linked to a sequence encoding a signal peptide on the 5' side)
TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCGCATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 4)
GLP-1分泌機能の細胞への付与工程で用いる細胞が多能性幹細胞の場合は、その後の拡大培養における細胞培養は、多能性幹細胞培養用の培地中で実施することが好ましい。多能性幹細胞用の培地としては公知のものを用いることができ、多能性幹細胞の増殖を阻害しない限り特に限定されないが、例えばDMEM、DMEMHG、EMEM、IMDM(Iscove's Modified Dulbecco's Medium)、GMEM(Glasgow's MEM)、RPMI-1640、α-MEM、Ham's Medium F-12、Ham's Medium F-10、Ham's Medium F12K、Medium 199、ATCC-CRCM30、DM-160、DM-201、BME、Fischer、McCoy's 5A、Leibovitz's L-15、RITC80-7、MCDB105、MCDB107、MCDB131、MCDB153、MCDB201、NCTC109、NCTC135、Waymouth's MB752/1、CMRL-1066、Williams' medium E、Brinster's BMOC-3 Medium、E8 medium(Nature Methods, 2011, 8, 424-429)、ReproFF2培地(リプロセル社)、StemFit(登録商標)AK培地(味の素)及びこれらの混合培地等が挙げられる。
かくして得られたGLP-1分泌機能を有する多能性幹細胞を分化誘導し、所望の分化細胞を得ることができる。分化誘導は目的とする分化細胞の種類に応じて適宜決定され、既知の材料/方法によって実施される。例えばGLP-1分泌機能を有する多能性幹細胞を特許文献1~3の記載に従って分化誘導することで、角膜内皮様の細胞である、角膜内皮代替細胞を得ることができる。得られた角膜内皮代替細胞は、GLP-1分泌機能を有する。 When the cells used in the step of imparting GLP-1 secretion function to the cells are pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably carried out in a medium for culturing pluripotent stem cells. As a medium for pluripotent stem cells, a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of pluripotent stem cells. For example, DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's Examples of the medium include BMOC-3 Medium, E8 medium (Nature Methods, 2011, 8, 424-429), ReproFF2 medium (ReproCell), StemFit (registered trademark) AK medium (Ajinomoto), and a mixture of these.
The thus obtained pluripotent stem cells having the function of secreting GLP-1 can be induced to differentiate into desired differentiated cells. Differentiation induction is appropriately determined depending on the type of differentiated cells to be obtained, and is carried out using known materials and methods. For example, corneal endothelial replacement cells, which are corneal endothelial-like cells, can be obtained by inducing differentiation of pluripotent stem cells having the function of secreting GLP-1 according to the descriptions in Patent Documents 1 to 3. The obtained corneal endothelial replacement cells have the function of secreting GLP-1.
かくして得られたGLP-1分泌機能を有する多能性幹細胞を分化誘導し、所望の分化細胞を得ることができる。分化誘導は目的とする分化細胞の種類に応じて適宜決定され、既知の材料/方法によって実施される。例えばGLP-1分泌機能を有する多能性幹細胞を特許文献1~3の記載に従って分化誘導することで、角膜内皮様の細胞である、角膜内皮代替細胞を得ることができる。得られた角膜内皮代替細胞は、GLP-1分泌機能を有する。 When the cells used in the step of imparting GLP-1 secretion function to the cells are pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably carried out in a medium for culturing pluripotent stem cells. As a medium for pluripotent stem cells, a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of pluripotent stem cells. For example, DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's Examples of the medium include BMOC-3 Medium, E8 medium (Nature Methods, 2011, 8, 424-429), ReproFF2 medium (ReproCell), StemFit (registered trademark) AK medium (Ajinomoto), and a mixture of these.
The thus obtained pluripotent stem cells having the function of secreting GLP-1 can be induced to differentiate into desired differentiated cells. Differentiation induction is appropriately determined depending on the type of differentiated cells to be obtained, and is carried out using known materials and methods. For example, corneal endothelial replacement cells, which are corneal endothelial-like cells, can be obtained by inducing differentiation of pluripotent stem cells having the function of secreting GLP-1 according to the descriptions in Patent Documents 1 to 3. The obtained corneal endothelial replacement cells have the function of secreting GLP-1.
GLP-1分泌機能の細胞への付与工程で用いる細胞が多能性幹細胞から分化誘導した分化細胞、好ましくは内皮細胞、特に角膜内皮代替細胞である場合には、その後の拡大培養における細胞培養は、分化細胞、好ましくは内皮細胞、特に角膜内皮代替細胞培養用の培地中で実施することが好ましい。このような培地としては、公知のものを用いることができ、分化細胞の増殖を阻害しない限り特に限定されないが、例えばDMEM、DMEMHG、EMEM、IMDM(Iscove's Modified Dulbecco's Medium)、GMEM(Glasgow's MEM)、RPMI-1640、α-MEM、Ham's Medium F-12、Ham's Medium F-10、Ham's Medium F12K、Medium 199、ATCC-CRCM30、DM-160、DM-201、BME、Fischer、McCoy's 5A、Leibovitz's L-15、RITC80-7、MCDB105、MCDB107、MCDB131、MCDB153、MCDB201、NCTC109、NCTC135、Waymouth's MB752/1、CMRL-1066、Williams' medium E、Brinster's BMOC-3 Medium及びこれらの混合培地等が挙げられる。
なお、多能性幹細胞から分化細胞への分化誘導自体は公知の方法で実施することができる。例えば多能性幹細胞から角膜内皮代替細胞への分化誘導は特許文献1~3の記載に従って実施することができる。 When the cells used in the step of imparting GLP-1 secretion function to cells are differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells, which have been induced to differentiate from pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably performed in a medium for culturing differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells. As such a medium, a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of differentiated cells. For example, DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's BMOC-3 Examples of the medium include medium and mixed mediums thereof.
The differentiation of pluripotent stem cells into differentiated cells can be carried out by known methods. For example, the differentiation of pluripotent stem cells into corneal endothelial replacement cells can be carried out according to the descriptions in Patent Documents 1 to 3.
なお、多能性幹細胞から分化細胞への分化誘導自体は公知の方法で実施することができる。例えば多能性幹細胞から角膜内皮代替細胞への分化誘導は特許文献1~3の記載に従って実施することができる。 When the cells used in the step of imparting GLP-1 secretion function to cells are differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells, which have been induced to differentiate from pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably performed in a medium for culturing differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells. As such a medium, a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of differentiated cells. For example, DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's BMOC-3 Examples of the medium include medium and mixed mediums thereof.
The differentiation of pluripotent stem cells into differentiated cells can be carried out by known methods. For example, the differentiation of pluripotent stem cells into corneal endothelial replacement cells can be carried out according to the descriptions in Patent Documents 1 to 3.
3.医薬組成物
本発明は、GLP-1分泌機能を有する細胞を有効成分として含む医薬組成物(以下、単に本発明の医薬組成物とも称する)を提供する。本発明の医薬組成物に有効成分として含められるGLP-1分泌機能を有する細胞は、上記「1.細胞」の項に記載した細胞であり、上記「2.細胞の製造方法」の項に記載した方法により製造された細胞である。 3. Pharmaceutical composition The present invention provides a pharmaceutical composition comprising cells capable of secreting GLP-1 as an active ingredient (hereinafter, also simply referred to as the pharmaceutical composition of the present invention). The cells capable of secreting GLP-1 contained as an active ingredient in the pharmaceutical composition of the present invention are the cells described above in "1. Cells" and are cells produced by the method described above in "2. Method for producing cells".
本発明は、GLP-1分泌機能を有する細胞を有効成分として含む医薬組成物(以下、単に本発明の医薬組成物とも称する)を提供する。本発明の医薬組成物に有効成分として含められるGLP-1分泌機能を有する細胞は、上記「1.細胞」の項に記載した細胞であり、上記「2.細胞の製造方法」の項に記載した方法により製造された細胞である。 3. Pharmaceutical composition The present invention provides a pharmaceutical composition comprising cells capable of secreting GLP-1 as an active ingredient (hereinafter, also simply referred to as the pharmaceutical composition of the present invention). The cells capable of secreting GLP-1 contained as an active ingredient in the pharmaceutical composition of the present invention are the cells described above in "1. Cells" and are cells produced by the method described above in "2. Method for producing cells".
本発明の医薬組成物は、通常、有効成分である本発明の細胞と医薬上許容され得る担体とを混合して製することができる。「医薬上許容され得る担体」という用語は、本明細書で使用される場合、使用される投与量及び濃度でそれにさらされる細胞に対して非毒性である希釈剤、アジュバント、賦形剤、安定剤、ビヒクル又は支持体(セルファイバ等(アルギン酸ハイドロゲル))を包含する。多くの場合、担体は、水性pH緩衝溶液、抗酸化剤、低分子量(約10残基未満)のポリペプチド、親水性ポリマー、アミノ酸、単糖、二糖、EDTAなどのキレート化剤、ナトリウムなどの塩形成性対イオン;並びにツイーン(TWEEN)(登録商標)、ポリエチレングリコール(PEG)、及びプルロニック(PLURONICS)(登録商標)などの非イオン性界面活性物質であり、特に、静脈内に投与される組成物に関しては、好ましい担体は生理食塩水溶液である。
The pharmaceutical compositions of the present invention can be prepared by mixing the cells of the present invention, which are the active ingredient, with a pharma-
ceutical acceptable carrier. The term "pharma-ceutical acceptable carrier" as used herein includes diluents, adjuvants, excipients, stabilizers, vehicles, or supports (such as cell fiber (alginate hydrogel)) that are non-toxic to cells exposed thereto at the doses and concentrations used. In many cases, the carrier is an aqueous pH buffer solution, an antioxidant, a low molecular weight (less than about 10 residues) polypeptide, a hydrophilic polymer, an amino acid, a monosaccharide, a disaccharide, a chelating agent such as EDTA, a salt-forming counterion such as sodium; and a non-ionic surfactant such as TWEEN (registered trademark), polyethylene glycol (PEG), and PLURONICS (registered trademark). In particular, for compositions administered intravenously, a preferred carrier is a saline solution.
本発明の医薬組成物は、有効成分として、本発明の細胞を含有し、該本発明の細胞は、治療有効量のGLP-1を分泌し得る。治療有効量とは、本発明の医薬組成物を被験体に投与した場合に、該医薬組成物を投与していない被験体と比較して前記のような疾患に対して治療効果を得ることができる量である。具体的な治療有効量としては、投与方法、使用目的および被験体の年齢、体重、症状等によって適宜決定される。
The pharmaceutical composition of the present invention contains the cells of the present invention as an active ingredient, and the cells of the present invention are capable of secreting a therapeutically effective amount of GLP-1. A therapeutically effective amount is an amount that, when the pharmaceutical composition of the present invention is administered to a subject, can provide a therapeutic effect against the above-mentioned diseases, compared to a subject not administered the pharmaceutical composition. A specific therapeutically effective amount is appropriately determined depending on the administration method, the purpose of use, and the age, weight, symptoms, etc. of the subject.
本発明の一実施態様において、GLP-1分泌機能を有する細胞を使用してヒト体内でのGLP-1分泌が可能になる。分泌されたGLP-1は、膵臓のβ細胞に存在するGLP-1受容体に作用し、インスリンの分泌を促す。本発明の医薬組成物が適用可能な疾患としGLP-1がその予防や治療に有用であることが知られている各種疾患であり、具体的には糖尿病(1型糖尿病、2型糖尿病)、肥満、中枢神経障害、末梢神経障害、腎機能障害、心不全、虚血性心疾患、肝機能障害(非アルコール性肝疾患(NASH)等)、味覚障害、肺炎等が挙げられるが、これらに限定されない。
In one embodiment of the present invention, GLP-1 can be secreted in the human body using cells having GLP-1 secretion function. The secreted GLP-1 acts on the GLP-1 receptor present in the β cells of the pancreas, promoting the secretion of insulin. Diseases to which the pharmaceutical composition of the present invention can be applied include various diseases for which GLP-1 is known to be useful for the prevention and treatment of, and specific examples include, but are not limited to, diabetes (type 1 diabetes, type 2 diabetes), obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction (non-alcoholic liver disease (NASH), etc.), taste disorders, pneumonia, etc.
以下に実験例及び実施例を用いて本発明を詳述するが、本発明はそれらに何ら限定されるものではない。また、使用する試薬及び材料は特に限定されない限り商業的に入手可能である。本明細書中で用いた略語は特に断りの無い限り、当分野で通常用いられるものと同様である。
The present invention will be described in detail below using experimental examples and examples, but the present invention is not limited thereto. Furthermore, unless otherwise specified, the reagents and materials used are commercially available. Abbreviations used in this specification are the same as those commonly used in this field.
実験例1:GLP-1の発現ベクターの構築
pRPの哺乳類のベクターにおいて、EF1Aプロモーターを使用し、開始コドンATGを付加したIL2由来のシグナル配列(TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCG)(配列番号5)とGLP-1の配列(CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA)(配列番号6)を付加したものを構築した。T2AリンカーでEGFP(Enhanced Green Fluorescent Protein)に連結することで、翻訳時にEGFPは細胞内に残り、培養上清中には目的のタンパク質(GLP-1)が分泌される構造とした(sig-GLP-1、図6A(b))。すなわち、EGFPの発現はGLP-1を発現している状態と同等とみなされる。ただし、T2Aリンカーの切断効率は100%ではないので、融合タンパク質として培養上清中に分泌されるものもある。従って培養上清中のGFPを検出することで、細胞内にGLP-1が発現されていることを確認することが可能である。コントロールには、シグナルペプチドのみが分泌される構造のものを作製した(sig-peptide、図6A(a))。加えて、T2Aリンカーの代わりに3xGGGGS(配列番号11)を用いたEGFP融合型シグナル配列付加GLP-1の構築も行った(sig-GLP-1-GFP、図6A(d))。この場合のコントロールにはシグナル配列とEGFPを直接的に繋げた配列を用いた(sig-GFP、図6A(c))。
pAAVのウイルス発現ベクターにおいては、プラスミドベクターと同様にEF1Aプロモーターを使用し、T2Aリンカーの代わりにIRESを用いて目的のベクターを構築した(sig-GLP-1、図6A(b))。融合タンパク質型については、pAAVウイルスベクターにプラスミドベクターと同様の配列(T2Aリンカーの代わりに3xGGGGSを用いたEGFP融合型シグナル配列付加GLP-1)を用いて構築した(sig-GLP-1-GFP、図6A(d))。 Experimental Example 1: Construction of an expression vector for GLP-1 A vector for mammals, pRP, was constructed using the EF1A promoter, and was added with a signal sequence (TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCG) (SEQ ID NO: 5) derived from IL2 with the start codon ATG added, and a sequence of GLP-1 (CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA) (SEQ ID NO: 6). By linking to EGFP (Enhanced Green Fluorescent Protein) with a T2A linker, EGFP remains in the cell during translation, and the target protein (GLP-1) is secreted into the culture supernatant (sig-GLP-1, FIG. 6A(b)). In other words, the expression of EGFP is considered to be equivalent to the state in which GLP-1 is expressed. However, since the cleavage efficiency of the T2A linker is not 100%, some of the fusion protein is secreted into the culture supernatant. Therefore, it is possible to confirm that GLP-1 is expressed in the cells by detecting GFP in the culture supernatant. As a control, a structure in which only the signal peptide is secreted was prepared (sig-peptide, FIG. 6A(a)). In addition, an EGFP-fused signal sequence-added GLP-1 was also constructed using 3xGGGGS (SEQ ID NO: 11) instead of the T2A linker (sig-GLP-1-GFP, FIG. 6A(d)). In this case, a sequence in which the signal sequence and EGFP were directly linked was used as a control (sig-GFP, FIG. 6A(c)).
In the pAAV viral expression vector, the EF1A promoter was used as in the plasmid vector, and the target vector was constructed using IRES instead of the T2A linker (sig-GLP-1, FIG. 6A(b)). The fusion protein type was constructed using the same sequence as in the plasmid vector (EGFP fusion signal sequence-added GLP-1 using 3xGGGGS instead of the T2A linker) in the pAAV viral vector (sig-GLP-1-GFP, FIG. 6A(d)).
pRPの哺乳類のベクターにおいて、EF1Aプロモーターを使用し、開始コドンATGを付加したIL2由来のシグナル配列(TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCG)(配列番号5)とGLP-1の配列(CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA)(配列番号6)を付加したものを構築した。T2AリンカーでEGFP(Enhanced Green Fluorescent Protein)に連結することで、翻訳時にEGFPは細胞内に残り、培養上清中には目的のタンパク質(GLP-1)が分泌される構造とした(sig-GLP-1、図6A(b))。すなわち、EGFPの発現はGLP-1を発現している状態と同等とみなされる。ただし、T2Aリンカーの切断効率は100%ではないので、融合タンパク質として培養上清中に分泌されるものもある。従って培養上清中のGFPを検出することで、細胞内にGLP-1が発現されていることを確認することが可能である。コントロールには、シグナルペプチドのみが分泌される構造のものを作製した(sig-peptide、図6A(a))。加えて、T2Aリンカーの代わりに3xGGGGS(配列番号11)を用いたEGFP融合型シグナル配列付加GLP-1の構築も行った(sig-GLP-1-GFP、図6A(d))。この場合のコントロールにはシグナル配列とEGFPを直接的に繋げた配列を用いた(sig-GFP、図6A(c))。
pAAVのウイルス発現ベクターにおいては、プラスミドベクターと同様にEF1Aプロモーターを使用し、T2Aリンカーの代わりにIRESを用いて目的のベクターを構築した(sig-GLP-1、図6A(b))。融合タンパク質型については、pAAVウイルスベクターにプラスミドベクターと同様の配列(T2Aリンカーの代わりに3xGGGGSを用いたEGFP融合型シグナル配列付加GLP-1)を用いて構築した(sig-GLP-1-GFP、図6A(d))。 Experimental Example 1: Construction of an expression vector for GLP-1 A vector for mammals, pRP, was constructed using the EF1A promoter, and was added with a signal sequence (TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCG) (SEQ ID NO: 5) derived from IL2 with the start codon ATG added, and a sequence of GLP-1 (CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA) (SEQ ID NO: 6). By linking to EGFP (Enhanced Green Fluorescent Protein) with a T2A linker, EGFP remains in the cell during translation, and the target protein (GLP-1) is secreted into the culture supernatant (sig-GLP-1, FIG. 6A(b)). In other words, the expression of EGFP is considered to be equivalent to the state in which GLP-1 is expressed. However, since the cleavage efficiency of the T2A linker is not 100%, some of the fusion protein is secreted into the culture supernatant. Therefore, it is possible to confirm that GLP-1 is expressed in the cells by detecting GFP in the culture supernatant. As a control, a structure in which only the signal peptide is secreted was prepared (sig-peptide, FIG. 6A(a)). In addition, an EGFP-fused signal sequence-added GLP-1 was also constructed using 3xGGGGS (SEQ ID NO: 11) instead of the T2A linker (sig-GLP-1-GFP, FIG. 6A(d)). In this case, a sequence in which the signal sequence and EGFP were directly linked was used as a control (sig-GFP, FIG. 6A(c)).
In the pAAV viral expression vector, the EF1A promoter was used as in the plasmid vector, and the target vector was constructed using IRES instead of the T2A linker (sig-GLP-1, FIG. 6A(b)). The fusion protein type was constructed using the same sequence as in the plasmid vector (EGFP fusion signal sequence-added GLP-1 using 3xGGGGS instead of the T2A linker) in the pAAV viral vector (sig-GLP-1-GFP, FIG. 6A(d)).
実験例2:iPS細胞へのGLP-1発現プラスミドの遺伝子導入
ATCC社より購入したiPS細胞(ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026))に実験例1で調製した各プラスミド2μgをLipofectamineTM Stem Transfection Reagent (サーモフィッシャー STEM00001)で遺伝子導入し、24時間後のEGFPの蛍光を蛍光顕微鏡(KEYENCE BZ-X810、倍率x40、露光時間は6sec)で確認した。
iPS細胞にプラスミドを導入することにより細胞内でEGFPの発現が確認された。 Experimental Example 2: Genetic transfer of GLP-1 expression plasmid into iPS cells [0223] 2 µg of each of the plasmids prepared in Experimental Example 1 was transfected into iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) using Lipofectamine ™ Stem Transfection Reagent (Thermo Fisher STEM00001), and the EGFP fluorescence after 24 hours was observed using a fluorescence microscope (KEYENCE BZ-X810, magnification x40, exposure time 6 sec).
By introducing the plasmid into iPS cells, expression of EGFP was confirmed in the cells.
ATCC社より購入したiPS細胞(ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026))に実験例1で調製した各プラスミド2μgをLipofectamineTM Stem Transfection Reagent (サーモフィッシャー STEM00001)で遺伝子導入し、24時間後のEGFPの蛍光を蛍光顕微鏡(KEYENCE BZ-X810、倍率x40、露光時間は6sec)で確認した。
iPS細胞にプラスミドを導入することにより細胞内でEGFPの発現が確認された。 Experimental Example 2: Genetic transfer of GLP-1 expression plasmid into iPS cells [0223] 2 µg of each of the plasmids prepared in Experimental Example 1 was transfected into iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) using Lipofectamine ™ Stem Transfection Reagent (Thermo Fisher STEM00001), and the EGFP fluorescence after 24 hours was observed using a fluorescence microscope (KEYENCE BZ-X810, magnification x40, exposure time 6 sec).
By introducing the plasmid into iPS cells, expression of EGFP was confirmed in the cells.
実験例(実施例)3:CECSi細胞でのGLP-1発現の確認(プラスミドベクター)
3-1:プラスミド導入後48時間での、細胞における蛍光タンパク質の検出
ATCCより購入したiPS細胞(ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026))をiMatrix-511(0.6μg/cm2)をコートした培養皿で、StemFit(登録商標)AK03N培地(味の素)を用いて1週間培養した。その後、iMatrix-511(0.3μg/cm2)をコートした培養皿に播種しなおして、表1の分化誘導培地を用いて、iPS細胞から角膜内皮代替細胞への分化誘導培養を14日間行い、その後細胞を凍結した。分化誘導後14日目で凍結した細胞を融解し、接着培養開始後3日目の状態を蛍光顕微鏡で観察し、角膜内皮代替細胞(CECSi細胞)に分化誘導していることを確認し、以降の実験に供した。ATCCより購入したiPS細胞から誘導して得られたCECSi細胞をATCC CECSI細胞と称する場合がある。
CECSi細胞に分化誘導されたことの確認はZO-1、N-Cadherin、及びPITX2の蛍光免疫染色により行った。具体的には以下の通り。
6ウェルで培養されている細胞において、培養上清を除去した後、1xDPBS(-)500μlで2回洗浄した。1xDPBS(-)を除去し、4%PFAを500μl加え、室温で15分静置した。4%PFAをウェルから除去し、新しい1xDPBS(-)で2回洗浄し、抗体希釈液(0.1%Triton/1%ロバ血清/1xDPBS(-))でブロッキング処理を30分以上行った。ブロッキング処理後の細胞を1次抗体で12時間、室温で反応させた。各1次抗体はそれぞれ抗体希釈液で希釈した;ZO-1(500倍希釈)、N-Cadherin(100倍希釈)、PITX2(200倍希釈)。反応後の細胞を1xDPBS(-)で1回リンスした後、1xDPBS(-)を加え5分間静置する洗浄を2回繰り返した。各2次抗体を抗体希釈液で希釈し、DAPI(1000倍希釈)を加えた2次抗体液を調製した;Alexa488-Rabbit IgG(150希釈)、Cy3-mouse IgG(200倍希釈)。2次抗体液と細胞とを反応させ、1時間半静置した。1xDPBS(-)で1回リンスした後、1xDPBS(-)を加え5分間静置する洗浄を2回繰り返した後、1xDPBS(-)除去後にマウント剤を用いて細胞の表面を覆い、蛍光顕微鏡による観察を行った(図1)。 Experimental Example (Example) 3: Confirmation of GLP-1 expression in CECSi cells (plasmid vector)
3-1: Detection of fluorescent protein in cells 48 hours after plasmid introduction iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were cultured for 1 week on a culture dish coated with iMatrix-511 (0.6 μg/cm 2 ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells were seeded again on a culture dish coated with iMatrix-511 (0.3 μg/cm 2 ) and differentiation induction culture of the iPS cells into corneal endothelial substitute cells was performed for 14 days using the differentiation induction medium in Table 1, and the cells were then frozen. The frozen cells were thawed on the 14th day after the induction of differentiation, and the state was observed under a fluorescent microscope on the 3rd day after the start of adhesion culture to confirm that the cells had been induced to differentiate into corneal endothelial replacement cells (CECSi cells), and the cells were then subjected to the subsequent experiments. The CECSi cells obtained by induction from the iPS cells purchased from ATCC may be referred to as ATCC CECSI cells.
The confirmation of induction of differentiation into CECSi cells was carried out by fluorescent immunostaining of ZO-1, N-Cadherin, and PITX2.
For cells cultured in 6 wells, the culture supernatant was removed, and the wells were washed twice with 500 μl of 1xDPBS(-). The 1xDPBS(-) was removed, 500 μl of 4% PFA was added, and the wells were left to stand at room temperature for 15 minutes. The 4% PFA was removed from the wells, and the wells were washed twice with fresh 1xDPBS(-), and blocked with antibody diluent (0.1% Triton/1% donkey serum/1xDPBS(-)) for 30 minutes or more. The blocked cells were reacted with primary antibodies at room temperature for 12 hours. Each primary antibody was diluted with antibody diluent; ZO-1 (500-fold dilution), N-Cadherin (100-fold dilution), and PITX2 (200-fold dilution). After the reaction, the cells were rinsed once with 1xDPBS(-), and then washed twice by adding 1xDPBS(-) and leaving the wells to stand for 5 minutes. Each secondary antibody was diluted with antibody diluent, and DAPI (1000-fold dilution) was added to prepare secondary antibody solutions; Alexa488-Rabbit IgG (150-fold dilution), Cy3-mouse IgG (200-fold dilution). The secondary antibody solutions were reacted with the cells and left to stand for 1.5 hours. After rinsing once with 1xDPBS(-), 1xDPBS(-) was added and left to stand for 5 minutes, this washing was repeated twice, and after removing 1xDPBS(-), the cell surface was covered with a mounting agent and observed under a fluorescent microscope (Figure 1).
3-1:プラスミド導入後48時間での、細胞における蛍光タンパク質の検出
ATCCより購入したiPS細胞(ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026))をiMatrix-511(0.6μg/cm2)をコートした培養皿で、StemFit(登録商標)AK03N培地(味の素)を用いて1週間培養した。その後、iMatrix-511(0.3μg/cm2)をコートした培養皿に播種しなおして、表1の分化誘導培地を用いて、iPS細胞から角膜内皮代替細胞への分化誘導培養を14日間行い、その後細胞を凍結した。分化誘導後14日目で凍結した細胞を融解し、接着培養開始後3日目の状態を蛍光顕微鏡で観察し、角膜内皮代替細胞(CECSi細胞)に分化誘導していることを確認し、以降の実験に供した。ATCCより購入したiPS細胞から誘導して得られたCECSi細胞をATCC CECSI細胞と称する場合がある。
CECSi細胞に分化誘導されたことの確認はZO-1、N-Cadherin、及びPITX2の蛍光免疫染色により行った。具体的には以下の通り。
6ウェルで培養されている細胞において、培養上清を除去した後、1xDPBS(-)500μlで2回洗浄した。1xDPBS(-)を除去し、4%PFAを500μl加え、室温で15分静置した。4%PFAをウェルから除去し、新しい1xDPBS(-)で2回洗浄し、抗体希釈液(0.1%Triton/1%ロバ血清/1xDPBS(-))でブロッキング処理を30分以上行った。ブロッキング処理後の細胞を1次抗体で12時間、室温で反応させた。各1次抗体はそれぞれ抗体希釈液で希釈した;ZO-1(500倍希釈)、N-Cadherin(100倍希釈)、PITX2(200倍希釈)。反応後の細胞を1xDPBS(-)で1回リンスした後、1xDPBS(-)を加え5分間静置する洗浄を2回繰り返した。各2次抗体を抗体希釈液で希釈し、DAPI(1000倍希釈)を加えた2次抗体液を調製した;Alexa488-Rabbit IgG(150希釈)、Cy3-mouse IgG(200倍希釈)。2次抗体液と細胞とを反応させ、1時間半静置した。1xDPBS(-)で1回リンスした後、1xDPBS(-)を加え5分間静置する洗浄を2回繰り返した後、1xDPBS(-)除去後にマウント剤を用いて細胞の表面を覆い、蛍光顕微鏡による観察を行った(図1)。 Experimental Example (Example) 3: Confirmation of GLP-1 expression in CECSi cells (plasmid vector)
3-1: Detection of fluorescent protein in cells 48 hours after plasmid introduction iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were cultured for 1 week on a culture dish coated with iMatrix-511 (0.6 μg/cm 2 ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells were seeded again on a culture dish coated with iMatrix-511 (0.3 μg/cm 2 ) and differentiation induction culture of the iPS cells into corneal endothelial substitute cells was performed for 14 days using the differentiation induction medium in Table 1, and the cells were then frozen. The frozen cells were thawed on the 14th day after the induction of differentiation, and the state was observed under a fluorescent microscope on the 3rd day after the start of adhesion culture to confirm that the cells had been induced to differentiate into corneal endothelial replacement cells (CECSi cells), and the cells were then subjected to the subsequent experiments. The CECSi cells obtained by induction from the iPS cells purchased from ATCC may be referred to as ATCC CECSI cells.
The confirmation of induction of differentiation into CECSi cells was carried out by fluorescent immunostaining of ZO-1, N-Cadherin, and PITX2.
For cells cultured in 6 wells, the culture supernatant was removed, and the wells were washed twice with 500 μl of 1xDPBS(-). The 1xDPBS(-) was removed, 500 μl of 4% PFA was added, and the wells were left to stand at room temperature for 15 minutes. The 4% PFA was removed from the wells, and the wells were washed twice with fresh 1xDPBS(-), and blocked with antibody diluent (0.1% Triton/1% donkey serum/1xDPBS(-)) for 30 minutes or more. The blocked cells were reacted with primary antibodies at room temperature for 12 hours. Each primary antibody was diluted with antibody diluent; ZO-1 (500-fold dilution), N-Cadherin (100-fold dilution), and PITX2 (200-fold dilution). After the reaction, the cells were rinsed once with 1xDPBS(-), and then washed twice by adding 1xDPBS(-) and leaving the wells to stand for 5 minutes. Each secondary antibody was diluted with antibody diluent, and DAPI (1000-fold dilution) was added to prepare secondary antibody solutions; Alexa488-Rabbit IgG (150-fold dilution), Cy3-mouse IgG (200-fold dilution). The secondary antibody solutions were reacted with the cells and left to stand for 1.5 hours. After rinsing once with 1xDPBS(-), 1xDPBS(-) was added and left to stand for 5 minutes, this washing was repeated twice, and after removing 1xDPBS(-), the cell surface was covered with a mounting agent and observed under a fluorescent microscope (Figure 1).
CECSi細胞であることが確認された細胞に、実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6A(b)))5μgをLipofectamineTM 3000 Transfection Reagent(サーモフィッシャー L3000001)で導入し48時間経過後のEGFPの蛍光を蛍光顕微鏡KEYENCE BZ-X810で確認した(露光時間は10秒)。また、培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーでEGFPの存在量を計測した。485nmの波長で励起し、535nmの吸収を測定した。
結果を図2に示す。図2Aは、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図2Bは細胞培養上清中のEGFP量を測定した結果を示す。遺伝子導入後48時間で細胞内のEGFPの発現が確認できたことから、上流のGLP-1も発現して培養上清中に分泌されていると考えられた。なお、培養上清中にもEGFPが検出されたので、融合タンパク質が分泌していることが確認できた。 5 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b))) prepared in Experimental Example 1 was introduced into the cells confirmed to be CECSi cells using Lipofectamine ™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the EGFP fluorescence after 48 hours was confirmed using a KEYENCE BZ-X810 fluorescence microscope (exposure time: 10 seconds). The amount of EGFP present in the secretory protein contained in the culture supernatant was measured using a TECAN Spark fluorescence plate reader. Excitation was performed at a wavelength of 485 nm, and absorption at 535 nm was measured.
The results are shown in Figure 2. Figure 2A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 2B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 48 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.
結果を図2に示す。図2Aは、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図2Bは細胞培養上清中のEGFP量を測定した結果を示す。遺伝子導入後48時間で細胞内のEGFPの発現が確認できたことから、上流のGLP-1も発現して培養上清中に分泌されていると考えられた。なお、培養上清中にもEGFPが検出されたので、融合タンパク質が分泌していることが確認できた。 5 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b))) prepared in Experimental Example 1 was introduced into the cells confirmed to be CECSi cells using Lipofectamine ™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the EGFP fluorescence after 48 hours was confirmed using a KEYENCE BZ-X810 fluorescence microscope (exposure time: 10 seconds). The amount of EGFP present in the secretory protein contained in the culture supernatant was measured using a TECAN Spark fluorescence plate reader. Excitation was performed at a wavelength of 485 nm, and absorption at 535 nm was measured.
The results are shown in Figure 2. Figure 2A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 2B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 48 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.
3-2:プラスミド導入後65時間での、細胞における蛍光タンパク質の検出
上記3-1と同様に、ATCC CECSi細胞を用い、実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6A(b)))2μgをLipofectamineTM 3000 Transfection Reagent(サーモフィッシャー L3000001)で遺伝子導入し65時間のEGFPの蛍光を蛍光顕微鏡KEYENCE BZ-X810で確認した(露光時間は10sec)。また、培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーでEGFPの存在量を計測した。485nmの波長で励起し、535nmの吸収を測定した。
結果を図3に示す。図3Aは、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図3Bは細胞培養上清中のEGFP量を測定した結果を示す。遺伝子導入後65時間で細胞内のEGFPの発現が確認できたことから、上流のGLP-1も発現して培養上清中に分泌されていると考えられた。なお、培養上清中にもEGFPが検出されたので、融合タンパク質が分泌していることが確認できた。 3-2: Detection of fluorescent protein in cells 65 hours after plasmid introduction As in 3-1 above, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b))) prepared in Experimental Example 1 was introduced using Lipofectamine ™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 65 hours was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 10 sec). In addition, the amount of EGFP present in the secretory protein contained in the culture supernatant was measured using a TECAN Spark fluorescent plate reader. Excitation was performed at a wavelength of 485 nm, and absorption at 535 nm was measured.
The results are shown in Figure 3. Figure 3A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 3B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 65 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.
上記3-1と同様に、ATCC CECSi細胞を用い、実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6A(b)))2μgをLipofectamineTM 3000 Transfection Reagent(サーモフィッシャー L3000001)で遺伝子導入し65時間のEGFPの蛍光を蛍光顕微鏡KEYENCE BZ-X810で確認した(露光時間は10sec)。また、培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーでEGFPの存在量を計測した。485nmの波長で励起し、535nmの吸収を測定した。
結果を図3に示す。図3Aは、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図3Bは細胞培養上清中のEGFP量を測定した結果を示す。遺伝子導入後65時間で細胞内のEGFPの発現が確認できたことから、上流のGLP-1も発現して培養上清中に分泌されていると考えられた。なお、培養上清中にもEGFPが検出されたので、融合タンパク質が分泌していることが確認できた。 3-2: Detection of fluorescent protein in cells 65 hours after plasmid introduction As in 3-1 above, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b))) prepared in Experimental Example 1 was introduced using Lipofectamine ™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 65 hours was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 10 sec). In addition, the amount of EGFP present in the secretory protein contained in the culture supernatant was measured using a TECAN Spark fluorescent plate reader. Excitation was performed at a wavelength of 485 nm, and absorption at 535 nm was measured.
The results are shown in Figure 3. Figure 3A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 3B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 65 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.
3-3:プラスミド導入後24時間又は5日目での、遺伝子発現の確認
上記3-1と同様に、ATCC CECSi細胞を用い、実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6(b)))2μgをLipofectamineTM 3000 Transfection Reagent(サーモフィッシャー L3000001)で遺伝子導入し24時間又は5日目のEGFPの蛍光を蛍光顕微鏡KEYENCE BZ-X810で確認した(露光時間は9sec)。また、細胞内の遺伝子発現の確認のため、細胞からRNeasy Plus Mini kit(Qiagen 74134)を用いてmRNAを抽出し、RevaTraAce逆転写酵素(Takara TRT-101)で0.5μgのmRNAを逆転写しcDNAを調製した。qPCR用プライマーは以下のものを使用した。
GLP-1 Forward Primer: TTATTTGGAAGGCCAAGCTGC(配列番号7)
GLP-1 Reverse Primer: TTAGAAGACTTCCCCTGCCCT(配列番号8)
GAPDH Forward Primer: GAAGGTGAAGGTCGGAGTC(配列番号9)
GAPDH Reverse Primer: GAAGATGGTGATGGGATTTC(配列番号10)
qPCRはCronoSTAR Portable 4を用いた2step法で行った。
結果を図4及び図5に示す。図4(A)は、遺伝子導入後24時間の、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図4(B)は遺伝子発現の結果を示すグラフである。遺伝子導入後24時間でmRNAレベルでの発現が確認された。図5Aは、遺伝子導入後5日目の、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図5Bは遺伝子発現の結果を示すグラフである。遺伝子導入後5日目の細胞でもmRNAレベルでの発現が確認された。 3-3: Confirmation of gene expression 24 hours or 5 days after plasmid introduction As in 3-1 above, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6(b))) prepared in Experimental Example 1 was introduced using Lipofectamine ™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 24 hours or 5 days was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 9 sec). In addition, to confirm intracellular gene expression, mRNA was extracted from the cells using RNeasy Plus Mini kit (Qiagen 74134), and 0.5 μg of mRNA was reverse transcribed with RevaTraAce reverse transcriptase (Takara TRT-101) to prepare cDNA. The following qPCR primers were used.
GLP-1 Forward Primer: TTATTTGGAAGGCCAAGCTGC (SEQ ID NO: 7)
GLP-1 Reverse Primer: TTAGAAGACTTCCCCTGCCCT (SEQ ID NO: 8)
GAPDH Forward Primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 9)
GAPDH Reverse Primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 10)
qPCR was performed using CronoSTAR Portable 4 by the 2-step method.
The results are shown in Figures 4 and 5. Figure 4(A) shows an image (fluorescence, bright field) observed by a fluorescent microscope 24 hours after gene transfer, and Figure 4(B) is a graph showing the results of gene expression. Expression at the mRNA level was confirmed 24 hours after gene transfer. Figure 5A shows an image (fluorescence, bright field) observed by a fluorescent microscope 5 days after gene transfer, and Figure 5B is a graph showing the results of gene expression. Expression at the mRNA level was also confirmed in cells 5 days after gene transfer.
上記3-1と同様に、ATCC CECSi細胞を用い、実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6(b)))2μgをLipofectamineTM 3000 Transfection Reagent(サーモフィッシャー L3000001)で遺伝子導入し24時間又は5日目のEGFPの蛍光を蛍光顕微鏡KEYENCE BZ-X810で確認した(露光時間は9sec)。また、細胞内の遺伝子発現の確認のため、細胞からRNeasy Plus Mini kit(Qiagen 74134)を用いてmRNAを抽出し、RevaTraAce逆転写酵素(Takara TRT-101)で0.5μgのmRNAを逆転写しcDNAを調製した。qPCR用プライマーは以下のものを使用した。
GLP-1 Forward Primer: TTATTTGGAAGGCCAAGCTGC(配列番号7)
GLP-1 Reverse Primer: TTAGAAGACTTCCCCTGCCCT(配列番号8)
GAPDH Forward Primer: GAAGGTGAAGGTCGGAGTC(配列番号9)
GAPDH Reverse Primer: GAAGATGGTGATGGGATTTC(配列番号10)
qPCRはCronoSTAR Portable 4を用いた2step法で行った。
結果を図4及び図5に示す。図4(A)は、遺伝子導入後24時間の、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図4(B)は遺伝子発現の結果を示すグラフである。遺伝子導入後24時間でmRNAレベルでの発現が確認された。図5Aは、遺伝子導入後5日目の、蛍光顕微鏡による観察像(蛍光、明視野)を示し、図5Bは遺伝子発現の結果を示すグラフである。遺伝子導入後5日目の細胞でもmRNAレベルでの発現が確認された。 3-3: Confirmation of gene expression 24 hours or 5 days after plasmid introduction As in 3-1 above, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6(b))) prepared in Experimental Example 1 was introduced using Lipofectamine ™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 24 hours or 5 days was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 9 sec). In addition, to confirm intracellular gene expression, mRNA was extracted from the cells using RNeasy Plus Mini kit (Qiagen 74134), and 0.5 μg of mRNA was reverse transcribed with RevaTraAce reverse transcriptase (Takara TRT-101) to prepare cDNA. The following qPCR primers were used.
GLP-1 Forward Primer: TTATTTGGAAGGCCAAGCTGC (SEQ ID NO: 7)
GLP-1 Reverse Primer: TTAGAAGACTTCCCCTGCCCT (SEQ ID NO: 8)
GAPDH Forward Primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 9)
GAPDH Reverse Primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 10)
qPCR was performed using CronoSTAR Portable 4 by the 2-step method.
The results are shown in Figures 4 and 5. Figure 4(A) shows an image (fluorescence, bright field) observed by a fluorescent microscope 24 hours after gene transfer, and Figure 4(B) is a graph showing the results of gene expression. Expression at the mRNA level was confirmed 24 hours after gene transfer. Figure 5A shows an image (fluorescence, bright field) observed by a fluorescent microscope 5 days after gene transfer, and Figure 5B is a graph showing the results of gene expression. Expression at the mRNA level was also confirmed in cells 5 days after gene transfer.
実験例(実施例)4:細胞培養上清中のGLP-1の検出
実験例3の3-1と同様に、ATCC CECSi細胞を用い実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6A(b))、sig-GFP(図6A(c))、sig-GLP-1-GFP(図6A(d)))2μgを導入し、19時間後に、培養上清中に分泌されるGLP-1のタンパク質(GFPとの融合型)をウエスタンブロッティングで検出した。培養上清500μlをVIVAspin 500 10kDa MWCOで濃縮後、1xDPBS(-)によりバッファーを置換した。約5~10μlの濃縮後サンプルに、6倍濃縮SDSサンプルバッファー(ナカライテスク)に還元剤(b-me)を加えたものを使用し、1倍のサンプルバッファーになるよう加え、サンプル調整を行った。ATTOパジェランAceにe-PAGEL ミニサイズ既製ゲル(5~20%)を準備し、サンプルを全量アプライし、スタンダード80の条件で80分電気泳動した。泳動終了後、ATTOパワードブロット2M(WSE-4125)を用い、25mV、30分間の条件でタンパク質をメンブレンに転写した。転写後のメンブレンはブロッキングワン(ナカライテスク03953-66)で室温、2時間のブロッキング処理を施した後、1次抗体Anti GFP(Green Fluorescent Protein)pAb(MBL 598)を1000倍に希釈し、4℃で一晩反応させた。1xTBS/0.05%Tweenバッファーでメンブレンを洗浄後、2000倍に希釈した2次抗体HRP-linked IgG (CST #7074S)とメンブレンを1時間反応させた。反応終了後、1xTBS/0.05%Tweenバッファーでメンブレンを洗浄し、Nacalai Chemi-lumi One Ultra(ナカライテスク 11644)でELC反応を行った。メンブレンの化学発光はiBright FL1000 Imaging Systemsにて検出した。
結果を図6Bに示す。上段は各ペプチド及びそれをコードするプラスミドの模式図を示す。下段は、抗GFP抗体を用いたウエスタンブロッティングの結果を示す。
培養上清中にsig-GLP-1-GFPと想定されるタンパク質をGFPにより確認できたことにより、sig-GLP-1も培養上清中に存在することが想定される。 Experimental Example (Example) 4: Detection of GLP-1 in cell culture supernatant As in Experimental Example 3, 3-1, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b)), sig-GFP (FIG. 6A(c)), sig-GLP-1-GFP (FIG. 6A(d))) prepared in Experimental Example 1 was introduced, and after 19 hours, the GLP-1 protein (GFP fusion type) secreted into the culture supernatant was detected by Western blotting. 500 μl of the culture supernatant was concentrated with VIVAspin 500 10 kDa MWCO, and the buffer was replaced with 1×DPBS(−). About 5 to 10 μl of the concentrated sample was added with 6-fold concentrated SDS sample buffer (Nacalai Tesque) to which a reducing agent (b-me) was added to make the sample 1-fold sample buffer, and the sample was adjusted. A mini-sized e-PAGEL gel (5-20%) was prepared on ATTO Pagelan Ace, the entire sample was applied, and electrophoresis was performed for 80 minutes under standard 80 conditions. After electrophoresis, the protein was transferred to the membrane using ATTO Powered Blot 2M (WSE-4125) at 25 mV for 30 minutes. The membrane after transfer was subjected to blocking treatment with Blocking One (Nacalai Tesque 03953-66) at room temperature for 2 hours, and then the primary antibody Anti GFP (Green Fluorescent Protein) pAb (MBL 598) was diluted 1000 times and reacted overnight at 4 ° C. After washing the membrane with 1x TBS / 0.05% Tween buffer, the membrane was reacted with the secondary antibody HRP-linked IgG (CST # 7074S) diluted 2000 times for 1 hour. After the reaction, the membrane was washed with 1x TBS/0.05% Tween buffer and subjected to ELC reaction using Nacalai Chemi-lumi One Ultra (Nacalai Tesque 11644). The chemiluminescence of the membrane was detected using iBright FL1000 Imaging Systems.
The results are shown in Figure 6B. The upper panel shows schematic diagrams of each peptide and the plasmids encoding them. The lower panel shows the results of Western blotting using an anti-GFP antibody.
Since the protein assumed to be sig-GLP-1-GFP was confirmed in the culture supernatant by GFP, it is assumed that sig-GLP-1 is also present in the culture supernatant.
実験例3の3-1と同様に、ATCC CECSi細胞を用い実験例1で調製した各プラスミド(sig-peptide(図6A(a))、sig-GLP-1(図6A(b))、sig-GFP(図6A(c))、sig-GLP-1-GFP(図6A(d)))2μgを導入し、19時間後に、培養上清中に分泌されるGLP-1のタンパク質(GFPとの融合型)をウエスタンブロッティングで検出した。培養上清500μlをVIVAspin 500 10kDa MWCOで濃縮後、1xDPBS(-)によりバッファーを置換した。約5~10μlの濃縮後サンプルに、6倍濃縮SDSサンプルバッファー(ナカライテスク)に還元剤(b-me)を加えたものを使用し、1倍のサンプルバッファーになるよう加え、サンプル調整を行った。ATTOパジェランAceにe-PAGEL ミニサイズ既製ゲル(5~20%)を準備し、サンプルを全量アプライし、スタンダード80の条件で80分電気泳動した。泳動終了後、ATTOパワードブロット2M(WSE-4125)を用い、25mV、30分間の条件でタンパク質をメンブレンに転写した。転写後のメンブレンはブロッキングワン(ナカライテスク03953-66)で室温、2時間のブロッキング処理を施した後、1次抗体Anti GFP(Green Fluorescent Protein)pAb(MBL 598)を1000倍に希釈し、4℃で一晩反応させた。1xTBS/0.05%Tweenバッファーでメンブレンを洗浄後、2000倍に希釈した2次抗体HRP-linked IgG (CST #7074S)とメンブレンを1時間反応させた。反応終了後、1xTBS/0.05%Tweenバッファーでメンブレンを洗浄し、Nacalai Chemi-lumi One Ultra(ナカライテスク 11644)でELC反応を行った。メンブレンの化学発光はiBright FL1000 Imaging Systemsにて検出した。
結果を図6Bに示す。上段は各ペプチド及びそれをコードするプラスミドの模式図を示す。下段は、抗GFP抗体を用いたウエスタンブロッティングの結果を示す。
培養上清中にsig-GLP-1-GFPと想定されるタンパク質をGFPにより確認できたことにより、sig-GLP-1も培養上清中に存在することが想定される。 Experimental Example (Example) 4: Detection of GLP-1 in cell culture supernatant As in Experimental Example 3, 3-1, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b)), sig-GFP (FIG. 6A(c)), sig-GLP-1-GFP (FIG. 6A(d))) prepared in Experimental Example 1 was introduced, and after 19 hours, the GLP-1 protein (GFP fusion type) secreted into the culture supernatant was detected by Western blotting. 500 μl of the culture supernatant was concentrated with VIVAspin 500 10 kDa MWCO, and the buffer was replaced with 1×DPBS(−). About 5 to 10 μl of the concentrated sample was added with 6-fold concentrated SDS sample buffer (Nacalai Tesque) to which a reducing agent (b-me) was added to make the sample 1-fold sample buffer, and the sample was adjusted. A mini-sized e-PAGEL gel (5-20%) was prepared on ATTO Pagelan Ace, the entire sample was applied, and electrophoresis was performed for 80 minutes under standard 80 conditions. After electrophoresis, the protein was transferred to the membrane using ATTO Powered Blot 2M (WSE-4125) at 25 mV for 30 minutes. The membrane after transfer was subjected to blocking treatment with Blocking One (Nacalai Tesque 03953-66) at room temperature for 2 hours, and then the primary antibody Anti GFP (Green Fluorescent Protein) pAb (MBL 598) was diluted 1000 times and reacted overnight at 4 ° C. After washing the membrane with 1x TBS / 0.05% Tween buffer, the membrane was reacted with the secondary antibody HRP-linked IgG (CST # 7074S) diluted 2000 times for 1 hour. After the reaction, the membrane was washed with 1x TBS/0.05% Tween buffer and subjected to ELC reaction using Nacalai Chemi-lumi One Ultra (Nacalai Tesque 11644). The chemiluminescence of the membrane was detected using iBright FL1000 Imaging Systems.
The results are shown in Figure 6B. The upper panel shows schematic diagrams of each peptide and the plasmids encoding them. The lower panel shows the results of Western blotting using an anti-GFP antibody.
Since the protein assumed to be sig-GLP-1-GFP was confirmed in the culture supernatant by GFP, it is assumed that sig-GLP-1 is also present in the culture supernatant.
実験例(実施例)5:CECSi細胞でのGLP-1発現の確認(AAVベクター)
5-1:AAVウイルス感染後での、細胞における蛍光タンパク質の検出
ベクタービルダー社にて構築したベクターと該ベクターを用いて作製されたAAVウイルスを購入した(ウイルスは感染効率2×111を保証する製品)。ベクター構築に用いたプラスミドは、sig-GFP(コントロール、図6A(c))及びsig-GLP-1-GFP(図6A(d))である。
一方、ATCCより購入したiPS細胞(ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026))から、実験例3の方法に従い分化誘導した。分化誘導後14日目で細胞を凍結した(凍結ATCC CECSi細胞)。
凍結ATCC CECSi細胞を24ウェルに5.5x104個で播種し、約MOI:100000で感染を行った。翌日ウイルスを除去し、培地(表1の培地組成中、DMEM/F12,ITS,IGF-1のみを含む培地)に培地交換後、24時間後に蛍光顕微鏡KEYENCE BZ-X810で蛍光観察を行った。ウイルス感染後13日目の、細胞におけるGFP発現状況を図7Aに示す(蛍光、明視野)。培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーにてEGFPの蛍光強度を計測した。培養上清を黒透明底96ウェルプレートに、100μlずつアプライし、485nmの波長で励起し、535nmの吸収を測定した。結果を図7Bに示す。
感染後13日目でも細胞内でのEGFPの発現が確認でき、培養上清中でもEGFPとして検出された。18日目においても同様の結果が得られた。 Experimental Example (Example) 5: Confirmation of GLP-1 expression in CECSi cells (AAV vector)
5-1: Detection of fluorescent protein in cells after AAV virus infection A vector constructed by Vector Builder and an AAV virus produced using the vector were purchased (the virus is a product with a guaranteed infection efficiency of 2× 111 ). The plasmids used in the vector construction were sig-GFP (control, FIG. 6A(c)) and sig-GLP-1-GFP (FIG. 6A(d)).
Meanwhile, iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were induced to differentiate according to the method of Experimental Example 3. The cells were frozen 14 days after differentiation induction (frozen ATCC CECSi cells).
Frozen ATCC CECSi cells were seeded at 5.5x104 cells in 24 wells and infected at approximately MOI: 100,000. The virus was removed the next day, and the medium was replaced with a medium (medium containing only DMEM/F12, ITS, and IGF-1 in the medium composition in Table 1), and then fluorescence observation was performed 24 hours later using a fluorescent microscope KEYENCE BZ-X810. GFP expression in the cells 13 days after virus infection is shown in Figure 7A (fluorescence, bright field). The secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 μl of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in Figure 7B.
Even on day 13 after infection, intracellular expression of EGFP was confirmed, and EGFP was also detected in the culture supernatant. Similar results were obtained on day 18.
5-1:AAVウイルス感染後での、細胞における蛍光タンパク質の検出
ベクタービルダー社にて構築したベクターと該ベクターを用いて作製されたAAVウイルスを購入した(ウイルスは感染効率2×111を保証する製品)。ベクター構築に用いたプラスミドは、sig-GFP(コントロール、図6A(c))及びsig-GLP-1-GFP(図6A(d))である。
一方、ATCCより購入したiPS細胞(ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026))から、実験例3の方法に従い分化誘導した。分化誘導後14日目で細胞を凍結した(凍結ATCC CECSi細胞)。
凍結ATCC CECSi細胞を24ウェルに5.5x104個で播種し、約MOI:100000で感染を行った。翌日ウイルスを除去し、培地(表1の培地組成中、DMEM/F12,ITS,IGF-1のみを含む培地)に培地交換後、24時間後に蛍光顕微鏡KEYENCE BZ-X810で蛍光観察を行った。ウイルス感染後13日目の、細胞におけるGFP発現状況を図7Aに示す(蛍光、明視野)。培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーにてEGFPの蛍光強度を計測した。培養上清を黒透明底96ウェルプレートに、100μlずつアプライし、485nmの波長で励起し、535nmの吸収を測定した。結果を図7Bに示す。
感染後13日目でも細胞内でのEGFPの発現が確認でき、培養上清中でもEGFPとして検出された。18日目においても同様の結果が得られた。 Experimental Example (Example) 5: Confirmation of GLP-1 expression in CECSi cells (AAV vector)
5-1: Detection of fluorescent protein in cells after AAV virus infection A vector constructed by Vector Builder and an AAV virus produced using the vector were purchased (the virus is a product with a guaranteed infection efficiency of 2× 111 ). The plasmids used in the vector construction were sig-GFP (control, FIG. 6A(c)) and sig-GLP-1-GFP (FIG. 6A(d)).
Meanwhile, iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were induced to differentiate according to the method of Experimental Example 3. The cells were frozen 14 days after differentiation induction (frozen ATCC CECSi cells).
Frozen ATCC CECSi cells were seeded at 5.5x104 cells in 24 wells and infected at approximately MOI: 100,000. The virus was removed the next day, and the medium was replaced with a medium (medium containing only DMEM/F12, ITS, and IGF-1 in the medium composition in Table 1), and then fluorescence observation was performed 24 hours later using a fluorescent microscope KEYENCE BZ-X810. GFP expression in the cells 13 days after virus infection is shown in Figure 7A (fluorescence, bright field). The secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 μl of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in Figure 7B.
Even on day 13 after infection, intracellular expression of EGFP was confirmed, and EGFP was also detected in the culture supernatant. Similar results were obtained on day 18.
5-2:AAVウイルス感染後での、細胞における蛍光タンパク質の検出
ベクター構築に用いたプラスミドをsig-peptide(コントロール、図6A(a))及びsig-GLP-1(図6A(b))に替えた以外は、上記5-1と同様にAAVウイルス感染後の蛍光タンパク質の発現を調べた。AAV感染後2日目の結果を図8に示す。ウイルス感染後2日目の、細胞におけるGFP発現状況を図8Aに示す(蛍光、明視野)。培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーにてEGFPの蛍光強度を計測した。培養上清を黒透明底96ウェルプレートに、100μlずつアプライし、485nmの波長で励起し、535nmの吸収を測定した。結果を図8Bに示す。
感染後2日目でも細胞内でのEGFPの発現が確認でき、培養上清中でもGFPとして検出された。3日目、7日目、18日目、22日目、32日目(図9)においても細胞内のEGFPの発現が確認できた。つまり、構築したベクターにおいてEGFPの上流にある、GLP-1が発現しているといえる。 5-2: Detection of fluorescent protein in cells after AAV virus infection The expression of fluorescent protein after AAV virus infection was examined in the same manner as in 5-1 above, except that the plasmid used for vector construction was replaced with sig-peptide (control, FIG. 6A(a)) and sig-GLP-1 (FIG. 6A(b)). The results on the second day after AAV infection are shown in FIG. 8. The state of GFP expression in cells on the second day after virus infection is shown in FIG. 8A (fluorescence, bright field). The secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 μl of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in FIG. 8B.
Expression of EGFP in the cells was confirmed even on day 2 after infection, and was also detected as GFP in the culture supernatant. Expression of EGFP in the cells was also confirmed on days 3, 7, 18, 22, and 32 (FIG. 9). In other words, it can be said that GLP-1, which is located upstream of EGFP in the constructed vector, is expressed.
ベクター構築に用いたプラスミドをsig-peptide(コントロール、図6A(a))及びsig-GLP-1(図6A(b))に替えた以外は、上記5-1と同様にAAVウイルス感染後の蛍光タンパク質の発現を調べた。AAV感染後2日目の結果を図8に示す。ウイルス感染後2日目の、細胞におけるGFP発現状況を図8Aに示す(蛍光、明視野)。培養上清中に含まれる分泌タンパク質は、TECAN Spark 蛍光プレードリーダーにてEGFPの蛍光強度を計測した。培養上清を黒透明底96ウェルプレートに、100μlずつアプライし、485nmの波長で励起し、535nmの吸収を測定した。結果を図8Bに示す。
感染後2日目でも細胞内でのEGFPの発現が確認でき、培養上清中でもGFPとして検出された。3日目、7日目、18日目、22日目、32日目(図9)においても細胞内のEGFPの発現が確認できた。つまり、構築したベクターにおいてEGFPの上流にある、GLP-1が発現しているといえる。 5-2: Detection of fluorescent protein in cells after AAV virus infection The expression of fluorescent protein after AAV virus infection was examined in the same manner as in 5-1 above, except that the plasmid used for vector construction was replaced with sig-peptide (control, FIG. 6A(a)) and sig-GLP-1 (FIG. 6A(b)). The results on the second day after AAV infection are shown in FIG. 8. The state of GFP expression in cells on the second day after virus infection is shown in FIG. 8A (fluorescence, bright field). The secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 μl of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in FIG. 8B.
Expression of EGFP in the cells was confirmed even on day 2 after infection, and was also detected as GFP in the culture supernatant. Expression of EGFP in the cells was also confirmed on days 3, 7, 18, 22, and 32 (FIG. 9). In other words, it can be said that GLP-1, which is located upstream of EGFP in the constructed vector, is expressed.
実験例(実施例)6:CECSi細胞で発現・分泌されたGLP-1の機能評価(c-peptide分泌促進効果)
ヒトiPS由来β細胞を用いsig-GLP-1-GFP(融合型、図6A(d))のin vitroでの機能を確認した。ヒトiPS細胞由来膵β細胞とsig-GLP-1-GFPを分泌するATCC CECSi細胞はそれぞれ別に準備し、適当なタイミングで共培養を実施した。それぞれの方法を示す。
ヒトiPS細胞由来膵β細胞であるCellartis(登録商標) hiPS Beta Cells(ChiPS22)(Takara Bio:Y10100)を、付属のコーティング剤でコーティングした24ウェル用カルチャーインサート(ファルコン CBP 353104)に起眠し、キットに添付のBasal Medium1にサプリメントを加えた培地で8日間培養後、9日目からBasal Medium2にサプリメントを加えた培地に変え3日目の細胞を用いて、共培養を開始した。
分化誘導10日後に凍結した、凍結ATCC CECSi細胞を24ウェルプレートに1.1×105個/ウェルで起眠したのち、4日目にAAV2-sigGLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターをMOI:100000で感染させ、翌日ウイルスベクターを除去した。2日おきに培地交換をしながら、感染後4日目に共培養を行った。共培養を開始する際、クレブスバッファー(KRB)に置換し、24時間後に膵β細胞の上清を回収後再びKRBを加え24時間経過した後の上清中に含まれるc-peptideの量をc-peptide ELISA kit(エービークローン RKO3588)を用いて測定した。コントロールGFPウイルスベクターを感染させた条件に比べ、AAV2-sigGLP-1-GFP(融合型)ウイルスベクターを感染させた条件との共培養では培地中に分泌されたc-peptideの増加が確認された(図10B)。
AAV2-sigGLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターは、セロタイプ2のAAV(AAV2)を用い、それぞれプラスミドsig-GLP-1-GFP(図6A(d))又はEGFP配列のみ有するプラスミドを用いてベクター構築することによって作製した。 Experimental Example (Example) 6: Functional evaluation of GLP-1 expressed and secreted in CECSi cells (c-peptide secretion promoting effect)
The in vitro function of sig-GLP-1-GFP (fusion type, FIG. 6A(d)) was confirmed using human iPS cell-derived β cells. Human iPS cell-derived pancreatic β cells and ATCC CECSi cells secreting sig-GLP-1-GFP were prepared separately, and co-cultured at appropriate times. The methods for each are described below.
Cellartis (registered trademark) hiPS Beta Cells (ChiPS22) (Takara Bio: Y10100), which are pancreatic β cells derived from human iPS cells, were placed on a 24-well culture insert (Falcon CBP 353104) coated with the attached coating agent, and cultured for 8 days in Basal Medium 1 supplemented with supplements provided in the kit. From the 9th day, the medium was changed to Basal Medium 2 supplemented with supplements, and co-culture was initiated using cells on the 3rd day.
Frozen ATCC CECSi cells, frozen 10 days after differentiation induction, were placed in a 24-well plate at 1.1 x 105 cells/well and then infected with AAV2-sigGLP-1-GFP (fusion type) viral vector and control GFP viral vector at MOI: 100,000 on the fourth day, and the viral vector was removed the next day. The medium was replaced every two days, and co-culture was performed on the fourth day after infection. When co-culture was started, the medium was replaced with Krebs buffer (KRB), and after 24 hours, the supernatant of pancreatic β cells was collected, KRB was added again, and the amount of c-peptide contained in the supernatant after 24 hours was measured using a c-peptide ELISA kit (AB Clone RKO3588). Compared to infection with the control GFP viral vector, co-culture with the AAV2-sigGLP-1-GFP (fusion type) viral vector showed an increase in the amount of c-peptide secreted into the medium (Figure 10B).
The AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the plasmid sig-GLP-1-GFP (Figure 6A(d)) or a plasmid containing only the EGFP sequence, respectively.
ヒトiPS由来β細胞を用いsig-GLP-1-GFP(融合型、図6A(d))のin vitroでの機能を確認した。ヒトiPS細胞由来膵β細胞とsig-GLP-1-GFPを分泌するATCC CECSi細胞はそれぞれ別に準備し、適当なタイミングで共培養を実施した。それぞれの方法を示す。
ヒトiPS細胞由来膵β細胞であるCellartis(登録商標) hiPS Beta Cells(ChiPS22)(Takara Bio:Y10100)を、付属のコーティング剤でコーティングした24ウェル用カルチャーインサート(ファルコン CBP 353104)に起眠し、キットに添付のBasal Medium1にサプリメントを加えた培地で8日間培養後、9日目からBasal Medium2にサプリメントを加えた培地に変え3日目の細胞を用いて、共培養を開始した。
分化誘導10日後に凍結した、凍結ATCC CECSi細胞を24ウェルプレートに1.1×105個/ウェルで起眠したのち、4日目にAAV2-sigGLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターをMOI:100000で感染させ、翌日ウイルスベクターを除去した。2日おきに培地交換をしながら、感染後4日目に共培養を行った。共培養を開始する際、クレブスバッファー(KRB)に置換し、24時間後に膵β細胞の上清を回収後再びKRBを加え24時間経過した後の上清中に含まれるc-peptideの量をc-peptide ELISA kit(エービークローン RKO3588)を用いて測定した。コントロールGFPウイルスベクターを感染させた条件に比べ、AAV2-sigGLP-1-GFP(融合型)ウイルスベクターを感染させた条件との共培養では培地中に分泌されたc-peptideの増加が確認された(図10B)。
AAV2-sigGLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターは、セロタイプ2のAAV(AAV2)を用い、それぞれプラスミドsig-GLP-1-GFP(図6A(d))又はEGFP配列のみ有するプラスミドを用いてベクター構築することによって作製した。 Experimental Example (Example) 6: Functional evaluation of GLP-1 expressed and secreted in CECSi cells (c-peptide secretion promoting effect)
The in vitro function of sig-GLP-1-GFP (fusion type, FIG. 6A(d)) was confirmed using human iPS cell-derived β cells. Human iPS cell-derived pancreatic β cells and ATCC CECSi cells secreting sig-GLP-1-GFP were prepared separately, and co-cultured at appropriate times. The methods for each are described below.
Cellartis (registered trademark) hiPS Beta Cells (ChiPS22) (Takara Bio: Y10100), which are pancreatic β cells derived from human iPS cells, were placed on a 24-well culture insert (Falcon CBP 353104) coated with the attached coating agent, and cultured for 8 days in Basal Medium 1 supplemented with supplements provided in the kit. From the 9th day, the medium was changed to Basal Medium 2 supplemented with supplements, and co-culture was initiated using cells on the 3rd day.
Frozen ATCC CECSi cells, frozen 10 days after differentiation induction, were placed in a 24-well plate at 1.1 x 105 cells/well and then infected with AAV2-sigGLP-1-GFP (fusion type) viral vector and control GFP viral vector at MOI: 100,000 on the fourth day, and the viral vector was removed the next day. The medium was replaced every two days, and co-culture was performed on the fourth day after infection. When co-culture was started, the medium was replaced with Krebs buffer (KRB), and after 24 hours, the supernatant of pancreatic β cells was collected, KRB was added again, and the amount of c-peptide contained in the supernatant after 24 hours was measured using a c-peptide ELISA kit (AB Clone RKO3588). Compared to infection with the control GFP viral vector, co-culture with the AAV2-sigGLP-1-GFP (fusion type) viral vector showed an increase in the amount of c-peptide secreted into the medium (Figure 10B).
The AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the plasmid sig-GLP-1-GFP (Figure 6A(d)) or a plasmid containing only the EGFP sequence, respectively.
実験例(実施例)7:GLP-1を発現・分泌するCECSi細胞を健常SCIDマウスに腹腔内移植した場合の血糖コントロールに及ぼす影響
GLP-1を発現・分泌するCECSi細胞のin vivoでの機能を検討するために、健常SCIDマウスにおける血糖コントロールへの影響を検証した。
6ウェルに5×105/ウェルで起眠した凍結ATCC CECSI細胞にAAV2-sig-GLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターをMOI:100000で24時間感染させた。ウイルス除去後2日間後に細胞を回収し、生理食塩水100μlに懸濁した状態で調製する。SCIDマウスを4時間絶食にし、調製した細胞を腹腔内に移植した。1匹目から5分間隔で、20%グルコース溶液を体重当たり2g/kgになるようにゾンデでマウスの胃に直接糖負荷した。血糖値測定は糖負荷直前を0時間とし、糖負荷後から30分、1時間、1.5時間、2時間測定したところ、コントロールでは30分で血糖の上昇がみられるが、sig-GLP-1-GFP存在下では血糖の上昇の抑制が確認された(図11A)。体内におけるsig-GLP-1-GFPの分布については、糖負荷直前と30分後に採血したサンプルを用いてELISAを実施したところ、糖負荷後30分の血液中にsigGLP-1が存在することが確認された(図11B)。
AAV2-sigGLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターは、セロタイプ2のAAV(AAV2)を用い、それぞれプラスミドsig-GLP-1(図6A(b))又はEGFP配列のみ有するプラスミドを用いてベクター構築することによって作製した。 Experimental Example (Example) 7: Effect on glycemic control when CECSi cells expressing and secreting GLP-1 are intraperitoneally transplanted into healthy SCID mice In order to investigate the in vivo function of CECSi cells expressing and secreting GLP-1, the effect on glycemic control in healthy SCID mice was examined.
Frozen ATCC CECSI cells, 5x105 /well, were infected with AAV2-sig-GLP-1-GFP (fusion type) virus vector and control GFP virus vector at MOI: 100000 for 24 hours. After 2 days of virus removal, the cells were collected and suspended in 100μl of physiological saline. SCID mice were fasted for 4 hours, and the prepared cells were transplanted into the abdominal cavity. Starting from the first mouse, 20% glucose solution was directly loaded into the stomach of the mouse with a sonde at 5-minute intervals to 2g/kg of body weight. Blood glucose levels were measured immediately before the glucose load, with the time set as 0 hours, and 30 minutes, 1 hour, 1.5 hours, and 2 hours after the glucose load. In the control, blood glucose increased at 30 minutes, but in the presence of sig-GLP-1-GFP, the increase in blood glucose was suppressed (Fig. 11A). Regarding the distribution of sig-GLP-1-GFP in the body, ELISA was performed using blood samples taken immediately before and 30 minutes after glucose loading, and it was confirmed that sigGLP-1 was present in the blood 30 minutes after glucose loading (Figure 11B).
The AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the sig-GLP-1 plasmid (Figure 6A(b)) or a plasmid carrying only the EGFP sequence, respectively.
GLP-1を発現・分泌するCECSi細胞のin vivoでの機能を検討するために、健常SCIDマウスにおける血糖コントロールへの影響を検証した。
6ウェルに5×105/ウェルで起眠した凍結ATCC CECSI細胞にAAV2-sig-GLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターをMOI:100000で24時間感染させた。ウイルス除去後2日間後に細胞を回収し、生理食塩水100μlに懸濁した状態で調製する。SCIDマウスを4時間絶食にし、調製した細胞を腹腔内に移植した。1匹目から5分間隔で、20%グルコース溶液を体重当たり2g/kgになるようにゾンデでマウスの胃に直接糖負荷した。血糖値測定は糖負荷直前を0時間とし、糖負荷後から30分、1時間、1.5時間、2時間測定したところ、コントロールでは30分で血糖の上昇がみられるが、sig-GLP-1-GFP存在下では血糖の上昇の抑制が確認された(図11A)。体内におけるsig-GLP-1-GFPの分布については、糖負荷直前と30分後に採血したサンプルを用いてELISAを実施したところ、糖負荷後30分の血液中にsigGLP-1が存在することが確認された(図11B)。
AAV2-sigGLP-1-GFP(融合型)ウイルスベクターとコントロールGFPウイルスベクターは、セロタイプ2のAAV(AAV2)を用い、それぞれプラスミドsig-GLP-1(図6A(b))又はEGFP配列のみ有するプラスミドを用いてベクター構築することによって作製した。 Experimental Example (Example) 7: Effect on glycemic control when CECSi cells expressing and secreting GLP-1 are intraperitoneally transplanted into healthy SCID mice In order to investigate the in vivo function of CECSi cells expressing and secreting GLP-1, the effect on glycemic control in healthy SCID mice was examined.
Frozen ATCC CECSI cells, 5x105 /well, were infected with AAV2-sig-GLP-1-GFP (fusion type) virus vector and control GFP virus vector at MOI: 100000 for 24 hours. After 2 days of virus removal, the cells were collected and suspended in 100μl of physiological saline. SCID mice were fasted for 4 hours, and the prepared cells were transplanted into the abdominal cavity. Starting from the first mouse, 20% glucose solution was directly loaded into the stomach of the mouse with a sonde at 5-minute intervals to 2g/kg of body weight. Blood glucose levels were measured immediately before the glucose load, with the time set as 0 hours, and 30 minutes, 1 hour, 1.5 hours, and 2 hours after the glucose load. In the control, blood glucose increased at 30 minutes, but in the presence of sig-GLP-1-GFP, the increase in blood glucose was suppressed (Fig. 11A). Regarding the distribution of sig-GLP-1-GFP in the body, ELISA was performed using blood samples taken immediately before and 30 minutes after glucose loading, and it was confirmed that sigGLP-1 was present in the blood 30 minutes after glucose loading (Figure 11B).
The AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the sig-GLP-1 plasmid (Figure 6A(b)) or a plasmid carrying only the EGFP sequence, respectively.
本発明によれば、角膜内皮代替細胞にGLP-1分泌機能を持たせることができた。GLP-1分泌機能を有する角膜内皮代替細胞によりGLP-1が関与する疾患、特にGLP-1分泌が有用な疾患(糖尿病、等)に対する新規細胞治療の開発が可能となる。
本出願は、日本で出願された特願2022-158838(出願日:2022年9月30日)を基礎としておりその内容は本明細書に全て包含されるものである。 According to the present invention, it has become possible to impart a GLP-1 secretion function to corneal endothelial substitute cells. Corneal endothelial substitute cells having a GLP-1 secretion function make it possible to develop a new cell therapy for diseases involving GLP-1, particularly diseases in which GLP-1 secretion is useful (diabetes, etc.).
This application is based on Patent Application No. 2022-158838 filed in Japan (filing date: September 30, 2022), the contents of which are incorporated in their entirety herein.
本出願は、日本で出願された特願2022-158838(出願日:2022年9月30日)を基礎としておりその内容は本明細書に全て包含されるものである。 According to the present invention, it has become possible to impart a GLP-1 secretion function to corneal endothelial substitute cells. Corneal endothelial substitute cells having a GLP-1 secretion function make it possible to develop a new cell therapy for diseases involving GLP-1, particularly diseases in which GLP-1 secretion is useful (diabetes, etc.).
This application is based on Patent Application No. 2022-158838 filed in Japan (filing date: September 30, 2022), the contents of which are incorporated in their entirety herein.
Claims (16)
- GLP-1分泌機能を有する、多能性幹細胞、又は幹細胞から分化誘導された細胞。 Pluripotent stem cells or cells induced to differentiate from stem cells that have the ability to secrete GLP-1.
- 幹細胞から分化誘導された細胞が内皮細胞である、請求項1記載の細胞。 The cell according to claim 1, wherein the cell induced to differentiate from a stem cell is an endothelial cell.
- 多能性幹細胞又は幹細胞がiPS細胞である、請求項1記載の細胞。 The cell according to claim 1, wherein the pluripotent stem cell or stem cell is an iPS cell.
- 内皮細胞が角膜内皮細胞である、請求項2記載の細胞。 The cells according to claim 2, wherein the endothelial cells are corneal endothelial cells.
- GLP-1を発現する請求項1記載の細胞。 The cell according to claim 1, which expresses GLP-1.
- GLP-1をコードする核酸を細胞に導入する工程を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞、又は幹細胞から分化誘導された細胞である、方法。 A method for producing cells that express GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into cells, the cells being pluripotent stem cells or cells induced to differentiate from stem cells.
- 核酸の細胞への導入が遺伝子導入又はゲノム編集によるものである、請求項6記載の方法。 The method according to claim 6, wherein the introduction of the nucleic acid into the cell is by gene introduction or genome editing.
- 幹細胞から分化誘導された細胞が内皮細胞である、請求項6記載の方法。 The method according to claim 6, wherein the cells induced to differentiate from stem cells are endothelial cells.
- 多能性幹細胞又は幹細胞がiPS細胞である、請求項6記載の方法。 The method according to claim 6, wherein the pluripotent stem cells or stem cells are iPS cells.
- 内皮細胞が角膜内皮細胞である、請求項8記載の方法。 The method of claim 8, wherein the endothelial cells are corneal endothelial cells.
- (1)GLP-1をコードする核酸を発現ベクターに挿入し、該核酸を含む発現ベクターを作製する工程、
(2)前記核酸を含む発現ベクターを用いて細胞に該核酸を導入し、発現ベクターを含む細胞を作製する工程、及び
(3)前記発現ベクターを含む細胞を培養する工程、
を含む、GLP-1を発現する細胞の製造方法であって、該細胞が多能性幹細胞、又は幹細胞から分化誘導された細胞である、方法。 (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising: - 幹細胞から分化誘導された細胞が内皮細胞である、請求項11記載の方法。 The method according to claim 11, wherein the cells induced to differentiate from stem cells are endothelial cells.
- 多能性幹細胞又は幹細胞がiPS細胞である、請求項11記載の方法。 The method according to claim 11, wherein the pluripotent stem cells or stem cells are iPS cells.
- 内皮細胞が角膜内皮細胞である、請求項12記載の方法。 The method of claim 12, wherein the endothelial cells are corneal endothelial cells.
- 請求項1~5のいずれか1項に記載の細胞を含む医薬組成物。 A pharmaceutical composition comprising the cells according to any one of claims 1 to 5.
- 糖尿病、肥満、中枢神経障害、末梢神経障害、腎機能障害、心不全、虚血性心疾患、肝機能障害、味覚障害、肺炎の治療用である、請求項15記載の医薬組成物。 The pharmaceutical composition according to claim 15, which is for treating diabetes, obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction, taste disorders, and pneumonia.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2022-158838 | 2022-09-30 | ||
JP2022158838 | 2022-09-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024071382A1 true WO2024071382A1 (en) | 2024-04-04 |
Family
ID=90478132
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2023/035640 WO2024071382A1 (en) | 2022-09-30 | 2023-09-29 | Pluripotential stem cell and cell differentiated and induced from stem cell having glp-1 secretion function |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024071382A1 (en) |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005509409A (en) * | 2001-08-08 | 2005-04-14 | ジェンザイム・コーポレーション | Methods for treating diabetes and other glycemic disorders |
JP2010528614A (en) * | 2007-06-01 | 2010-08-26 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Modified nucleotide sequence encoding glucagon-like peptide-1 (GLP-1) |
WO2011136378A1 (en) * | 2010-04-30 | 2011-11-03 | 京都府公立大学法人 | Grafting material for genetic and cell therapy |
WO2013051722A1 (en) * | 2011-10-06 | 2013-04-11 | 学校法人 慶應義塾 | Method for producing corneal endothelial cell |
CN104805058A (en) * | 2014-01-23 | 2015-07-29 | 上海欧易生物医学科技有限公司 | Human umbilical cord-derived mesenchymal stem cell strain stably expressing exogenous GLP-1 gene |
JP2018523477A (en) * | 2015-08-06 | 2018-08-23 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | GLP-1 and its use in compositions for treating metabolic diseases |
WO2019142833A1 (en) * | 2018-01-16 | 2019-07-25 | 学校法人慶應義塾 | METHOD FOR DERIVING CORNEAL ENDOTHELIUM REPLACEMENT CELLS FROM iPS CELLS |
CN112138025A (en) * | 2020-09-27 | 2020-12-29 | 东北师范大学 | Long-acting GLP-1 gene modified stem cell preparation, preparation method and application thereof |
JP2022513736A (en) * | 2018-12-12 | 2022-02-09 | ザ ジョンズ ホプキンス ユニバーシティ | Preparation and cryopreservation of clinical-grade corneal endothelial cells derived from pluripotent stem cells |
-
2023
- 2023-09-29 WO PCT/JP2023/035640 patent/WO2024071382A1/en unknown
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005509409A (en) * | 2001-08-08 | 2005-04-14 | ジェンザイム・コーポレーション | Methods for treating diabetes and other glycemic disorders |
JP2010528614A (en) * | 2007-06-01 | 2010-08-26 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Modified nucleotide sequence encoding glucagon-like peptide-1 (GLP-1) |
WO2011136378A1 (en) * | 2010-04-30 | 2011-11-03 | 京都府公立大学法人 | Grafting material for genetic and cell therapy |
WO2013051722A1 (en) * | 2011-10-06 | 2013-04-11 | 学校法人 慶應義塾 | Method for producing corneal endothelial cell |
CN104805058A (en) * | 2014-01-23 | 2015-07-29 | 上海欧易生物医学科技有限公司 | Human umbilical cord-derived mesenchymal stem cell strain stably expressing exogenous GLP-1 gene |
JP2018523477A (en) * | 2015-08-06 | 2018-08-23 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | GLP-1 and its use in compositions for treating metabolic diseases |
WO2019142833A1 (en) * | 2018-01-16 | 2019-07-25 | 学校法人慶應義塾 | METHOD FOR DERIVING CORNEAL ENDOTHELIUM REPLACEMENT CELLS FROM iPS CELLS |
JP2022513736A (en) * | 2018-12-12 | 2022-02-09 | ザ ジョンズ ホプキンス ユニバーシティ | Preparation and cryopreservation of clinical-grade corneal endothelial cells derived from pluripotent stem cells |
CN112138025A (en) * | 2020-09-27 | 2020-12-29 | 东北师范大学 | Long-acting GLP-1 gene modified stem cell preparation, preparation method and application thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6005666B2 (en) | Production of hematopoietic progenitor cells by programming | |
US20220090078A1 (en) | Compositions and methods for induced tissue regeneration in mammalian species | |
JP6603529B2 (en) | Generation of reprogrammed pluripotent cells | |
US11015211B2 (en) | Cardiac cell reprogramming with myocardin and ASCL1 | |
JP2015523065A (en) | Heterologous protein expression method using recombinant minus-strand RNA viral vector | |
Jin et al. | Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology | |
JP6960396B2 (en) | Methods for Inducing Cell Division in End-Division Cells | |
US20120141441A1 (en) | Methods and Compositions for Treatment of Muscular Dystrophy | |
JP2024519218A (en) | Method for producing mature corneal endothelial cells | |
WO2024071382A1 (en) | Pluripotential stem cell and cell differentiated and induced from stem cell having glp-1 secretion function | |
US20200306296A1 (en) | Induced tissue regeneration using extracellular vesicles | |
CN114615985A (en) | Compositions comprising molecules that modify mRNA and methods of use thereof | |
JP7300764B2 (en) | Gene and cell therapy agents using cell fusion technology, and uses thereof | |
Loperfido | PiggyBac transposons for stem cell-based gene therapy of Duchenne muscular dystrophy | |
CN117716020A (en) | Method for producing mature hepatocytes | |
WO2023079105A1 (en) | Extracellular vesicles or composition thereof for use as a medicament, such as for the treatment of ocular surface disorders | |
Modarresi | Improved Plasmid-based Muscle Gene Therapy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23872601 Country of ref document: EP Kind code of ref document: A1 |